Sample records for target genes expression

  1. Multi-targeted priming for genome-wide gene expression assays.

    PubMed

    Adomas, Aleksandra B; Lopez-Giraldez, Francesc; Clark, Travis A; Wang, Zheng; Townsend, Jeffrey P

    2010-08-17

    Complementary approaches to assaying global gene expression are needed to assess gene expression in regions that are poorly assayed by current methodologies. A key component of nearly all gene expression assays is the reverse transcription of transcribed sequences that has traditionally been performed by priming the poly-A tails on many of the transcribed genes in eukaryotes with oligo-dT, or by priming RNA indiscriminately with random hexamers. We designed an algorithm to find common sequence motifs that were present within most protein-coding genes of Saccharomyces cerevisiae and of Neurospora crassa, but that were not present within their ribosomal RNA or transfer RNA genes. We then experimentally tested whether degenerately priming these motifs with multi-targeted primers improved the accuracy and completeness of transcriptomic assays. We discovered two multi-targeted primers that would prime a preponderance of genes in the genomes of Saccharomyces cerevisiae and Neurospora crassa while avoiding priming ribosomal RNA or transfer RNA. Examining the response of Saccharomyces cerevisiae to nitrogen deficiency and profiling Neurospora crassa early sexual development, we demonstrated that using multi-targeted primers in reverse transcription led to superior performance of microarray profiling and next-generation RNA tag sequencing. Priming with multi-targeted primers in addition to oligo-dT resulted in higher sensitivity, a larger number of well-measured genes and greater power to detect differences in gene expression. Our results provide the most complete and detailed expression profiles of the yeast nitrogen starvation response and N. crassa early sexual development to date. Furthermore, our multi-targeting priming methodology for genome-wide gene expression assays provides selective targeting of multiple sequences and counter-selection against undesirable sequences, facilitating a more complete and precise assay of the transcribed sequences within the genome.

  2. Characteristics of functional enrichment and gene expression level of human putative transcriptional target genes.

    PubMed

    Osato, Naoki

    2018-01-19

    Transcriptional target genes show functional enrichment of genes. However, how many and how significantly transcriptional target genes include functional enrichments are still unclear. To address these issues, I predicted human transcriptional target genes using open chromatin regions, ChIP-seq data and DNA binding sequences of transcription factors in databases, and examined functional enrichment and gene expression level of putative transcriptional target genes. Gene Ontology annotations showed four times larger numbers of functional enrichments in putative transcriptional target genes than gene expression information alone, independent of transcriptional target genes. To compare the number of functional enrichments of putative transcriptional target genes between cells or search conditions, I normalized the number of functional enrichment by calculating its ratios in the total number of transcriptional target genes. With this analysis, native putative transcriptional target genes showed the largest normalized number of functional enrichments, compared with target genes including 5-60% of randomly selected genes. The normalized number of functional enrichments was changed according to the criteria of enhancer-promoter interactions such as distance from transcriptional start sites and orientation of CTCF-binding sites. Forward-reverse orientation of CTCF-binding sites showed significantly higher normalized number of functional enrichments than the other orientations. Journal papers showed that the top five frequent functional enrichments were related to the cellular functions in the three cell types. The median expression level of transcriptional target genes changed according to the criteria of enhancer-promoter assignments (i.e. interactions) and was correlated with the changes of the normalized number of functional enrichments of transcriptional target genes. Human putative transcriptional target genes showed significant functional enrichments. Functional

  3. Identification and consequences of miRNA-target interactions--beyond repression of gene expression.

    PubMed

    Hausser, Jean; Zavolan, Mihaela

    2014-09-01

    Comparative genomics analyses and high-throughput experimental studies indicate that a microRNA (miRNA) binds to hundreds of sites across the transcriptome. Although the knockout of components of the miRNA biogenesis pathway has profound phenotypic consequences, most predicted miRNA targets undergo small changes at the mRNA and protein levels when the expression of the miRNA is perturbed. Alternatively, miRNAs can establish thresholds in and increase the coherence of the expression of their target genes, as well as reduce the cell-to-cell variability in target gene expression. Here, we review the recent progress in identifying miRNA targets and the emerging paradigms of how miRNAs shape the dynamics of target gene expression.

  4. MicroRNA expression, target genes, and signaling pathways in infants with a ventricular septal defect.

    PubMed

    Chai, Hui; Yan, Zhaoyuan; Huang, Ke; Jiang, Yuanqing; Zhang, Lin

    2018-02-01

    This study aimed to systematically investigate the relationship between miRNA expression and the occurrence of ventricular septal defect (VSD), and characterize the miRNA target genes and pathways that can lead to VSD. The miRNAs that were differentially expressed in blood samples from VSD and normal infants were screened and validated by implementing miRNA microarrays and qRT-PCR. The target genes regulated by differentially expressed miRNAs were predicted using three target gene databases. The functions and signaling pathways of the target genes were enriched using the GO database and KEGG database, respectively. The transcription and protein expression of specific target genes in critical pathways were compared in the VSD and normal control groups using qRT-PCR and western blotting, respectively. Compared with the normal control group, the VSD group had 22 differentially expressed miRNAs; 19 were downregulated and three were upregulated. The 10,677 predicted target genes participated in many biological functions related to cardiac development and morphogenesis. Four target genes (mGLUR, Gq, PLC, and PKC) were involved in the PKC pathway and four (ECM, FAK, PI3 K, and PDK1) were involved in the PI3 K-Akt pathway. The transcription and protein expression of these eight target genes were significantly upregulated in the VSD group. The 22 miRNAs that were dysregulated in the VSD group were mainly downregulated, which may result in the dysregulation of several key genes and biological functions related to cardiac development. These effects could also be exerted via the upregulation of eight specific target genes, the subsequent over-activation of the PKC and PI3 K-Akt pathways, and the eventual abnormal cardiac development and VSD.

  5. Quantification of differential gene expression by multiplexed targeted resequencing of cDNA

    PubMed Central

    Arts, Peer; van der Raadt, Jori; van Gestel, Sebastianus H.C.; Steehouwer, Marloes; Shendure, Jay; Hoischen, Alexander; Albers, Cornelis A.

    2017-01-01

    Whole-transcriptome or RNA sequencing (RNA-Seq) is a powerful and versatile tool for functional analysis of different types of RNA molecules, but sample reagent and sequencing cost can be prohibitive for hypothesis-driven studies where the aim is to quantify differential expression of a limited number of genes. Here we present an approach for quantification of differential mRNA expression by targeted resequencing of complementary DNA using single-molecule molecular inversion probes (cDNA-smMIPs) that enable highly multiplexed resequencing of cDNA target regions of ∼100 nucleotides and counting of individual molecules. We show that accurate estimates of differential expression can be obtained from molecule counts for hundreds of smMIPs per reaction and that smMIPs are also suitable for quantification of relative gene expression and allele-specific expression. Compared with low-coverage RNA-Seq and a hybridization-based targeted RNA-Seq method, cDNA-smMIPs are a cost-effective high-throughput tool for hypothesis-driven expression analysis in large numbers of genes (10 to 500) and samples (hundreds to thousands). PMID:28474677

  6. EBF factors drive expression of multiple classes of target genes governing neuronal development.

    PubMed

    Green, Yangsook S; Vetter, Monica L

    2011-04-30

    Early B cell factor (EBF) family members are transcription factors known to have important roles in several aspects of vertebrate neurogenesis, including commitment, migration and differentiation. Knowledge of how EBF family members contribute to neurogenesis is limited by a lack of detailed understanding of genes that are transcriptionally regulated by these factors. We performed a microarray screen in Xenopus animal caps to search for targets of EBF transcriptional activity, and identified candidate targets with multiple roles, including transcription factors of several classes. We determined that, among the most upregulated candidate genes with expected neuronal functions, most require EBF activity for some or all of their expression, and most have overlapping expression with ebf genes. We also found that the candidate target genes that had the most strongly overlapping expression patterns with ebf genes were predicted to be direct transcriptional targets of EBF transcriptional activity. The identification of candidate targets that are transcription factor genes, including nscl-1, emx1 and aml1, improves our understanding of how EBF proteins participate in the hierarchy of transcription control during neuronal development, and suggests novel mechanisms by which EBF activity promotes migration and differentiation. Other candidate targets, including pcdh8 and kcnk5, expand our knowledge of the types of terminal differentiated neuronal functions that EBF proteins regulate.

  7. [Construction and expression of the targeting super-antigen EGF-SEA fusion gene].

    PubMed

    Xie, Yang; Peng, Shaoping; Liao, Zhiying; Liu, Jiafeng; Liu, Xuemei; Chen, Weifeng

    2014-05-01

    To construct expression vector for the SEA-EGF fusion gene. Clone the SEA gene and the EGF gene segment with PCR and RT-PCR independently, and connect this two genes by the bridge PCR. Insert the fusion gene EGF-SEA into the expression vector PET-44. Induced the secretion of the fusion protein SEA-EGF by the antileptic. The gene fragment encoding EGF and SEA mature peptide was successfully cloned. The fusion gene EGF-SEA was successfully constructed and was inserted into expression vector. The new recombinant expression vector for fusion gene EGF-SEA is specific for head and neck cancer, laid the foundation for the further study of fusion protein SEA-EGF targeting immune therapy in head and neck tumors.

  8. Generation of TALE nickase-mediated gene-targeted cows expressing human serum albumin in mammary glands.

    PubMed

    Luo, Yan; Wang, Yongsheng; Liu, Jun; Cui, Chenchen; Wu, Yongyan; Lan, Hui; Chen, Qi; Liu, Xu; Quan, Fusheng; Guo, Zekun; Zhang, Yong

    2016-02-08

    Targeting exogenous genes at milk protein loci via gene-targeting technology is an ideal strategy for producing large quantities of pharmaceutical proteins. Transcription-activator-like effector (TALE) nucleases (TALENs) are an efficient genome-editing tool. However, the off-target effects may lead to unintended gene mutations. In this study, we constructed TALENs and TALE nickases directed against exon 2 of the bovine β-lactoglobulin (BLG) locus. The nickases can induce a site-specific DNA single-strand break, without inducing double-strand break and nonhomologous end joining mediated gene mutation, and lower cell apoptosis rate than TALENs. After co-transfecting the bovine fetal fibroblasts with human serum albumin (HSA) gene-targeting vector and TALE nickase expression vectors, approximately 4.8% (40/835) of the cell clones contained HSA at BLG locus. Unexpectedly, one homozygous gene-targeted cell clone (1/835, 0.1%) was obtained by targeting both alleles of BLG in a single round of transfection. The recombinant protein mimicking the endogenous BLG was highly expressed and correctly folded in the mammary glands of the targeted cows, and the expression level of HSA was significantly increased in the homozygous targeted cows. Results suggested that the combination of TALE nickase-mediated gene targeting and somatic cell nuclear transfer is a feasible and safe approach in producing gene-targeted livestock.

  9. Targeting gene expression selectively in cancer cells by using the progression-elevated gene-3 promoter.

    PubMed

    Su, Zhao-Zhong; Sarkar, Devanand; Emdad, Luni; Duigou, Gregory J; Young, Charles S H; Ware, Joy; Randolph, Aaron; Valerie, Kristoffer; Fisher, Paul B

    2005-01-25

    One impediment to effective cancer-specific gene therapy is the rarity of regulatory sequences targeting gene expression selectively in tumor cells. Although many tissue-specific promoters are recognized, few cancer-selective gene promoters are available. Progression-elevated gene-3 (PEG-3) is a rodent gene identified by subtraction hybridization that displays elevated expression as a function of transformation by diversely acting oncogenes, DNA damage, and cancer cell progression. The promoter of PEG-3, PEG-Prom, displays robust expression in a broad spectrum of human cancer cell lines with marginal expression in normal cellular counterparts. Whereas GFP expression, when under the control of a CMV promoter, is detected in both normal and cancer cells, when GFP is expressed under the control of the PEG-Prom, cancer-selective expression is evident. Mutational analysis identifies the AP-1 and PEA-3 transcription factors as primary mediators of selective, cancer-specific expression of the PEG-Prom. Synthesis of apoptosis-inducing genes, under the control of the CMV promoter, inhibits the growth of both normal and cancer cells, whereas PEG-Prom-mediated expression of these genes kills only cancer cells and spares normal cells. The efficacy of the PEG-Prom as part of a cancer gene therapeutic regimen is further documented by in vivo experiments in which PEG-Prom-controlled expression of an apoptosis-inducing gene completely inhibited prostate cancer xenograft growth in nude mice. These compelling observations indicate that the PEG-Prom, with its cancer-specific expression, provides a means of selectively delivering genes to cancer cells, thereby providing a crucial component in developing effective cancer gene therapies.

  10. Recombinant adeno-associated virus targets passenger gene expression to cones in primate retina

    NASA Astrophysics Data System (ADS)

    Mancuso, Katherine; Hendrickson, Anita E.; Connor, Thomas B., Jr.; Mauck, Matthew C.; Kinsella, James J.; Hauswirth, William W.; Neitz, Jay; Neitz, Maureen

    2007-05-01

    Recombinant adeno-associated virus (rAAV) is a promising vector for gene therapy of photoreceptor-based diseases. Previous studies have demonstrated that rAAV serotypes 2 and 5 can transduce both rod and cone photoreceptors in rodents and dogs, and it can target rods, but not cones in primates. Here we report that using a human cone-specific enhancer and promoter to regulate expression of a green fluorescent protein (GFP) reporter gene in an rAAV-5 vector successfully targeted expression of the reporter gene to primate cones, and the time course of GFP expression was able to be monitored in a living animal using the RetCam II digital imaging system.

  11. Changes in gene expression in PBMCs profiles of PPARα target genes in obese and non-obese individuals during fasting.

    PubMed

    Felicidade, Ingrid; Marcarini, Juliana Cristina; Carreira, Clísia Mara; Amarante, Marla Karine; Afman, Lydia A; Mantovani, Mário Sérgio; Ribeiro, Lúcia Regina

    2015-01-01

    The prevalence of obesity has risen dramatically and the World Health Organization estimates that 700 million people will be obese worldwide by 2015. Approximately, 50% of the Brazilian population above 20 years of age is overweight, and 16% is obese. This study aimed to evaluate the differences in the expression of PPARα target genes in human peripheral blood mononuclear cells (PBMCs) and free fatty acids (FFA) in obese and non-obese individuals after 24 h of fasting. We first presented evidence that Brazilian people exhibit expression changes in PPARα target genes in PBMCs under fasting conditions. Q-PCR was utilized to assess the mRNA expression levels of target genes. In both groups, the FFA concentrations increased significantly after 24 h of fasting. The basal FFA mean concentration was two-fold higher in the obese group compared with the non-obese group. After fasting, all genes evaluated in this study showed increased expression levels compared with basal expression in both groups. However, our results reveal no differences in gene expression between the obese and non-obese, more studies are necessary to precisely delineate the associated mechanisms, particularly those that include groups with different degrees of obesity and patients with diabetes mellitus type 2 because the expression of the main genes that are involved in β-oxidation and glucose level maintenance are affected by these factors. © 2014 S. Karger AG, Basel.

  12. Biological effects of RNAi targeted inhibiting Tiam1 gene expression on cholangiocarcinoma cells.

    PubMed

    Cheng, Wei; Liu, Yaling; Zuo, Zhi; Yin, Xinmin; Jiang, Bo; Chen, Daojin; Peng, Chuang; Yang, Jianhui

    2015-01-01

    To investigate the characteristics of Tiam1 gene expression in human cholangiocarcinoma tissues and benign bile duct tissues, and to analyze the correlations between Tiam1 gene expression and the degree of tumor differentiation, invasive and metastatic abilities. To explore the effect of targeted inhibiting Tiam1 gene expression on proliferation and migration activity of human cholangiocarcinoma cells. Expression of Tiam1 in 83 cases of cholangiocarcinoma tissues and 25 cases of benign bile tissues was detected using immunohistochemistry. The clinical data of patients with cholangiocarcinoma were collected. The correlations between Tiam1 gene expression and the clinicopathologic features in patients with cholangiocarcinoma were analyzed. The human cholangiocarcinoma RBE cells were divided into 3 groups. Cells in experimental group and control group were respectively transfected with Tiam1 shRNA lentiviral vectors and negative shRNA lentiviral control vectors. Cells in blank group received no treatment. Real-time PCR endogenesis was used to verify Tiam1 gene expression. Cell cycle experiments and MTT assay were used to measure cell proliferation activity. Transwell test was used to detect cell migration activity. The negative rate Tiam1 protein expression in cholangiocarcinoma tissues was significantly higher than that in benign bile tissues (P<0.001). Tiam1 protein expression in cholangiocarcinoma tissues had correlations with cholangiocarcinoma differentiation degree, TNM stage and lymph node metastasis (P<0.05), and had no significant correlations with gender, age and distant metastasis (P>0.05). Real-time PCR detection indicated that Tiam1 expression of experimental group was significantly lower than that in control group and blank group (P<0.05), demonstrating that Tiam1 shRNA was effective on Tiam1 gene silencing in RBE cells. Cell cycle experiment showed that the percentage of S phase in cell cycle in experimental group was lower than that in control group

  13. Biological effects of RNAi targeted inhibiting Tiam1 gene expression on cholangiocarcinoma cells

    PubMed Central

    Cheng, Wei; Liu, Yaling; Zuo, Zhi; Yin, Xinmin; Jiang, Bo; Chen, Daojin; Peng, Chuang; Yang, Jianhui

    2015-01-01

    Objective: To investigate the characteristics of Tiam1 gene expression in human cholangiocarcinoma tissues and benign bile duct tissues, and to analyze the correlations between Tiam1 gene expression and the degree of tumor differentiation, invasive and metastatic abilities. To explore the effect of targeted inhibiting Tiam1 gene expression on proliferation and migration activity of human cholangiocarcinoma cells. Methods: Expression of Tiam1 in 83 cases of cholangiocarcinoma tissues and 25 cases of benign bile tissues was detected using immunohistochemistry. The clinical data of patients with cholangiocarcinoma were collected. The correlations between Tiam1 gene expression and the clinicopathologic features in patients with cholangiocarcinoma were analyzed. The human cholangiocarcinoma RBE cells were divided into 3 groups. Cells in experimental group and control group were respectively transfected with Tiam1 shRNA lentiviral vectors and negative shRNA lentiviral control vectors. Cells in blank group received no treatment. Real-time PCR endogenesis was used to verify Tiam1 gene expression. Cell cycle experiments and MTT assay were used to measure cell proliferation activity. Transwell test was used to detect cell migration activity. Results: The negative rate Tiam1 protein expression in cholangiocarcinoma tissues was significantly higher than that in benign bile tissues (P<0.001). Tiam1 protein expression in cholangiocarcinoma tissues had correlations with cholangiocarcinoma differentiation degree, TNM stage and lymph node metastasis (P<0.05), and had no significant correlations with gender, age and distant metastasis (P>0.05). Real-time PCR detection indicated that Tiam1 expression of experimental group was significantly lower than that in control group and blank group (P<0.05), demonstrating that Tiam1 shRNA was effective on Tiam1 gene silencing in RBE cells. Cell cycle experiment showed that the percentage of S phase in cell cycle in experimental group was lower

  14. Golden Gate Assembly of CRISPR gRNA expression array for simultaneously targeting multiple genes.

    PubMed

    Vad-Nielsen, Johan; Lin, Lin; Bolund, Lars; Nielsen, Anders Lade; Luo, Yonglun

    2016-11-01

    The engineered CRISPR/Cas9 technology has developed as the most efficient and broadly used genome editing tool. However, simultaneously targeting multiple genes (or genomic loci) in the same individual cells using CRISPR/Cas9 remain one technical challenge. In this article, we have developed a Golden Gate Assembly method for the generation of CRISPR gRNA expression arrays, thus enabling simultaneous gene targeting. Using this method, the generation of CRISPR gRNA expression array can be accomplished in 2 weeks, and contains up to 30 gRNA expression cassettes. We demonstrated in the study that simultaneously targeting 10 genomic loci or simultaneously inhibition of multiple endogenous genes could be achieved using the multiplexed gRNA expression array vector in human cells. The complete set of plasmids is available through the non-profit plasmid repository Addgene.

  15. Tumor-targeted inhibition by a novel strategy - mimoretrovirus expressing siRNA targeting the Pokemon gene.

    PubMed

    Tian, Zhiqiang; Wang, Huaizhi; Jia, Zhengcai; Shi, Jinglei; Tang, Jun; Mao, Liwei; Liu, Hongli; Deng, Yijing; He, Yangdong; Ruan, Zhihua; Li, Jintao; Wu, Yuzhang; Ni, Bing

    2010-12-01

    Pokemon gene has crucial but versatile functions in cell differentiation, proliferation and tumorigenesis. It is a master regulator of the ARF-HDM2-p53 and Rb-E2F pathways. The facts that the expression of Pokemon is essential for tumor formation and many kinds of tumors over-express the Pokemon gene make it an attractive target for therapeutic intervention for cancer treatment. In this study, we used an RNAi strategy to silence the Pokemon gene in a cervical cancer model. To address the issues involving tumor specific delivery and durable expression of siRNA, we applied the Arg-Gly-Asp (RGD) peptide ligand and polylysine (K(18)) fusion peptide to encapsulate a recombinant retrovirus plasmid expressing a siRNA targeting the Pokemon gene and produced the 'mimoretrovirus'. At charge ratio 2.0 of fusion peptide/plasmid, the mimoretrovirus formed stable and homogenous nanoparticles, and provided complete DNase I protection and complete gel retardation. This nanoparticle inhibited SiHa cell proliferation and invasion, while it promoted SiHa cell apoptosis. The binding of the nanoparticle to SiHa cells was mediated via the RGD-integrin α(v)β(3) interaction, as evidenced by the finding that unconjugated RGD peptide inhibited this binding significantly. This tumor-targeting mimoretrovirus exhibited excellent anti-tumor capacity in vivo in a nude mouse model. Moreover, the mimoretrovirus inhibited tumor growth with a much higher efficiency than recombinant retrovirus expressing siRNA or the K(18)/P4 nanoparticle lacking the RGD peptide. Results suggest that the RNAi/RGD-based mimoretrovirus developed in this study represents a novel anti-tumor strategy that may be applicable to most research involving cancer therapy and, thus, has promising potential as a cervical cancer treatment.

  16. Targeted repression of AXIN2 and MYC gene expression using designer TALEs

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Rennoll, Sherri A.; Scott, Samantha A.; Yochum, Gregory S., E-mail: gsy3@psu.edu

    Highlights: • We designed TALE–SID fusion proteins to target AXIN2 and MYC. • TALE–SIDs bound the chromosomal AXIN2 and MYC genes and repressed their expression. • TALE–SIDs repress β-catenin{sup S45F}-dependent AXIN2 and MYC transcription. - Abstract: Designer TALEs (dTALEs) are chimeric transcription factors that can be engineered to regulate gene expression in mammalian cells. Whether dTALEs can block gene transcription downstream of signal transduction cascades, however, has yet to be fully explored. Here we tested whether dTALEs can be used to target genes whose expression is controlled by Wnt/β-catenin signaling. TALE DNA binding domains were engineered to recognize sequences adjacentmore » to Wnt responsive enhancer elements (WREs) that control expression of axis inhibition protein 2 (AXIN2) and c-MYC (MYC). These custom DNA binding domains were linked to the mSin3A interaction domain (SID) to generate TALE–SID chimeric repressors. The TALE–SIDs repressed luciferase reporter activity, bound their genomic target sites, and repressed AXIN2 and MYC expression in HEK293 cells. We generated a novel HEK293 cell line to determine whether the TALE–SIDs could function downstream of oncogenic Wnt/β-catenin signaling. Treating these cells with doxycycline and tamoxifen stimulates nuclear accumulation of a stabilized form of β-catenin found in a subset of colorectal cancers. The TALE–SIDs repressed AXIN2 and MYC expression in these cells, which suggests that dTALEs could offer an effective therapeutic strategy for the treatment of colorectal cancer.« less

  17. Tumor targeting of gene expression through metal-coordinated conjugation with dextran.

    PubMed

    Hosseinkhani, Hossein; Aoyama, Teruyoshi; Ogawa, Osamu; Tabata, Yasuhiko

    2003-03-07

    Tumor targeting of plasmid DNA was achieved through the conjugation of dextran derivatives with chelate residues based on metal coordination. Diethylenetriamine pentaacetic acid (DTPA), spermidine (Sd), and spermine (Sm) were chemically introduced to the hydroxyl groups of dextran to obtain dextran-DTPA, dextran-Sd and dextran-Sm derivatives. Conjugation of the dextran derivative by Zn(2+) coordination decreased the apparent size of the plasmid DNA, depending on the derivative type. The negative zeta potential of plasmid DNA became almost 0 mV after Zn(2+)-coordinated conjugation with dextran-Sm. When the dextran derivative-plasmid DNA conjugates with Zn(2+) coordination were intravenously injected subcutaneously into mice bearing Meth-AR-1 fibrosarcoma, the dextran-Sm-plasmid DNA conjugate significantly enhanced the level of gene expression in the tumor, in contrast to the conjugate of other dextran derivatives and free plasmid DNA. The enhanced gene expression produced by the Zn(2+)-coordinated dextran-Sm-plasmid DNA conjugate was specific to the tumor, whereas a simple mixture of dextran-Sm and plasmid DNA was not effective. The level of gene expression depended on the percentage of chelate residues introduced, the mixing weight ratio of the plasmid DNA/Sm residue used for conjugate preparation, and the plasmid DNA dose. A fluorescent microscopic study revealed that localization of plasmid DNA in the tumor tissue was observed only after injection of the dextran-Sm-plasmid DNA conjugate with Zn(2+) coordination. In addition, the gene expression induced by the conjugate lasted for more than 10 days after the injection. We conclude that Zn(2+)-coordinated dextran-Sm conjugation is a promising way to enable plasmid DNA to target the tumor in gene expression as well as to prolong the duration of gene expression.

  18. ARNetMiT R Package: association rules based gene co-expression networks of miRNA targets.

    PubMed

    Özgür Cingiz, M; Biricik, G; Diri, B

    2017-03-31

    miRNAs are key regulators that bind to target genes to suppress their gene expression level. The relations between miRNA-target genes enable users to derive co-expressed genes that may be involved in similar biological processes and functions in cells. We hypothesize that target genes of miRNAs are co-expressed, when they are regulated by multiple miRNAs. With the usage of these co-expressed genes, we can theoretically construct co-expression networks (GCNs) related to 152 diseases. In this study, we introduce ARNetMiT that utilize a hash based association rule algorithm in a novel way to infer the GCNs on miRNA-target genes data. We also present R package of ARNetMiT, which infers and visualizes GCNs of diseases that are selected by users. Our approach assumes miRNAs as transactions and target genes as their items. Support and confidence values are used to prune association rules on miRNA-target genes data to construct support based GCNs (sGCNs) along with support and confidence based GCNs (scGCNs). We use overlap analysis and the topological features for the performance analysis of GCNs. We also infer GCNs with popular GNI algorithms for comparison with the GCNs of ARNetMiT. Overlap analysis results show that ARNetMiT outperforms the compared GNI algorithms. We see that using high confidence values in scGCNs increase the ratio of the overlapped gene-gene interactions between the compared methods. According to the evaluation of the topological features of ARNetMiT based GCNs, the degrees of nodes have power-law distribution. The hub genes discovered by ARNetMiT based GCNs are consistent with the literature.

  19. Phage-mediated Delivery of Targeted sRNA Constructs to Knock Down Gene Expression in E. coli.

    PubMed

    Bernheim, Aude G; Libis, Vincent K; Lindner, Ariel B; Wintermute, Edwin H

    2016-03-20

    RNA-mediated knockdowns are widely used to control gene expression. This versatile family of techniques makes use of short RNA (sRNA) that can be synthesized with any sequence and designed to complement any gene targeted for silencing. Because sRNA constructs can be introduced to many cell types directly or using a variety of vectors, gene expression can be repressed in living cells without laborious genetic modification. The most common RNA knockdown technology, RNA interference (RNAi), makes use of the endogenous RNA-induced silencing complex (RISC) to mediate sequence recognition and cleavage of the target mRNA. Applications of this technique are therefore limited to RISC-expressing organisms, primarily eukaryotes. Recently, a new generation of RNA biotechnologists have developed alternative mechanisms for controlling gene expression through RNA, and so made possible RNA-mediated gene knockdowns in bacteria. Here we describe a method for silencing gene expression in E. coli that functionally resembles RNAi. In this system a synthetic phagemid is designed to express sRNA, which may designed to target any sequence. The expression construct is delivered to a population of E. coli cells with non-lytic M13 phage, after which it is able to stably replicate as a plasmid. Antisense recognition and silencing of the target mRNA is mediated by the Hfq protein, endogenous to E. coli. This protocol includes methods for designing the antisense sRNA, constructing the phagemid vector, packaging the phagemid into M13 bacteriophage, preparing a live cell population for infection, and performing the infection itself. The fluorescent protein mKate2 and the antibiotic resistance gene chloramphenicol acetyltransferase (CAT) are targeted to generate representative data and to quantify knockdown effectiveness.

  20. Regulation of p53 Target Gene Expression by Peptidylarginine Deiminase 4 ▿ †

    PubMed Central

    Li, Pingxin; Yao, Hongjie; Zhang, Zhiqiang; Li, Ming; Luo, Yuan; Thompson, Paul R.; Gilmour, David S.; Wang, Yanming

    2008-01-01

    Histone Arg methylation has been correlated with transcriptional activation of p53 target genes. However, whether this modification is reversed to repress the expression of p53 target genes is unclear. Here, we report that peptidylarginine deiminase 4, a histone citrullination enzyme, is involved in the repression of p53 target genes. Inhibition or depletion of PAD4 elevated the expression of a subset of p53 target genes, including p21/CIP1/WAF1, leading to cell cycle arrest and apoptosis. Moreover, the induction of p21, cell cycle arrest, and apoptosis by PAD4 depletion is p53 dependent. Protein-protein interaction studies showed an interaction between p53 and PAD4. Chromatin immunoprecipitation assays showed that PAD4 is recruited to the p21 promoter in a p53-dependent manner. RNA polymerase II (Pol II) activities and the association of PAD4 are dynamically regulated at the p21 promoter during UV irradiation. Paused RNA Pol II and high levels of PAD4 were detected before UV treatment. At early time points after UV treatment, an increase of histone Arg methylation and a decrease of citrullination were correlated with a transient activation of p21. At later times after UV irradiation, a loss of RNA Pol II and an increase of PAD4 were detected at the p21 promoter. The dynamics of RNA Pol II activities after UV treatment were further corroborated by permanganate footprinting. Together, these results suggest a role of PAD4 in the regulation of p53 target gene expression. PMID:18505818

  1. Alpha-fetoprotein-targeted reporter gene expression imaging in hepatocellular carcinoma.

    PubMed

    Kim, Kwang Il; Chung, Hye Kyung; Park, Ju Hui; Lee, Yong Jin; Kang, Joo Hyun

    2016-07-21

    Hepatocellular carcinoma (HCC) is one of the most common cancers in Eastern Asia, and its incidence is increasing globally. Numerous experimental models have been developed to better our understanding of the pathogenic mechanism of HCC and to evaluate novel therapeutic approaches. Molecular imaging is a convenient and up-to-date biomedical tool that enables the visualization, characterization and quantification of biologic processes in a living subject. Molecular imaging based on reporter gene expression, in particular, can elucidate tumor-specific events or processes by acquiring images of a reporter gene's expression driven by tumor-specific enhancers/promoters. In this review, we discuss the advantages and disadvantages of various experimental HCC mouse models and we present in vivo images of tumor-specific reporter gene expression driven by an alpha-fetoprotein (AFP) enhancer/promoter system in a mouse model of HCC. The current mouse models of HCC development are established by xenograft, carcinogen induction and genetic engineering, representing the spectrum of tumor-inducing factors and tumor locations. The imaging analysis approach of reporter genes driven by AFP enhancer/promoter is presented for these different HCC mouse models. Such molecular imaging can provide longitudinal information about carcinogenesis and tumor progression. We expect that clinical application of AFP-targeted reporter gene expression imaging systems will be useful for the detection of AFP-expressing HCC tumors and screening of increased/decreased AFP levels due to disease or drug treatment.

  2. Tuning Gene Activity by Inducible and Targeted Regulation of Gene Expression in Minimal Bacterial Cells.

    PubMed

    Mariscal, Ana M; Kakizawa, Shigeyuki; Hsu, Jonathan Y; Tanaka, Kazuki; González-González, Luis; Broto, Alicia; Querol, Enrique; Lluch-Senar, Maria; Piñero-Lambea, Carlos; Sun, Lijie; Weyman, Philip D; Wise, Kim S; Merryman, Chuck; Tse, Gavin; Moore, Adam J; Hutchison, Clyde A; Smith, Hamilton O; Tomita, Masaru; Venter, J Craig; Glass, John I; Piñol, Jaume; Suzuki, Yo

    2018-05-22

    Functional genomics studies in minimal mycoplasma cells enable unobstructed access to some of the most fundamental processes in biology. Conventional transposon bombardment and gene knockout approaches often fail to reveal functions of genes that are essential for viability, where lethality precludes phenotypic characterization. Conditional inactivation of genes is effective for characterizing functions central to cell growth and division, but tools are limited for this purpose in mycoplasmas. Here we demonstrate systems for inducible repression of gene expression based on clustered regularly interspaced short palindromic repeats-mediated interference (CRISPRi) in Mycoplasma pneumoniae and synthetic Mycoplasma mycoides, two organisms with reduced genomes actively used in systems biology studies. In the synthetic cell, we also demonstrate inducible gene expression for the first time. Time-course data suggest rapid kinetics and reversible engagement of CRISPRi. Targeting of six selected endogenous genes with this system results in lowered transcript levels or reduced growth rates that agree with lack or shortage of data in previous transposon bombardment studies, and now produces actual cells to analyze. The ksgA gene encodes a methylase that modifies 16S rRNA, rendering it vulnerable to inhibition by the antibiotic kasugamycin. Targeting the ksgA gene with CRISPRi removes the lethal effect of kasugamycin and enables cell growth, thereby establishing specific and effective gene modulation with our system. The facile methods for conditional gene activation and inactivation in mycoplasmas open the door to systematic dissection of genetic programs at the core of cellular life.

  3. Targeted expression of suicide gene by tissue-specific promoter and microRNA regulation for cancer gene therapy.

    PubMed

    Danda, Ravikanth; Krishnan, Gopinath; Ganapathy, Kalaivani; Krishnan, Uma Maheswari; Vikas, Khetan; Elchuri, Sailaja; Chatterjee, Nivedita; Krishnakumar, Subramanian

    2013-01-01

    In order to realise the full potential of cancer suicide gene therapy that allows the precise expression of suicide gene in cancer cells, we used a tissue specific Epithelial cell adhesion molecule (EpCAM) promoter (EGP-2) that directs transgene Herpes simplex virus-thymidine kinase (HSV-TK) expression preferentially in EpCAM over expressing cancer cells. EpCAM levels are considerably higher in retinoblastoma (RB), a childhood eye cancer with limited expression in normal cells. Use of miRNA regulation, adjacent to the use of the tissue-specific promoter, would provide the second layer of control to the transgene expression only in the tumor cells while sparing the normal cells. To test this hypothesis we cloned let-7b miRNA targets in the 3'UTR region of HSV-TK suicide gene driven by EpCAM promoter because let-7 family miRNAs, including let-7b, were found to be down regulated in the RB tumors and cell lines. We used EpCAM over expressing and let-7 down regulated RB cell lines Y79, WERI-Rb1 (EpCAM (+ve)/let-7b(down-regulated)), EpCAM down regulated, let-7 over expressing normal retinal Müller glial cell line MIO-M1(EpCAM (-ve)/let-7b(up-regulated)), and EpCAM up regulated, let-7b up-regulated normal thyroid cell line N-Thy-Ori-3.1(EpCAM (+ve)/let-7b(up-regulated)) in the study. The cell proliferation was measured by MTT assay, apoptosis was measured by probing cleaved Caspase3, EpCAM and TK expression were quantified by Western blot. Our results showed that the EGP2-promoter HSV-TK (EGP2-TK) construct with 2 or 4 copies of let-7b miRNA targets expressed TK gene only in Y79, WERI-Rb-1, while the TK gene did not express in MIO-M1. In summary, we have developed a tissue-specific, miRNA-regulated dual control vector, which selectively expresses the suicide gene in EpCAM over expressing cells.

  4. An Oomycete CRN Effector Reprograms Expression of Plant HSP Genes by Targeting their Promoters

    PubMed Central

    Song, Tianqiao; Ma, Zhenchuan; Shen, Danyu; Li, Qi; Li, Wanlin; Su, Liming; Ye, Tingyue; Zhang, Meixiang; Wang, Yuanchao; Dou, Daolong

    2015-01-01

    Oomycete pathogens produce a large number of CRN effectors to manipulate plant immune responses and promote infection. However, their functional mechanisms are largely unknown. Here, we identified a Phytophthora sojae CRN effector PsCRN108 which contains a putative DNA-binding helix-hairpin-helix (HhH) motif and acts in the plant cell nucleus. Silencing of the PsCRN108 gene reduced P. sojae virulence to soybean, while expression of the gene in Nicotiana benthamiana and Arabidopsis thaliana enhanced plant susceptibility to P. capsici. Moreover, PsCRN108 could inhibit expression of HSP genes in A. thaliana, N. benthamiana and soybean. Both the HhH motif and nuclear localization signal of this effector were required for its contribution to virulence and its suppression of HSP gene expression. Furthermore, we found that PsCRN108 targeted HSP promoters in an HSE- and HhH motif-dependent manner. PsCRN108 could inhibit the association of the HSE with the plant heat shock transcription factor AtHsfA1a, which initializes HSP gene expression in response to stress. Therefore, our data support a role for PsCRN108 as a nucleomodulin in down-regulating the expression of plant defense-related genes by directly targeting specific plant promoters. PMID:26714171

  5. An Oomycete CRN Effector Reprograms Expression of Plant HSP Genes by Targeting their Promoters.

    PubMed

    Song, Tianqiao; Ma, Zhenchuan; Shen, Danyu; Li, Qi; Li, Wanlin; Su, Liming; Ye, Tingyue; Zhang, Meixiang; Wang, Yuanchao; Dou, Daolong

    2015-12-01

    Oomycete pathogens produce a large number of CRN effectors to manipulate plant immune responses and promote infection. However, their functional mechanisms are largely unknown. Here, we identified a Phytophthora sojae CRN effector PsCRN108 which contains a putative DNA-binding helix-hairpin-helix (HhH) motif and acts in the plant cell nucleus. Silencing of the PsCRN108 gene reduced P. sojae virulence to soybean, while expression of the gene in Nicotiana benthamiana and Arabidopsis thaliana enhanced plant susceptibility to P. capsici. Moreover, PsCRN108 could inhibit expression of HSP genes in A. thaliana, N. benthamiana and soybean. Both the HhH motif and nuclear localization signal of this effector were required for its contribution to virulence and its suppression of HSP gene expression. Furthermore, we found that PsCRN108 targeted HSP promoters in an HSE- and HhH motif-dependent manner. PsCRN108 could inhibit the association of the HSE with the plant heat shock transcription factor AtHsfA1a, which initializes HSP gene expression in response to stress. Therefore, our data support a role for PsCRN108 as a nucleomodulin in down-regulating the expression of plant defense-related genes by directly targeting specific plant promoters.

  6. Self-focusing therapeutic gene delivery with intelligent gene vector swarms: intra-swarm signalling through receptor transgene expression in targeted cells.

    PubMed

    Tolmachov, Oleg E

    2015-01-01

    Gene delivery in vivo that is tightly focused on the intended target cells is essential to maximize the benefits of gene therapy and to reduce unwanted side-effects. Cell surface markers are immediately available for probing by therapeutic gene vectors and are often used to direct gene transfer with these vectors to specific target cell populations. However, it is not unusual for the choice of available extra-cellular markers to be too scarce to provide a reliable definition of the desired therapeutically relevant set of target cells. Therefore, interrogation of intra-cellular determinants of cell-specificity, such as tissue-specific transcription factors, can be vital in order to provide detailed cell-guiding information to gene vector particles. An important improvement in cell-specific gene delivery can be achieved through auto-buildup in vector homing efficiency using intelligent 'self-focusing' of swarms of vector particles on target cells. Vector self-focusing was previously suggested to rely on the release of diffusible chemo-attractants after a successful target-specific hit by 'scout' vector particles. I hypothesize that intelligent self-focusing behaviour of swarms of cell-targeted therapeutic gene vectors can be accomplished without the employment of difficult-to-use diffusible chemo-attractants, instead relying on the intra-swarm signalling through cells expressing a non-diffusible extra-cellular receptor for the gene vectors. In the proposed model, cell-guiding information is gathered by the 'scout' gene vector particles, which: (1) attach to a variety of cells via a weakly binding (low affinity) receptor; (2) successfully facilitate gene transfer into these cells; (3) query intra-cellular determinants of cell-specificity with their transgene expression control elements and (4) direct the cell-specific biosynthesis of a vector-encoded strongly binding (high affinity) cell-surface receptor. Free members of the vector swarm loaded with therapeutic cargo

  7. Alpha-fetoprotein-targeted reporter gene expression imaging in hepatocellular carcinoma

    PubMed Central

    Kim, Kwang Il; Chung, Hye Kyung; Park, Ju Hui; Lee, Yong Jin; Kang, Joo Hyun

    2016-01-01

    Hepatocellular carcinoma (HCC) is one of the most common cancers in Eastern Asia, and its incidence is increasing globally. Numerous experimental models have been developed to better our understanding of the pathogenic mechanism of HCC and to evaluate novel therapeutic approaches. Molecular imaging is a convenient and up-to-date biomedical tool that enables the visualization, characterization and quantification of biologic processes in a living subject. Molecular imaging based on reporter gene expression, in particular, can elucidate tumor-specific events or processes by acquiring images of a reporter gene’s expression driven by tumor-specific enhancers/promoters. In this review, we discuss the advantages and disadvantages of various experimental HCC mouse models and we present in vivo images of tumor-specific reporter gene expression driven by an alpha-fetoprotein (AFP) enhancer/promoter system in a mouse model of HCC. The current mouse models of HCC development are established by xenograft, carcinogen induction and genetic engineering, representing the spectrum of tumor-inducing factors and tumor locations. The imaging analysis approach of reporter genes driven by AFP enhancer/promoter is presented for these different HCC mouse models. Such molecular imaging can provide longitudinal information about carcinogenesis and tumor progression. We expect that clinical application of AFP-targeted reporter gene expression imaging systems will be useful for the detection of AFP-expressing HCC tumors and screening of increased/decreased AFP levels due to disease or drug treatment. PMID:27468205

  8. A gene expression profile indicative of early stage HER2 targeted therapy response.

    PubMed

    O'Neill, Fiona; Madden, Stephen F; Clynes, Martin; Crown, John; Doolan, Padraig; Aherne, Sinéad T; O'Connor, Robert

    2013-07-01

    Efficacious application of HER2-targetting agents requires the identification of novel predictive biomarkers. Lapatinib, afatinib and neratinib are tyrosine kinase inhibitors (TKIs) of HER2 and EGFR growth factor receptors. A panel of breast cancer cell lines was treated with these agents, trastuzumab, gefitinib and cytotoxic therapies and the expression pattern of a specific panel of genes using RT-PCR was investigated as a potential marker of early drug response to HER2-targeting therapies. Treatment of HER2 TKI-sensitive SKBR3 and BT474 cell lines with lapatinib, afatinib and neratinib induced an increase in the expression of RB1CC1, ERBB3, FOXO3a and NR3C1. The response directly correlated with the degree of sensitivity. This expression pattern switched from up-regulated to down-regulated in the HER2 expressing, HER2-TKI insensitive cell line MDAMB453. Expression of the CCND1 gene demonstrated an inversely proportional response to drug exposure. A similar expression pattern was observed following the treatment with both neratinib and afatinib. These patterns were retained following exposure to traztuzumab and lapatinib plus capecitabine. In contrast, gefitinib, dasatinib and epirubicin treatment resulted in a completely different expression pattern change. In these HER2-expressing cell line models, lapatinib, neratinib, afatinib and trastuzumab treatment generated a characteristic and specific gene expression response, proportionate to the sensitivity of the cell lines to the HER2 inhibitor.Characterisation of the induced changes in expression levels of these genes may therefore give a valuable, very early predictor of the likely extent and specificity of tumour HER2 inhibitor response in patients, potentially guiding more specific use of these agents.

  9. The LIM-homeodomain transcription factor LMX1B regulates expression of NF-kappa B target genes

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Rascle, Anne; Neumann, Tanja; Raschta, Anne-Sarah

    2009-01-01

    LMX1B is a LIM-homeodomain transcription factor essential for development. Putative LMX1B target genes have been identified through mouse gene targeting studies, but their identity as direct LMX1B targets remains hypothetical. We describe here the first molecular characterization of LMX1B target gene regulation. Microarray analysis using a tetracycline-inducible LMX1B expression system in HeLa cells revealed that a subset of NF-{kappa}B target genes, including IL-6 and IL-8, are upregulated in LMX1B-expressing cells. Inhibition of NF-{kappa}B activity by short interfering RNA-mediated knock-down of p65 impairs, while activation of NF-{kappa}B activity by TNF-{alpha} synergizes induction of NF-{kappa}B target genes by LMX1B. Chromatin immunoprecipitation demonstratedmore » that LMX1B binds to the proximal promoter of IL-6 and IL-8 in vivo, in the vicinity of the characterized {kappa}B site, and that LMX1B recruitment correlates with increased NF-{kappa}B DNA association. IL-6 promoter-reporter assays showed that the {kappa}B site and an adjacent putative LMX1B binding motif are both involved in LMX1B-mediated transcription. Expression of NF-{kappa}B target genes is affected in the kidney of Lmx1b{sup -/-} knock-out mice, thus supporting the biological relevance of our findings. Together, these data demonstrate for the first time that LMX1B directly regulates transcription of a subset of NF-{kappa}B target genes in cooperation with nuclear p50/p65 NF-{kappa}B.« less

  10. Gene expression profiling in multiple myeloma--reporting of entities, risk, and targets in clinical routine.

    PubMed

    Meissner, Tobias; Seckinger, Anja; Rème, Thierry; Hielscher, Thomas; Möhler, Thomas; Neben, Kai; Goldschmidt, Hartmut; Klein, Bernard; Hose, Dirk

    2011-12-01

    Multiple myeloma is an incurable malignant plasma cell disease characterized by survival ranging from several months to more than 15 years. Assessment of risk and underlying molecular heterogeneity can be excellently done by gene expression profiling (GEP), but its way into clinical routine is hampered by the lack of an appropriate reporting tool and the integration with other prognostic factors into a single "meta" risk stratification. The GEP-report (GEP-R) was built as an open-source software developed in R for gene expression reporting in clinical practice using Affymetrix microarrays. GEP-R processes new samples by applying a documentation-by-value strategy to the raw data to be able to assign thresholds and grouping algorithms defined on a reference cohort of 262 patients with multiple myeloma. Furthermore, we integrated expression-based and conventional prognostic factors within one risk stratification (HM-metascore). The GEP-R comprises (i) quality control, (ii) sample identity control, (iii) biologic classification, (iv) risk stratification, and (v) assessment of target genes. The resulting HM-metascore is defined as the sum over the weighted factors gene expression-based risk-assessment (UAMS-, IFM-score), proliferation, International Staging System (ISS) stage, t(4;14), and expression of prognostic target genes (AURKA, IGF1R) for which clinical grade inhibitors exist. The HM-score delineates three significantly different groups of 13.1%, 72.1%, and 14.7% of patients with a 6-year survival rate of 89.3%, 60.6%, and 18.6%, respectively. GEP reporting allows prospective assessment of risk and target gene expression and integration of current prognostic factors in clinical routine, being customizable about novel parameters or other cancer entities. ©2011 AACR.

  11. Zinc-finger nuclease-mediated targeted insertion of reporter genes for quantitative imaging of gene expression in sea urchin embryos

    PubMed Central

    Ochiai, Hiroshi; Sakamoto, Naoaki; Fujita, Kazumasa; Nishikawa, Masatoshi; Suzuki, Ken-ichi; Matsuura, Shinya; Miyamoto, Tatsuo; Sakuma, Tetsushi; Shibata, Tatsuo; Yamamoto, Takashi

    2012-01-01

    To understand complex biological systems, such as the development of multicellular organisms, it is important to characterize the gene expression dynamics. However, there is currently no universal technique for targeted insertion of reporter genes and quantitative imaging in multicellular model systems. Recently, genome editing using zinc-finger nucleases (ZFNs) has been reported in several models. ZFNs consist of a zinc-finger DNA-binding array with the nuclease domain of the restriction enzyme FokI and facilitate targeted transgene insertion. In this study, we successfully inserted a GFP reporter cassette into the HpEts1 gene locus of the sea urchin, Hemicentrotus pulcherrimus. We achieved this insertion by injecting eggs with a pair of ZFNs for HpEts1 with a targeting donor construct that contained ∼1-kb homology arms and a 2A-histone H2B–GFP cassette. We increased the efficiency of the ZFN-mediated targeted transgene insertion by in situ linearization of the targeting donor construct and cointroduction of an mRNA for a dominant-negative form of HpLig4, which encodes the H. pulcherrimus homolog of DNA ligase IV required for error-prone nonhomologous end joining. We measured the fluorescence intensity of GFP at the single-cell level in living embryos during development and found that there was variation in HpEts1 expression among the primary mesenchyme cells. These findings demonstrate the feasibility of ZFN-mediated targeted transgene insertion to enable quantification of the expression levels of endogenous genes during development in living sea urchin embryos. PMID:22711830

  12. A gene expression profile indicative of early stage HER2 targeted therapy response

    PubMed Central

    2013-01-01

    Background Efficacious application of HER2-targetting agents requires the identification of novel predictive biomarkers. Lapatinib, afatinib and neratinib are tyrosine kinase inhibitors (TKIs) of HER2 and EGFR growth factor receptors. A panel of breast cancer cell lines was treated with these agents, trastuzumab, gefitinib and cytotoxic therapies and the expression pattern of a specific panel of genes using RT-PCR was investigated as a potential marker of early drug response to HER2-targeting therapies. Results Treatment of HER2 TKI-sensitive SKBR3 and BT474 cell lines with lapatinib, afatinib and neratinib induced an increase in the expression of RB1CC1, ERBB3, FOXO3a and NR3C1. The response directly correlated with the degree of sensitivity. This expression pattern switched from up-regulated to down-regulated in the HER2 expressing, HER2-TKI insensitive cell line MDAMB453. Expression of the CCND1 gene demonstrated an inversely proportional response to drug exposure. A similar expression pattern was observed following the treatment with both neratinib and afatinib. These patterns were retained following exposure to traztuzumab and lapatinib plus capecitabine. In contrast, gefitinib, dasatinib and epirubicin treatment resulted in a completely different expression pattern change. Conclusions In these HER2-expressing cell line models, lapatinib, neratinib, afatinib and trastuzumab treatment generated a characteristic and specific gene expression response, proportionate to the sensitivity of the cell lines to the HER2 inhibitor. Characterisation of the induced changes in expression levels of these genes may therefore give a valuable, very early predictor of the likely extent and specificity of tumour HER2 inhibitor response in patients, potentially guiding more specific use of these agents. PMID:23816254

  13. Expression profiling and bioinformatic analyses suggest new target genes and pathways for human hair follicle related microRNAs.

    PubMed

    Hochfeld, Lara M; Anhalt, Thomas; Reinbold, Céline S; Herrera-Rivero, Marisol; Fricker, Nadine; Nöthen, Markus M; Heilmann-Heimbach, Stefanie

    2017-02-22

    Human hair follicle (HF) cycling is characterised by the tight orchestration and regulation of signalling cascades. Research shows that micro(mi)RNAs are potent regulators of these pathways. However, knowledge of the expression of miRNAs and their target genes and pathways in the human HF is limited. The objective of this study was to improve understanding of the role of miRNAs and their regulatory interactions in the human HF. Expression levels of ten candidate miRNAs with reported functions in hair biology were assessed in HFs from 25 healthy male donors. MiRNA expression levels were correlated with mRNA-expression levels from the same samples. Identified target genes were tested for enrichment in biological pathways and accumulation in protein-protein interaction (PPI) networks. Expression in the human HF was confirmed for seven of the ten candidate miRNAs, and numerous target genes for miR-24, miR-31, and miR-106a were identified. While the latter include several genes with known functions in hair biology (e.g., ITGB1, SOX9), the majority have not been previously implicated (e.g., PHF1). Target genes were enriched in pathways of interest to hair biology, such as integrin and GnRH signalling, and the respective gene products showed accumulation in PPIs. Further investigation of miRNA expression in the human HF, and the identification of novel miRNA target genes and pathways via the systematic integration of miRNA and mRNA expression data, may facilitate the delineation of tissue-specific regulatory interactions, and improve our understanding of both normal hair growth and the pathobiology of hair loss disorders.

  14. Small Changes in Gene Expression of Targeted Osmoregulatory Genes When Exposing Marine and Freshwater Threespine Stickleback (Gasterosteus aculeatus) to Abrupt Salinity Transfers

    PubMed Central

    Taugbøl, Annette; Arntsen, Tina; Østbye, Kjartan; Vøllestad, Leif Asbjørn

    2014-01-01

    Salinity is one of the key factors that affects metabolism, survival and distribution of fish species, as all fish osmoregulate and euryhaline fish maintain osmotic differences between their extracellular fluid and either freshwater or seawater. The threespine stickleback (Gasterosteus aculeatus) is a euryhaline species with populations in both marine and freshwater environments, where the physiological and genomic basis for salinity tolerance adaptation is not fully understood. Therefore, our main objective in this study was to investigate gene expression of three targeted osmoregulatory genes (Na+/K+-ATPase (ATPA13), cystic fibrosis transmembrane regulator (CFTR) and a voltage gated potassium channel gene (KCNH4) and one stress related heat shock protein gene (HSP70)) in gill tissue from marine and freshwater populations when exposed to non-native salinity for periods ranging from five minutes to three weeks. Overall, the targeted genes showed highly plastic expression profiles, in addition the expression of ATP1A3 was slightly higher in saltwater adapted fish and KCNH4 and HSP70 had slightly higher expression in freshwater. As no pronounced changes were observed in the expression profiles of the targeted genes, this indicates that the osmoregulatory apparatuses of both the marine and landlocked freshwater stickleback population have not been environmentally canalized, but are able to respond plastically to abrupt salinity challenges. PMID:25265477

  15. Small changes in gene expression of targeted osmoregulatory genes when exposing marine and freshwater threespine stickleback (Gasterosteus aculeatus) to abrupt salinity transfers.

    PubMed

    Taugbøl, Annette; Arntsen, Tina; Ostbye, Kjartan; Vøllestad, Leif Asbjørn

    2014-01-01

    Salinity is one of the key factors that affects metabolism, survival and distribution of fish species, as all fish osmoregulate and euryhaline fish maintain osmotic differences between their extracellular fluid and either freshwater or seawater. The threespine stickleback (Gasterosteus aculeatus) is a euryhaline species with populations in both marine and freshwater environments, where the physiological and genomic basis for salinity tolerance adaptation is not fully understood. Therefore, our main objective in this study was to investigate gene expression of three targeted osmoregulatory genes (Na+/K+-ATPase (ATPA13), cystic fibrosis transmembrane regulator (CFTR) and a voltage gated potassium channel gene (KCNH4) and one stress related heat shock protein gene (HSP70)) in gill tissue from marine and freshwater populations when exposed to non-native salinity for periods ranging from five minutes to three weeks. Overall, the targeted genes showed highly plastic expression profiles, in addition the expression of ATP1A3 was slightly higher in saltwater adapted fish and KCNH4 and HSP70 had slightly higher expression in freshwater. As no pronounced changes were observed in the expression profiles of the targeted genes, this indicates that the osmoregulatory apparatuses of both the marine and landlocked freshwater stickleback population have not been environmentally canalized, but are able to respond plastically to abrupt salinity challenges.

  16. Gene expression inference with deep learning

    PubMed Central

    Chen, Yifei; Li, Yi; Narayan, Rajiv; Subramanian, Aravind; Xie, Xiaohui

    2016-01-01

    Motivation: Large-scale gene expression profiling has been widely used to characterize cellular states in response to various disease conditions, genetic perturbations, etc. Although the cost of whole-genome expression profiles has been dropping steadily, generating a compendium of expression profiling over thousands of samples is still very expensive. Recognizing that gene expressions are often highly correlated, researchers from the NIH LINCS program have developed a cost-effective strategy of profiling only ∼1000 carefully selected landmark genes and relying on computational methods to infer the expression of remaining target genes. However, the computational approach adopted by the LINCS program is currently based on linear regression (LR), limiting its accuracy since it does not capture complex nonlinear relationship between expressions of genes. Results: We present a deep learning method (abbreviated as D-GEX) to infer the expression of target genes from the expression of landmark genes. We used the microarray-based Gene Expression Omnibus dataset, consisting of 111K expression profiles, to train our model and compare its performance to those from other methods. In terms of mean absolute error averaged across all genes, deep learning significantly outperforms LR with 15.33% relative improvement. A gene-wise comparative analysis shows that deep learning achieves lower error than LR in 99.97% of the target genes. We also tested the performance of our learned model on an independent RNA-Seq-based GTEx dataset, which consists of 2921 expression profiles. Deep learning still outperforms LR with 6.57% relative improvement, and achieves lower error in 81.31% of the target genes. Availability and implementation: D-GEX is available at https://github.com/uci-cbcl/D-GEX. Contact: xhx@ics.uci.edu Supplementary information: Supplementary data are available at Bioinformatics online. PMID:26873929

  17. Gene expression inference with deep learning.

    PubMed

    Chen, Yifei; Li, Yi; Narayan, Rajiv; Subramanian, Aravind; Xie, Xiaohui

    2016-06-15

    Large-scale gene expression profiling has been widely used to characterize cellular states in response to various disease conditions, genetic perturbations, etc. Although the cost of whole-genome expression profiles has been dropping steadily, generating a compendium of expression profiling over thousands of samples is still very expensive. Recognizing that gene expressions are often highly correlated, researchers from the NIH LINCS program have developed a cost-effective strategy of profiling only ∼1000 carefully selected landmark genes and relying on computational methods to infer the expression of remaining target genes. However, the computational approach adopted by the LINCS program is currently based on linear regression (LR), limiting its accuracy since it does not capture complex nonlinear relationship between expressions of genes. We present a deep learning method (abbreviated as D-GEX) to infer the expression of target genes from the expression of landmark genes. We used the microarray-based Gene Expression Omnibus dataset, consisting of 111K expression profiles, to train our model and compare its performance to those from other methods. In terms of mean absolute error averaged across all genes, deep learning significantly outperforms LR with 15.33% relative improvement. A gene-wise comparative analysis shows that deep learning achieves lower error than LR in 99.97% of the target genes. We also tested the performance of our learned model on an independent RNA-Seq-based GTEx dataset, which consists of 2921 expression profiles. Deep learning still outperforms LR with 6.57% relative improvement, and achieves lower error in 81.31% of the target genes. D-GEX is available at https://github.com/uci-cbcl/D-GEX CONTACT: xhx@ics.uci.edu Supplementary data are available at Bioinformatics online. © The Author 2016. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

  18. Chemopreventive agents alters global gene expression pattern: predicting their mode of action and targets.

    PubMed

    Narayanan, Bhagavathi A

    2006-12-01

    Chemoprevention has the potential to be a major component of colon, breast, prostate and lung cancer control. Epidemiological, experimental, and clinical studies provide evidence that antioxidants, anti-inflammatory agents, n-3 polyunsaturated fatty acids and several other phytochemicals possess unique modes of action against cancer growth. However, the mode of action of several of these agents at the gene transcription level is not completely understood. Completion of the human genome sequence and the advent of DNA microarrays using cDNAs enhanced the detection and identification of hundreds of differentially expressed genes in response to anticancer drugs or chemopreventive agents. In this review, we are presenting an extensive analysis of the key findings from studies using potential chemopreventive agents on global gene expression patterns, which lead to the identification of cancer drug targets. The summary of the study reports discussed in this review explains the extent of gene alterations mediated by more than 20 compounds including antioxidants, fatty acids, NSAIDs, phytochemicals, retinoids, selenium, vitamins, aromatase inhibitor, lovastatin, oltipraz, salvicine, and zinc. The findings from these studies further reveal the utility of DNA microarray in characterizing and quantifying the differentially expressed genes that are possibly reprogrammed by the above agents against colon, breast, prostate, lung, liver, pancreatic and other cancer types. Phenolic antioxidant resveratrol found in berries and grapes inhibits the formation of prostate tumors by acting on the regulatory genes such as p53 while activating a cascade of genes involved in cell cycle and apoptosis including p300, Apaf-1, cdk inhibitor p21, p57 (KIP2), p53 induced Pig 7, Pig 8, Pig 10, cyclin D, DNA fragmentation factor 45. The group of genes significantly altered by selenium includes cyclin D1, cdk5, cdk4, cdk2, cdc25A and GADD 153. Vitamine D shows impact on p21(Waf1/Cip1) p27 cyclin B

  19. Hybrid promoters directed tBid gene expression to breast cancer cells by transcriptional targeting.

    PubMed

    Farokhimanesh, Samila; Rahbarizadeh, Fatemeh; Rasaee, Mohammad J; Kamali, Abbas; Mashkani, Baratali

    2010-01-01

    Developing cancer gene therapy constructs based on transcriptional targeting of genes to cancer cells is a new and promising modality for treatment of cancer. Introducing truncated Bid (tBid), a recently known member of the Bcl-2 family, eradicates cancer cells efficiently. For transcriptional targeting of tBid, two dual-specificity promoters, combining cancer specific core promoters and response modules, were designed. These two core promoter modules contained cancer specific promoters of MUC1 and Survivin genes accompanied by hypoxia-responsive elements and estrogen responsive elements (microenvironment condition of breast cancer cells) which were employed to achieve a higher and more specific level of tBid expression in breast cancer cells. Correlation of the level of tBid expression in normal and cancer cell lines with promoter activity was measured by RT-PCR after treatment with hypoxia and estrogen. The level of tBid expression under control of new hybrid promoters was compared with its expression under control of cytomegalovirus (CMV) promoter as a control. Our data revealed that the level of tBid expression in breast cancer cells were nearly 11 times more than normal cells because of the cancer specific promoters, although tBid expression under control of CMV promoter was almost the same in normal and cancer cell lines. Increased apoptosis was detected in the transfected breast cancer cell lines by the Caspase-3 activity assay. The application of these promoters may prove to have the advantage of tumor selective gene therapy in breast cancer cells and low-potential toxicity for normal tissues.

  20. Selective elimination of long INterspersed element-1 expressing tumour cells by targeted expression of the HSV-TK suicide gene

    PubMed Central

    Chendeb, Mariam; Schneider, Robert; Davidson, Irwin; Fadloun, Anas

    2017-01-01

    In gene therapy, effective and selective suicide gene expression is crucial. We exploited the endogenous Long INterspersed Element-1 (L1) machinery often reactivated in human cancers to integrate the Herpes Simplex Virus Thymidine Kinase (HSV-TK) suicide gene selectively into the genome of cancer cells. We developed a plasmid-based system directing HSV-TK expression only when reverse transcribed and integrated in the host genome via the endogenous L1 ORF1/2 proteins and an Alu element. Delivery of these new constructs into cells followed by Ganciclovir (GCV) treatment selectively induced mortality of L1 ORF1/2 protein expressing cancer cells, but had no effect on primary cells that do not express L1 ORF1/2. This novel strategy for selective targeting of tumour cells provides high tolerability as the HSV-TK gene cannot be expressed without reverse transcription and integration, and high selectivity as these processes take place only in cancer cells expressing high levels of functional L1 ORF1/2. PMID:28415677

  1. Massive expression of germ cell-specific genes is a hallmark of cancer and a potential target for novel treatment development.

    PubMed

    Bruggeman, Jan Willem; Koster, Jan; Lodder, Paul; Repping, Sjoerd; Hamer, Geert

    2018-06-15

    Cancer cells have been found to frequently express genes that are normally restricted to the testis, often referred to as cancer/testis (CT) antigens or genes. Because germ cell-specific antigens are not recognized as "self" by the innate immune system, CT-genes have previously been suggested as ideal candidate targets for cancer therapy. The use of CT-genes in cancer therapy has thus far been unsuccessful, most likely because their identification has relied on gene expression in whole testis, including the testicular somatic cells, precluding the detection of true germ cell-specific genes. By comparing the transcriptomes of micro-dissected germ cell subtypes, representing the main developmental stages of human spermatogenesis, with the publicly accessible transcriptomes of 2617 samples from 49 different healthy somatic tissues and 9232 samples from 33 tumor types, we here discover hundreds of true germ cell-specific cancer expressed genes. Strikingly, we found these germ cell cancer genes (GC-genes) to be widely expressed in all analyzed tumors. Many GC-genes appeared to be involved in processes that are likely to actively promote tumor viability, proliferation and metastasis. Targeting these true GC-genes thus has the potential to inhibit tumor growth with infertility being the only possible side effect. Moreover, we identified a subset of GC-genes that are not expressed in spermatogonial stem cells. Targeting of this GC-gene subset is predicted to only lead to temporary infertility, as untargeted spermatogonial stem cells can recover spermatogenesis after treatment. Our GC-gene dataset enables improved understanding of tumor biology and provides multiple novel targets for cancer treatment.

  2. Use of an activated beta-catenin to identify Wnt pathway target genes in caenorhabditis elegans, including a subset of collagen genes expressed in late larval development.

    PubMed

    Jackson, Belinda M; Abete-Luzi, Patricia; Krause, Michael W; Eisenmann, David M

    2014-04-16

    The Wnt signaling pathway plays a fundamental role during metazoan development, where it regulates diverse processes, including cell fate specification, cell migration, and stem cell renewal. Activation of the beta-catenin-dependent/canonical Wnt pathway up-regulates expression of Wnt target genes to mediate a cellular response. In the nematode Caenorhabditis elegans, a canonical Wnt signaling pathway regulates several processes during larval development; however, few target genes of this pathway have been identified. To address this deficit, we used a novel approach of conditionally activated Wnt signaling during a defined stage of larval life by overexpressing an activated beta-catenin protein, then used microarray analysis to identify genes showing altered expression compared with control animals. We identified 166 differentially expressed genes, of which 104 were up-regulated. A subset of the up-regulated genes was shown to have altered expression in mutants with decreased or increased Wnt signaling; we consider these genes to be bona fide C. elegans Wnt pathway targets. Among these was a group of six genes, including the cuticular collagen genes, bli-1 col-38, col-49, and col-71. These genes show a peak of expression in the mid L4 stage during normal development, suggesting a role in adult cuticle formation. Consistent with this finding, reduction of function for several of the genes causes phenotypes suggestive of defects in cuticle function or integrity. Therefore, this work has identified a large number of putative Wnt pathway target genes during larval life, including a small subset of Wnt-regulated collagen genes that may function in synthesis of the adult cuticle.

  3. A Novel mRNA Level Subtraction Method for Quick Identification of Target-Orientated Uniquely Expressed Genes Between Peanut Immature Pod and Leaf

    PubMed Central

    2010-01-01

    Subtraction technique has been broadly applied for target gene discovery. However, most current protocols apply relative differential subtraction and result in great amount clone mixtures of unique and differentially expressed genes. This makes it more difficult to identify unique or target-orientated expressed genes. In this study, we developed a novel method for subtraction at mRNA level by integrating magnetic particle technology into driver preparation and tester–driver hybridization to facilitate uniquely expressed gene discovery between peanut immature pod and leaf through a single round subtraction. The resulting target clones were further validated through polymerase chain reaction screening using peanut immature pod and leaf cDNA libraries as templates. This study has resulted in identifying several genes expressed uniquely in immature peanut pod. These target genes can be used for future peanut functional genome and genetic engineering research. PMID:21406066

  4. Study on relationship between expression level and molecular conformations of gene drugs targeting to hepatoma cells in vitro

    PubMed Central

    Yang, Dong-Ye; Lu, Fang-Gen; Tang, Xi-Xiang; Zhao, Shui-Ping; Ouyang, Chun-Hui; Wu, Xiao-Ping; Liu, Xiao-Wei; Wu, Xiao-Ying

    2003-01-01

    AIM: To increase exogenous gene expression level by modulating molecular conformations of targeting gene drugs. METHODS: The full length cDNAs of both P40 and P35 subunits of human interleukin 12 were amplified through polymerase chain reaction (PCR) and cloned into eukaryotic expressing vectors pcDNA3.1 (±) to construct plasmids of P (+)/IL-12, P (+)/P40 and P (-)/P35. These plasmids were combined with ASOR-PLL to form two targeting gene drugs [ASOR-PLL-P (+)/IL-12 and ASOR-PLL-P (+)/P40 + ASOR-PLL-P (-)/P35] in optimal ratios. The conformations of these two drugs at various concentrations adjuvant were examined under electron microscope (EM) and the drugs were transfected into HepG2 (ASGr+) cells. Semi-quantitative reverse transcription polymerase chain reaction (RT-PCR) was performed with total RNA extracted from the transfected cells to determine the hIL12 mRNA transcript level. The hIL12 protein in the cultured supernatant was measured with enzyme-linked immunosorbent assay (ELISA) 48 hours after transfection. RESULTS: Targeting gene drugs, whose structures were granular and circle-like and diameters ranged from 25 nm to 150 nm, had the highest hIL-12 expression level. The hIL-12 expression level in the group co-transfected with ASOR-PLL-P (+)/P40 and ASOR-PLL-P (-)/P35 was higher than that of ASOR-PLL-P (+)/IL-12 transfected group. CONCLUSION: The molecular conformations of targeting gene drugs play an important role in exogenous gene expression level, the best structures are granular and circle-like and their diameters range from 25 nm to 150 nm. The sizes and linking styles of exogenous genes also have some effects on their expression level. PMID:12970883

  5. Targeting CTCF to Control Virus Gene Expression: A Common Theme amongst Diverse DNA Viruses.

    PubMed

    Pentland, Ieisha; Parish, Joanna L

    2015-07-06

    All viruses target host cell factors for successful life cycle completion. Transcriptional control of DNA viruses by host cell factors is important in the temporal and spatial regulation of virus gene expression. Many of these factors are recruited to enhance virus gene expression and thereby increase virus production, but host cell factors can also restrict virus gene expression and productivity of infection. CCCTC binding factor (CTCF) is a host cell DNA binding protein important for the regulation of genomic chromatin boundaries, transcriptional control and enhancer element usage. CTCF also functions in RNA polymerase II regulation and in doing so can influence co-transcriptional splicing events. Several DNA viruses, including Kaposi's sarcoma-associated herpesvirus (KSHV), Epstein-Barr virus (EBV) and human papillomavirus (HPV) utilize CTCF to control virus gene expression and many studies have highlighted a role for CTCF in the persistence of these diverse oncogenic viruses. CTCF can both enhance and repress virus gene expression and in some cases CTCF increases the complexity of alternatively spliced transcripts. This review article will discuss the function of CTCF in the life cycle of DNA viruses in the context of known host cell CTCF functions.

  6. Targeting gene therapy to cancer: a review.

    PubMed

    Dachs, G U; Dougherty, G J; Stratford, I J; Chaplin, D J

    1997-01-01

    In recent years the idea of using gene therapy as a modality in the treatment of diseases other than genetically inherited, monogenic disorders has taken root. This is particularly obvious in the field of oncology where currently more than 100 clinical trials have been approved worldwide. This report will summarize some of the exciting progress that has recently been made with respect to both targeting the delivery of potentially therapeutic genes to tumor sites and regulating their expression within the tumor microenvironment. In order to specifically target malignant cells while at the same time sparing normal tissue, cancer gene therapy will need to combine highly selective gene delivery with highly specific gene expression, specific gene product activity, and, possibly, specific drug activation. Although the efficient delivery of DNA to tumor sites remains a formidable task, progress has been made in recent years using both viral (retrovirus, adenovirus, adeno-associated virus) and nonviral (liposomes, gene gun, injection) methods. In this report emphasis will be placed on targeted rather than high-efficiency delivery, although those would need to be combined in the future for effective therapy. To date delivery has been targeted to tumor-specific and tissue-specific antigens, such as epithelial growth factor receptor, c-kit receptor, and folate receptor, and these will be described in some detail. To increase specificity and safety of gene therapy further, the expression of the therapeutic gene needs to be tightly controlled within the target tissue. Targeted gene expression has been analyzed using tissue-specific promoters (breast-, prostate-, and melanoma-specific promoters) and disease-specific promoters (carcinoembryonic antigen, HER-2/neu, Myc-Max response elements, DF3/MUC). Alternatively, expression could be regulated externally with the use of radiation-induced promoters or tetracycline-responsive elements. Another novel possibility that will be

  7. Hapten-derivatized nanoparticle targeting and imaging of gene expression by multimodality imaging systems.

    PubMed

    Cheng, C-M; Chu, P-Y; Chuang, K-H; Roffler, S R; Kao, C-H; Tseng, W-L; Shiea, J; Chang, W-D; Su, Y-C; Chen, B-M; Wang, Y-M; Cheng, T-L

    2009-01-01

    Non-invasive gene monitoring is important for most gene therapy applications to ensure selective gene transfer to specific cells or tissues. We developed a non-invasive imaging system to assess the location and persistence of gene expression by anchoring an anti-dansyl (DNS) single-chain antibody (DNS receptor) on the cell surface to trap DNS-derivatized imaging probes. DNS hapten was covalently attached to cross-linked iron oxide (CLIO) to form a 39+/-0.5 nm DNS-CLIO nanoparticle imaging probe. DNS-CLIO specifically bound to DNS receptors but not to a control single-chain antibody receptor. DNS-CLIO (100 microM Fe) was non-toxic to both B16/DNS (DNS receptor positive) and B16/phOx (control receptor positive) cells. Magnetic resonance (MR) imaging could detect as few as 10% B16/DNS cells in a mixture in vitro. Importantly, DNS-CLIO specifically bound to a B16/DNS tumor, which markedly reduced signal intensity. Similar results were also shown with DNS quantum dots, which specifically targeted CT26/DNS cells but not control CT26/phOx cells both in vitro and in vivo. These results demonstrate that DNS nanoparticles can systemically monitor the expression of DNS receptor in vivo by feasible imaging systems. This targeting strategy may provide a valuable tool to estimate the efficacy and specificity of different gene delivery systems and optimize gene therapy protocols in the clinic.

  8. Reconstructing directed gene regulatory network by only gene expression data.

    PubMed

    Zhang, Lu; Feng, Xi Kang; Ng, Yen Kaow; Li, Shuai Cheng

    2016-08-18

    Accurately identifying gene regulatory network is an important task in understanding in vivo biological activities. The inference of such networks is often accomplished through the use of gene expression data. Many methods have been developed to evaluate gene expression dependencies between transcription factor and its target genes, and some methods also eliminate transitive interactions. The regulatory (or edge) direction is undetermined if the target gene is also a transcription factor. Some methods predict the regulatory directions in the gene regulatory networks by locating the eQTL single nucleotide polymorphism, or by observing the gene expression changes when knocking out/down the candidate transcript factors; regrettably, these additional data are usually unavailable, especially for the samples deriving from human tissues. In this study, we propose the Context Based Dependency Network (CBDN), a method that is able to infer gene regulatory networks with the regulatory directions from gene expression data only. To determine the regulatory direction, CBDN computes the influence of source to target by evaluating the magnitude changes of expression dependencies between the target gene and the others with conditioning on the source gene. CBDN extends the data processing inequality by involving the dependency direction to distinguish between direct and transitive relationship between genes. We also define two types of important regulators which can influence a majority of the genes in the network directly or indirectly. CBDN can detect both of these two types of important regulators by averaging the influence functions of candidate regulator to the other genes. In our experiments with simulated and real data, even with the regulatory direction taken into account, CBDN outperforms the state-of-the-art approaches for inferring gene regulatory network. CBDN identifies the important regulators in the predicted network: 1. TYROBP influences a batch of genes that are

  9. Isolation and expression analysis of four HD-ZIP III family genes targeted by microRNA166 in peach.

    PubMed

    Zhang, C H; Zhang, B B; Ma, R J; Yu, M L; Guo, S L; Guo, L

    2015-10-30

    MicroRNA166 (miR166) is known to have highly conserved targets that encode proteins of the class III homeodomain-leucine zipper (HD-ZIP III) family, in a broad range of plant species. To further understand the relationship between HD-ZIP III genes and miR166, four HD-ZIP III family genes (PpHB14, PpHB15, PpHB8, and PpREV) were isolated from peach (Prunus persica) tissue and characterized. Spatio-temporal expression profiles of the genes were analyzed. Genes of the peach HD-ZIP III family were predicted to encode five conserved domains. Deduced amino acid sequences and tertiary structures of the four peach HD-ZIP III genes were highly conserved, with corresponding genes in Arabidopsis thaliana. The expression level of four targets displayed the opposite trend to that of miR166 throughout fruit development, with the exception of PpHB14 from 35 to 55 days after full bloom (DAFB). This finding indicates that miR166 may negatively regulate its four targets throughout fruit development. As for leaf and phloem, the same trend in expression level was observed between four targets and miR166 from 75 to 105 DAFB. However, the opposite trend was observed for the transcript level between four targets and miR166 from 35 to 55 DAFB. miRNA166 may negatively regulate four targets in some but not all developmental stages for a given tissue. The four genes studied were observed to have, exactly or generally, the same change tendency as individual tissue development, a finding that suggests genes of the HD-ZIP III family in peach may have complementary or cooperative functions in various tissues.

  10. Improved luciferase gene expression using ultrasound targeted microbubble destruction therapy in swine

    NASA Astrophysics Data System (ADS)

    Noble, Misty L.; Song, Shuxian; Sun, Ryan R.; Fan, Luping; DiBlasi, Robert M.; O'Kelly-Priddy, Colleen; Loeb, Keith R.; Miao, Carol H.

    2012-11-01

    Ultrasound (US) targeted microbubble (MB) destruction (UTMD) has been shown to be an effective method in delivering drugs and plasmid DNA (pDNA) into cells. We previously reported successful gene transfection of a reporter luciferase gene, pGL4, into livers of mice and rats using UTMD. The challenge is to translate and achieve similar gene expression in large animals, like swine, where the treated tissue volume is substantially larger. The scale-up study requires proportionally increased amount of pDNA/MBs delivered to tissues and an equivalent increase in US energy. We use different MBs and surgical strategies to retain most of pDNA/MB locally during US application in order to maximize the effect of UTMD in gene transfection. Our results show significant increase in luciferase expression in swine injected with MBs and exposed to 2.7 MPa US. We obtained up to 1800-fold enhancement in the pig experiment using Definity® MBs, and 2000-fold and 6300-fold enhancement in two pig studies using RN18 MBs compared to sham. These results represent an important developmental step towards US mediated gene delivery in large animals and clinical trials.

  11. Targeted gene expression without a tissue-specific promoter: creating mosaic embryos using laser-induced single-cell heat shock

    NASA Technical Reports Server (NTRS)

    Halfon, M. S.; Kose, H.; Chiba, A.; Keshishian, H.

    1997-01-01

    We have developed a method to target gene expression in the Drosophila embryo to a specific cell without having a promoter that directs expression in that particular cell. Using a digitally enhanced imaging system to identify single cells within the living embryo, we apply a heat shock to each cell individually by using a laser microbeam. A 1- to 2-min laser treatment is sufficient to induce a heat-shock response but is not lethal to the heat-shocked cells. Induction of heat shock was measured in a variety of cell types, including neurons and somatic muscles, by the expression of beta-galactosidase from an hsp26-lacZ reporter construct or by expression of a UAS target gene after induction of hsGAL4. We discuss the applicability of this technique to ectopic gene expression studies, lineage tracing, gene inactivation studies, and studies of cells in vitro. Laser heat shock is a versatile technique that can be adapted for use in a variety of research organisms and is useful for any studies in which it is desirable to express a given gene in only a distinct cell or clone of cells, either transiently or constitutively, at a time point of choice.

  12. A targeted gene expression platform allows for rapid analysis of chemical-induced antioxidant mRNA expression in zebrafish larvae.

    PubMed

    Mills, Margaret G; Gallagher, Evan P

    2017-01-01

    Chemical-induced oxidative stress and the biochemical pathways that protect against oxidative damage are of particular interest in the field of toxicology. To rapidly identify oxidative stress-responsive gene expression changes in zebrafish, we developed a targeted panel of antioxidant genes using the Affymetrix QuantiGene Plex (QGP) platform. The genes contained in our panel include eight putative Nrf2 (Nfe2l2a)-dependent antioxidant genes (hmox1a, gstp1, gclc, nqo1, prdx1, gpx1a, sod1, sod2), a stress response gene (hsp70), an inducible DNA damage repair gene (gadd45bb), and three reference genes (actb1, gapdh, hprt1). We tested this platform on larval zebrafish exposed to tert-butyl hydroperoxide (tBHP) and cadmium (Cd), two model oxidative stressors with different modes of action, and compared our results with those obtained using the more common quantitative PCR (qPCR) method. Both methods showed that exposure to tBHP and Cd induced expression of prdx1, gstp1, and hmox1a (2- to 12-fold increase via QGP), indicative of an activated Nrf2 response in larval zebrafish. Both compounds also elicited a general stress response as reflected by elevation of hsp70 and gadd45bb, with Cd being the more potent inducer. Transient changes were observed in sod2 and gpx1a expression, whereas nqo1, an Nrf2-responsive gene in mammalian cells, was minimally affected by either tBHP or Cd chemical exposures. Developmental expression analysis of the target genes by QGP revealed marked upregulation of sod2 between 0-96hpf, and to a lesser extent, of sod1 and gstp1. Once optimized, QGP analysis of these experiments was accomplished more rapidly, using far less tissue, and at lower total costs than qPCR analysis. In summary, the QGP platform as applied to higher-throughput zebrafish studies provides a reasonable cost-effective alternative to qPCR or more comprehensive transcriptomics approaches to rapidly assess the potential for chemicals to elicit oxidative stress as a mechanism of

  13. RNA interference can target pre-mRNA: consequences for gene expression in a Caenorhabditis elegans operon.

    PubMed Central

    Bosher, J M; Dufourcq, P; Sookhareea, S; Labouesse, M

    1999-01-01

    In nematodes, flies, trypanosomes, and planarians, introduction of double-stranded RNA results in sequence-specific inactivation of gene function, a process termed RNA interference (RNAi). We demonstrate that RNAi against the Caenorhabditis elegans gene lir-1, which is part of the lir-1/lin-26 operon, induced phenotypes very different from a newly isolated lir-1 null mutation. Specifically, lir-1(RNAi) induced embryonic lethality reminiscent of moderately strong lin-26 alleles, whereas the lir-1 null mutant was viable. We show that the lir-1(RNAi) phenotypes resulted from a severe loss of lin-26 gene expression. In addition, we found that RNAi directed against lir-1 or lin-26 introns induced similar phenotypes, so we conclude that lir-1(RNAi) targets the lir-1/lin-26 pre-mRNA. This provides direct evidence that RNA interference can prevent gene expression by targeting nuclear transcripts. Our results highlight that caution may be necessary when interpreting RNA interference without the benefit of mutant alleles. PMID:10545456

  14. Sulforaphane induces Nrf2 target genes and attenuates inflammatory gene expression in microglia from brain of young adult and aged mice.

    PubMed

    Townsend, Brigitte E; Johnson, Rodney W

    2016-01-01

    Increased neuroinflammation and oxidative stress resulting from heightened microglial activation are associated with age-related cognitive impairment. The objectives of this study were to examine the effects of the bioactive sulforaphane (SFN) on the nuclear factor E2-related factor 2 (Nrf2) pathway in BV2 microglia and primary microglia, and to evaluate proinflammatory cytokine expression in lipopolysaccharide (LPS)-stimulated primary microglia from adult and aged mice. BV2 microglia and primary microglia isolated from young adult and aged mice were treated with SFN and LPS. Changes in Nrf2 activity, expression of Nrf2 target genes, and levels of proinflammatory markers were assessed by quantitative PCR and immunoassay. SFN increased Nrf2 DNA-binding activity and upregulated Nrf2 target genes in BV2 microglia, while reducing LPS-induced interleukin (IL-)1β, IL-6, and inducible nitric oxide synthase (iNOS). In primary microglia from adult and aged mice, SFN increased expression of Nrf2 target genes and attenuated IL-1β, IL-6, and iNOS induced by LPS. These data indicate that SFN is a potential beneficial supplement that may be useful for reducing microglial mediated neuroinflammation and oxidative stress associated with aging. Copyright © 2015 Elsevier Inc. All rights reserved.

  15. Drosophila CLOCK target gene characterization: implications for circadian tissue-specific gene expression

    PubMed Central

    Abruzzi, Katharine Compton; Rodriguez, Joseph; Menet, Jerome S.; Desrochers, Jennifer; Zadina, Abigail; Luo, Weifei; Tkachev, Sasha; Rosbash, Michael

    2011-01-01

    CLOCK (CLK) is a master transcriptional regulator of the circadian clock in Drosophila. To identify CLK direct target genes and address circadian transcriptional regulation in Drosophila, we performed chromatin immunoprecipitation (ChIP) tiling array assays (ChIP–chip) with a number of circadian proteins. CLK binding cycles on at least 800 sites with maximal binding in the early night. The CLK partner protein CYCLE (CYC) is on most of these sites. The CLK/CYC heterodimer is joined 4–6 h later by the transcriptional repressor PERIOD (PER), indicating that the majority of CLK targets are regulated similarly to core circadian genes. About 30% of target genes also show cycling RNA polymerase II (Pol II) binding. Many of these generate cycling RNAs despite not being documented in prior RNA cycling studies. This is due in part to different RNA isoforms and to fly head tissue heterogeneity. CLK has specific targets in different tissues, implying that important CLK partner proteins and/or mechanisms contribute to gene-specific and tissue-specific regulation. PMID:22085964

  16. Zinc-finger protein-targeted gene regulation: Genomewide single-gene specificity

    PubMed Central

    Tan, Siyuan; Guschin, Dmitry; Davalos, Albert; Lee, Ya-Li; Snowden, Andrew W.; Jouvenot, Yann; Zhang, H. Steven; Howes, Katherine; McNamara, Andrew R.; Lai, Albert; Ullman, Chris; Reynolds, Lindsey; Moore, Michael; Isalan, Mark; Berg, Lutz-Peter; Campos, Bradley; Qi, Hong; Spratt, S. Kaye; Case, Casey C.; Pabo, Carl O.; Campisi, Judith; Gregory, Philip D.

    2003-01-01

    Zinc-finger protein transcription factors (ZFP TFs) can be designed to control the expression of any desired target gene, and thus provide potential therapeutic tools for the study and treatment of disease. Here we report that a ZFP TF can repress target gene expression with single-gene specificity within the human genome. A ZFP TF repressor that binds an 18-bp recognition sequence within the promoter of the endogenous CHK2 gene gives a >10-fold reduction in CHK2 mRNA and protein. This level of repression was sufficient to generate a functional phenotype, as demonstrated by the loss of DNA damage-induced CHK2-dependent p53 phosphorylation. We determined the specificity of repression by using DNA microarrays and found that the ZFP TF repressed a single gene (CHK2) within the monitored genome in two different cell types. These data demonstrate the utility of ZFP TFs as precise tools for target validation, and highlight their potential as clinical therapeutics. PMID:14514889

  17. Expression of anaesthetic and analgesic drug target genes in excised breast tumour tissue: Association with clinical disease recurrence or metastasis.

    PubMed

    Connolly, C; Madden, S F; Buggy, D J; Gallagher, H C

    2017-01-01

    Retrospective analyses suggest anaesthetic-analgesics technique during cancer surgery may affect recurrence/metastasis. This could involve direct effects of anaesthetic-analgesic drugs on cancer cells. While μ-opioid receptor over-expression in lung tumours is associated with greater metastasis, other anaesthetic-analgesic receptor targets in cancer recurrence/metastasis remain unexplored. Therefore, we evaluated the association between genetic expression of anaesthetic-analgesic receptor targets and recurrence/metastasis, using a repository of breast cancer gene expression and matching clinical data. A list of 23 genes encoding for the most prominent anaesthetic-analgesic receptor targets was compiled. This was processed through BreastMark- an algorithm integrating gene expression data from ~17,000 samples and clinical data from >4,500 breast cancer samples. Gene expression data was dichotomized using disease-free survival (survival without recurrence) and distant disease-free survival (survival without metastasis) as end points. Hazard ratios were calculated by Cox-regression analysis. Enrichment for prognostic markers was determined by randomly choosing 23-member gene lists from all available genes, calculating how often >5 significant markers were observed and adjusting p-values for multiple testing. This was repeated 10,000 times and an empirical p-value calculated. Of 23 selected genes, 9 were significantly associated with altered rates of metastasis and 4 with recurrence on univariate analysis. Adjusting for multiple testing, 5 of these 9 genes remained significantly associated with metastasis, non with recurrence. This ratio of genes (5/23) was not significantly enriched for markers of metastasis (p = 0.07). Several anaesthetic-analgesic receptor genes were associated with metastatic spread in breast cancer. Overall there was no significant enrichment in prognostic markers of metastasis, although a trend was observed.

  18. Core Promoter Functions in the Regulation of Gene Expression of Drosophila Dorsal Target Genes*

    PubMed Central

    Zehavi, Yonathan; Kuznetsov, Olga; Ovadia-Shochat, Avital; Juven-Gershon, Tamar

    2014-01-01

    Developmental processes are highly dependent on transcriptional regulation by RNA polymerase II. The RNA polymerase II core promoter is the ultimate target of a multitude of transcription factors that control transcription initiation. Core promoters consist of core promoter motifs, e.g. the initiator, TATA box, and the downstream core promoter element (DPE), which confer specific properties to the core promoter. Here, we explored the importance of core promoter functions in the dorsal-ventral developmental gene regulatory network. This network includes multiple genes that are activated by different nuclear concentrations of Dorsal, an NFκB homolog transcription factor, along the dorsal-ventral axis. We show that over two-thirds of Dorsal target genes contain DPE sequence motifs, which is significantly higher than the proportion of DPE-containing promoters in Drosophila genes. We demonstrate that multiple Dorsal target genes are evolutionarily conserved and functionally dependent on the DPE. Furthermore, we have analyzed the activation of key Dorsal target genes by Dorsal, as well as by another Rel family transcription factor, Relish, and the dependence of their activation on the DPE motif. Using hybrid enhancer-promoter constructs in Drosophila cells and embryo extracts, we have demonstrated that the core promoter composition is an important determinant of transcriptional activity of Dorsal target genes. Taken together, our results provide evidence for the importance of core promoter composition in the regulation of Dorsal target genes. PMID:24634215

  19. Ewing's Sarcoma: An Analysis of miRNA Expression Profiles and Target Genes in Paraffin-Embedded Primary Tumor Tissue.

    PubMed

    Parafioriti, Antonina; Bason, Caterina; Armiraglio, Elisabetta; Calciano, Lucia; Daolio, Primo Andrea; Berardocco, Martina; Di Bernardo, Andrea; Colosimo, Alessia; Luksch, Roberto; Berardi, Anna C

    2016-04-30

    The molecular mechanism responsible for Ewing's Sarcoma (ES) remains largely unknown. MicroRNAs (miRNAs), a class of small non-coding RNAs able to regulate gene expression, are deregulated in tumors and may serve as a tool for diagnosis and prediction. However, the status of miRNAs in ES has not yet been thoroughly investigated. This study compared global miRNAs expression in paraffin-embedded tumor tissue samples from 20 ES patients, affected by primary untreated tumors, with miRNAs expressed in normal human mesenchymal stromal cells (MSCs) by microarray analysis. A miRTarBase database was used to identify the predicted target genes for differentially expressed miRNAs. The miRNAs microarray analysis revealed distinct patterns of miRNAs expression between ES samples and normal MSCs. 58 of the 954 analyzed miRNAs were significantly differentially expressed in ES samples compared to MSCs. Moreover, the qRT-PCR analysis carried out on three selected miRNAs showed that miR-181b, miR-1915 and miR-1275 were significantly aberrantly regulated, confirming the microarray results. Bio-database analysis identified BCL-2 as a bona fide target gene of the miR-21, miR-181a, miR-181b, miR-29a, miR-29b, miR-497, miR-195, miR-let-7a, miR-34a and miR-1915. Using paraffin-embedded tissues from ES patients, this study has identified several potential target miRNAs and one gene that might be considered a novel critical biomarker for ES pathogenesis.

  20. Meta-analysis of human gene expression in response to Mycobacterium tuberculosis infection reveals potential therapeutic targets.

    PubMed

    Wang, Zhang; Arat, Seda; Magid-Slav, Michal; Brown, James R

    2018-01-10

    With the global emergence of multi-drug resistant strains of Mycobacterium tuberculosis, new strategies to treat tuberculosis are urgently needed such as therapeutics targeting potential human host factors. Here we performed a statistical meta-analysis of human gene expression in response to both latent and active pulmonary tuberculosis infections from nine published datasets. We found 1655 genes that were significantly differentially expressed during active tuberculosis infection. In contrast, no gene was significant for latent tuberculosis. Pathway enrichment analysis identified 90 significant canonical human pathways, including several pathways more commonly related to non-infectious diseases such as the LRRK2 pathway in Parkinson's disease, and PD-1/PD-L1 signaling pathway important for new immuno-oncology therapies. The analysis of human genome-wide association studies datasets revealed tuberculosis-associated genetic variants proximal to several genes in major histocompatibility complex for antigen presentation. We propose several new targets and drug-repurposing opportunities including intravenous immunoglobulin, ion-channel blockers and cancer immuno-therapeutics for development as combination therapeutics with anti-mycobacterial agents. Our meta-analysis provides novel insights into host genes and pathways important for tuberculosis and brings forth potential drug repurposing opportunities for host-directed therapies.

  1. Multicomponent DNA carrier with a vesicular stomatitis virus G-peptide greatly enhances liver-targeted gene expression in mice.

    PubMed

    Schuster, M J; Wu, G Y; Walton, C M; Wu, C H

    1999-01-01

    Genes can be targeted to hepatocytes in vitro and in vivo by the use of asialoorosomucoid-polylysine conjugates. After systemic application, this nonviral vector is recognized by highly selective asialoglycoprotein (AsGP) receptors on the sinusoidal liver cell membrane and is taken up via receptor-mediated endocytosis. As most of the DNA is rapidly transferred to lysosomes where it is degraded, transfection efficiency is low and gene expression transient. To address this problem, we incorporated a pH-dependent synthetic hemolytic peptide derived of the G-protein of Vesicular Stomatitis Virus (VSV) into the gene transfer system, to increase endosomal escape of internalized DNA. The multicomponent carrier binds DNA in a nondamaging way, is still recognized by the AsGP receptor, and is targeted to the liver in vivo. Injection of DNA complexes containing a luciferase marker gene resulted in luciferase expression of 29 000 pg/g liver which corresponded to an increase of a factor of 10(3) overexpression after injection of DNA complexes without endosomolytic peptide. Furthermore, the amount of intact transgene within isolated liver cell nuclei was increased by a factor of 10(1)-10(2) by the use of the multicomponent carriers. These results demonstrate that incorporation of a hemolytic peptide into a nonviral vector can greatly increase gene expression while retaining cell type targetability in vivo.

  2. AAV-mediated targeting of gene expression to the peri-infarct region in rat cortical stroke model.

    PubMed

    Mätlik, Kert; Abo-Ramadan, Usama; Harvey, Brandon K; Arumäe, Urmas; Airavaara, Mikko

    2014-10-30

    For stroke patients the recovery of cognitive and behavioral functions is often incomplete. Functional recovery is thought to be mediated largely by connectivity rearrangements in the peri-infarct region. A method for manipulating gene expression in this region would be useful for identifying new recovery-enhancing treatments. We have characterized a way of targeting adeno-associated virus (AAV) vectors to the peri-infarct region of cortical ischemic lesion in rats 2days after middle cerebral artery occlusion (MCAo). We used magnetic resonance imaging (MRI) to show that the altered properties of post-ischemic brain tissue facilitate the spreading of intrastriatally injected nanoparticles toward the infarct. We show that subcortical injection of green fluorescent protein-encoding dsAAV7-GFP resulted in transduction of cells in and around the white matter tract underlying the lesion, and in the cortex proximal to the lesion. A similar result was achieved with dsAAV7 vector encoding the cerebral dopamine neurotrophic factor (CDNF), a protein with therapeutic potential. Viral vector-mediated intracerebral gene delivery has been used before in rodent models of ischemic injury. However, the method of targeting gene expression to the peri-infarct region, after the initial phase of ischemic cell death, has not been described before. We demonstrate a straightforward and robust way to target AAV vector-mediated over-expression of genes to the peri-infarct region in a rat stroke model. This method will be useful for studying the action of specific proteins in peri-infarct region during the recovery process. Copyright © 2014 Elsevier B.V. All rights reserved.

  3. Glioma stem cells targeted by oncolytic virus carrying endostatin-angiostatin fusion gene and the expression of its exogenous gene in vitro.

    PubMed

    Zhu, Guidong; Su, Wei; Jin, Guishan; Xu, Fujian; Hao, Shuyu; Guan, Fangxia; Jia, William; Liu, Fusheng

    2011-05-16

    The development of the cancer stem cell (CSCs) niche theory has provided a new target for the treatment of gliomas. Gene therapy using oncolytic viral vectors has shown great potential for the therapeutic targeting of CSCs. To explore whether a viral vector carrying an exogenous Endo-Angio fusion gene (VAE) can infect and kill glioma stem cells (GSCs), as well as inhibit their vascular niche in vitro, we have collected surgical specimens of human high-grade glioma (world health organization, WHO Classes III-VI) from which we isolated and cultured GSCs under conditions originally designed for the selective expansion of neural stem cells. Our results demonstrate the following: (1) Four lines of GSCs (isolated from 20 surgical specimens) could grow in suspension, were multipotent, had the ability to self-renew and expressed the neural stem cell markers, CD133 and nestin. (2) VAE could infect GSCs and significantly inhibit their viability. (3) The Endo-Angio fusion gene was expressed in GSCs 48 h after VAE infection and could inhibit the proliferation of human brain microvascular endothelial cells (HBMEC). (4) Residual viable cells lose the ability of self-renewal and adherent differentiation. In conclusion, VAE can significantly inhibit the activity of GSCs in vitro and the expression of exogenous Endo-Angio fusion gene can inhibit HBMEC proliferation. VAE can be used as a novel virus-gene therapy strategy for glioma. Copyright © 2011 Elsevier B.V. All rights reserved.

  4. Evolving phage vectors for cell targeted gene delivery.

    PubMed

    Larocca, David; Burg, Michael A; Jensen-Pergakes, Kristen; Ravey, Edward Prenn; Gonzalez, Ana Maria; Baird, Andrew

    2002-03-01

    We adapted filamentous phage vectors for targeted gene delivery to mammalian cells by inserting a mammalian reporter gene expression cassette (GFP) into the vector backbone and fusing the pIII coat protein to a cell targeting ligand (i.e. FGF2, EGF). Like transfection with animal viral vectors, targeted phage gene delivery is concentration, time, and ligand dependent. Importantly, targeted phage particles are specific for the appropriate target cell surface receptor. Phage have distinct advantages over existing gene therapy vectors because they are simple, economical to produce at high titer, have no intrinsic tropism for mammalian cells, and are relatively simple to genetically modify and evolve. Initially transduction by targeted phage particles was low resulting in foreign gene expression in 1-2% of transfected cells. We increased transduction efficiency by modifying both the transfection protocol and vector design. For example, we stabilized the display of the targeting ligand to create multivalent phagemid-based vectors with transduction efficiencies of up to 45% in certain cell lines when combined with genotoxic treatment. Taken together, these studies establish that the efficiency of phage-mediated gene transfer can be significantly improved through genetic modification. We are currently evolving phage vectors with enhanced cell targeting, increased stability, reduced immunogenicity and other properties suitable for gene therapy.

  5. Genome-wide targeted prediction of ABA responsive genes in rice based on over-represented cis-motif in co-expressed genes.

    PubMed

    Lenka, Sangram K; Lohia, Bikash; Kumar, Abhay; Chinnusamy, Viswanathan; Bansal, Kailash C

    2009-02-01

    Abscisic acid (ABA), the popular plant stress hormone, plays a key role in regulation of sub-set of stress responsive genes. These genes respond to ABA through specific transcription factors which bind to cis-regulatory elements present in their promoters. We discovered the ABA Responsive Element (ABRE) core (ACGT) containing CGMCACGTGB motif as over-represented motif among the promoters of ABA responsive co-expressed genes in rice. Targeted gene prediction strategy using this motif led to the identification of 402 protein coding genes potentially regulated by ABA-dependent molecular genetic network. RT-PCR analysis of arbitrarily chosen 45 genes from the predicted 402 genes confirmed 80% accuracy of our prediction. Plant Gene Ontology (GO) analysis of ABA responsive genes showed enrichment of signal transduction and stress related genes among diverse functional categories.

  6. miR-24-2 controls H2AFX expression regardless of gene copy number alteration and induces apoptosis by targeting antiapoptotic gene BCL-2: a potential for therapeutic intervention.

    PubMed

    Srivastava, Niloo; Manvati, Siddharth; Srivastava, Archita; Pal, Ranjana; Kalaiarasan, Ponnusamy; Chattopadhyay, Shilpi; Gochhait, Sailesh; Dua, Raina; Bamezai, Rameshwar N K

    2011-04-04

    New levels of gene regulation with microRNA (miR) and gene copy number alterations (CNAs) have been identified as playing a role in various cancers. We have previously reported that sporadic breast cancer tissues exhibit significant alteration in H2AX gene copy number. However, how CNA affects gene expression and what is the role of miR, miR-24-2, known to regulate H2AX expression, in the background of the change in copy number, are not known. Further, many miRs, including miR-24-2, are implicated as playing a role in cell proliferation and apoptosis, but their specific target genes and the pathways contributing to them remain unexplored. Changes in gene copy number and mRNA/miR expression were estimated using real-time polymerase chain reaction assays in two mammalian cell lines, MCF-7 and HeLa, and in a set of sporadic breast cancer tissues. In silico analysis was performed to find the putative target for miR-24-2. MCF-7 cells were transfected with precursor miR-24-2 oligonucleotides, and the gene expression levels of BRCA1, BRCA2, ATM, MDM2, TP53, CHEK2, CYT-C, BCL-2, H2AFX and P21 were examined using TaqMan gene expression assays. Apoptosis was measured by flow cytometric detection using annexin V dye. A luciferase assay was performed to confirm BCL-2 as a valid cellular target of miR-24-2. It was observed that H2AX gene expression was negatively correlated with miR-24-2 expression and not in accordance with the gene copy number status, both in cell lines and in sporadic breast tumor tissues. Further, the cells overexpressing miR-24-2 were observed to be hypersensitive to DNA damaging drugs, undergoing apoptotic cell death, suggesting the potentiating effect of mir-24-2-mediated apoptotic induction in human cancer cell lines treated with anticancer drugs. BCL-2 was identified as a novel cellular target of miR-24-2. mir-24-2 is capable of inducing apoptosis by modulating different apoptotic pathways and targeting BCL-2, an antiapoptotic gene. The study suggests

  7. Transcriptome-Level Signatures in Gene Expression and Gene Expression Variability during Bacterial Adaptive Evolution.

    PubMed

    Erickson, Keesha E; Otoupal, Peter B; Chatterjee, Anushree

    2017-01-01

    Antibiotic-resistant bacteria are an increasingly serious public health concern, as strains emerge that demonstrate resistance to almost all available treatments. One factor that contributes to the crisis is the adaptive ability of bacteria, which exhibit remarkable phenotypic and gene expression heterogeneity in order to gain a survival advantage in damaging environments. This high degree of variability in gene expression across biological populations makes it a challenging task to identify key regulators of bacterial adaptation. Here, we research the regulation of adaptive resistance by investigating transcriptome profiles of Escherichia coli upon adaptation to disparate toxins, including antibiotics and biofuels. We locate potential target genes via conventional gene expression analysis as well as using a new analysis technique examining differential gene expression variability. By investigating trends across the diverse adaptation conditions, we identify a focused set of genes with conserved behavior, including those involved in cell motility, metabolism, membrane structure, and transport, and several genes of unknown function. To validate the biological relevance of the observed changes, we synthetically perturb gene expression using clustered regularly interspaced short palindromic repeat (CRISPR)-dCas9. Manipulation of select genes in combination with antibiotic treatment promotes adaptive resistance as demonstrated by an increased degree of antibiotic tolerance and heterogeneity in MICs. We study the mechanisms by which identified genes influence adaptation and find that select differentially variable genes have the potential to impact metabolic rates, mutation rates, and motility. Overall, this work provides evidence for a complex nongenetic response, encompassing shifts in gene expression and gene expression variability, which underlies adaptive resistance. IMPORTANCE Even initially sensitive bacteria can rapidly thwart antibiotic treatment through stress

  8. Renal Gene Expression Database (RGED): a relational database of gene expression profiles in kidney disease

    PubMed Central

    Zhang, Qingzhou; Yang, Bo; Chen, Xujiao; Xu, Jing; Mei, Changlin; Mao, Zhiguo

    2014-01-01

    We present a bioinformatics database named Renal Gene Expression Database (RGED), which contains comprehensive gene expression data sets from renal disease research. The web-based interface of RGED allows users to query the gene expression profiles in various kidney-related samples, including renal cell lines, human kidney tissues and murine model kidneys. Researchers can explore certain gene profiles, the relationships between genes of interests and identify biomarkers or even drug targets in kidney diseases. The aim of this work is to provide a user-friendly utility for the renal disease research community to query expression profiles of genes of their own interest without the requirement of advanced computational skills. Availability and implementation: Website is implemented in PHP, R, MySQL and Nginx and freely available from http://rged.wall-eva.net. Database URL: http://rged.wall-eva.net PMID:25252782

  9. Renal Gene Expression Database (RGED): a relational database of gene expression profiles in kidney disease.

    PubMed

    Zhang, Qingzhou; Yang, Bo; Chen, Xujiao; Xu, Jing; Mei, Changlin; Mao, Zhiguo

    2014-01-01

    We present a bioinformatics database named Renal Gene Expression Database (RGED), which contains comprehensive gene expression data sets from renal disease research. The web-based interface of RGED allows users to query the gene expression profiles in various kidney-related samples, including renal cell lines, human kidney tissues and murine model kidneys. Researchers can explore certain gene profiles, the relationships between genes of interests and identify biomarkers or even drug targets in kidney diseases. The aim of this work is to provide a user-friendly utility for the renal disease research community to query expression profiles of genes of their own interest without the requirement of advanced computational skills. Website is implemented in PHP, R, MySQL and Nginx and freely available from http://rged.wall-eva.net. http://rged.wall-eva.net. © The Author(s) 2014. Published by Oxford University Press.

  10. Identification and validation of reference genes for quantification of target gene expression with quantitative real-time PCR for tall fescue under four abiotic stresses.

    PubMed

    Yang, Zhimin; Chen, Yu; Hu, Baoyun; Tan, Zhiqun; Huang, Bingru

    2015-01-01

    Tall fescue (Festuca arundinacea Schreb.) is widely utilized as a major forage and turfgrass species in the temperate regions of the world and is a valuable plant material for studying molecular mechanisms of grass stress tolerance due to its superior drought and heat tolerance among cool-season species. Selection of suitable reference genes for quantification of target gene expression is important for the discovery of molecular mechanisms underlying improved growth traits and stress tolerance. The stability of nine potential reference genes (ACT, TUB, EF1a, GAPDH, SAND, CACS, F-box, PEPKR1 and TIP41) was evaluated using four programs, GeNorm, NormFinder, BestKeeper, and RefFinder. The combinations of SAND and TUB or TIP41 and TUB were most stably expressed in salt-treated roots or leaves. The combinations of GAPDH with TIP41 or TUB were stable in roots and leaves under drought stress. TIP41 and PEPKR1 exhibited stable expression in cold-treated roots, and the combination of F-box, TIP41 and TUB was also stable in cold-treated leaves. CACS and TUB were the two most stable reference genes in heat-stressed roots. TIP41 combined with TUB and ACT was stably expressed in heat-stressed leaves. Finally, quantitative real-time polymerase chain reaction (qRT-PCR) assays of the target gene FaWRKY1 using the identified most stable reference genes confirmed the reliability of selected reference genes. The selection of suitable reference genes in tall fescue will allow for more accurate identification of stress-tolerance genes and molecular mechanisms conferring stress tolerance in this stress-tolerant species.

  11. Differential Sensitivity of Target Genes to Translational Repression by miR-17~92

    PubMed Central

    Jin, Hyun Yong; Oda, Hiroyo; Chen, Pengda; Kang, Seung Goo; Valentine, Elizabeth; Liao, Lujian; Zhang, Yaoyang; Gonzalez-Martin, Alicia; Shepherd, Jovan; Head, Steven R.; Kim, Pyeung-Hyeun; Fu, Guo; Liu, Wen-Hsien; Han, Jiahuai

    2017-01-01

    MicroRNAs (miRNAs) are thought to exert their functions by modulating the expression of hundreds of target genes and each to a small degree, but it remains unclear how small changes in hundreds of target genes are translated into the specific function of a miRNA. Here, we conducted an integrated analysis of transcriptome and translatome of primary B cells from mutant mice expressing miR-17~92 at three different levels to address this issue. We found that target genes exhibit differential sensitivity to miRNA suppression and that only a small fraction of target genes are actually suppressed by a given concentration of miRNA under physiological conditions. Transgenic expression and deletion of the same miRNA gene regulate largely distinct sets of target genes. miR-17~92 controls target gene expression mainly through translational repression and 5’UTR plays an important role in regulating target gene sensitivity to miRNA suppression. These findings provide molecular insights into a model in which miRNAs exert their specific functions through a small number of key target genes. PMID:28241004

  12. Genotoxic chemical carcinogens target inducible genes in vivo

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Hamilton, J.W.; McCaffrey, J.; Caron, R.M.

    1994-12-31

    Our laboratory is interested in whether carcinogen-induced DNA damage is distributed nonrandomly in the genome - that is, {open_quotes}targeted{close_quotes} to specific genes or gene regions in vivo. As an indirect measure of whether targeting occurs at the gene level, we have examined whether carcinogens differentially alter the expression of individual genes. We have compared the effects of model genotoxic carcinogens that principally induce either strand breaks, simple alkylations, bulky lesions, or DNA cross-links on the expression of several constitutive and inducible genes in a simple in vivo system, the chick embryo. Each agent was examined for its effects on genemore » expression over a 24 hour period corresponding to the period of maximal DNA damage and repair induced by each compound. The doses used in these studies represented the maximum doses that caused no overt toxicity over a 96 hour period but that induced significant levels of DNA damage. Our results demonstrate that inducible genes are targeted by chemical carcinogens. We hypothesize that such effects may be a result of DNA damage specifically altering DNA-protein interactions within the promoters of inducible genes.« less

  13. Low-rank regularization for learning gene expression programs.

    PubMed

    Ye, Guibo; Tang, Mengfan; Cai, Jian-Feng; Nie, Qing; Xie, Xiaohui

    2013-01-01

    Learning gene expression programs directly from a set of observations is challenging due to the complexity of gene regulation, high noise of experimental measurements, and insufficient number of experimental measurements. Imposing additional constraints with strong and biologically motivated regularizations is critical in developing reliable and effective algorithms for inferring gene expression programs. Here we propose a new form of regulation that constrains the number of independent connectivity patterns between regulators and targets, motivated by the modular design of gene regulatory programs and the belief that the total number of independent regulatory modules should be small. We formulate a multi-target linear regression framework to incorporate this type of regulation, in which the number of independent connectivity patterns is expressed as the rank of the connectivity matrix between regulators and targets. We then generalize the linear framework to nonlinear cases, and prove that the generalized low-rank regularization model is still convex. Efficient algorithms are derived to solve both the linear and nonlinear low-rank regularized problems. Finally, we test the algorithms on three gene expression datasets, and show that the low-rank regularization improves the accuracy of gene expression prediction in these three datasets.

  14. Identification of Direct Target Genes Using Joint Sequence and Expression Likelihood with Application to DAF-16

    PubMed Central

    Yu, Ron X.; Liu, Jie; True, Nick; Wang, Wei

    2008-01-01

    A major challenge in the post-genome era is to reconstruct regulatory networks from the biological knowledge accumulated up to date. The development of tools for identifying direct target genes of transcription factors (TFs) is critical to this endeavor. Given a set of microarray experiments, a probabilistic model called TRANSMODIS has been developed which can infer the direct targets of a TF by integrating sequence motif, gene expression and ChIP-chip data. The performance of TRANSMODIS was first validated on a set of transcription factor perturbation experiments (TFPEs) involving Pho4p, a well studied TF in Saccharomyces cerevisiae. TRANSMODIS removed elements of arbitrariness in manual target gene selection process and produced results that concur with one's intuition. TRANSMODIS was further validated on a genome-wide scale by comparing it with two other methods in Saccharomyces cerevisiae. The usefulness of TRANSMODIS was then demonstrated by applying it to the identification of direct targets of DAF-16, a critical TF regulating ageing in Caenorhabditis elegans. We found that 189 genes were tightly regulated by DAF-16. In addition, DAF-16 has differential preference for motifs when acting as an activator or repressor, which awaits experimental verification. TRANSMODIS is computationally efficient and robust, making it a useful probabilistic framework for finding immediate targets. PMID:18350157

  15. Targeting Activation of Specific NF-κB Subunits Prevents Stress-Dependent Atherothrombotic Gene Expression

    PubMed Central

    Djuric, Zdenka; Kashif, Muhammed; Fleming, Thomas; Muhammad, Sajjad; Piel, David; von Bauer, Rüdiger; Bea, Florian; Herzig, Stephan; Zeier, Martin; Pizzi, Marina; Isermann, Berend; Hecker, Markus; Schwaninger, Markus; Bierhaus, Angelika; Nawroth, Peter P

    2012-01-01

    Psychosocial stress has been shown to be a contributing factor in the development of atherosclerosis. Although the underlying mechanisms have not been elucidated entirely, it has been shown previously that the transcription factor nuclear factor-κB (NF-κB) is an important component of stress-activated signaling pathway. In this study, we aimed to decipher the mechanisms of stress-induced NF-κB-mediated gene expression, using an in vitro and in vivo model of psychosocial stress. Induction of stress led to NF-κB-dependent expression of proinflammatory (tissue factor, intracellular adhesive molecule 1 [ICAM-1]) and protective genes (manganese superoxide dismutase [MnSOD]) via p50, p65 or cRel. Selective inhibition of the different subunits and the respective kinases showed that inhibition of cRel leads to the reduction of atherosclerotic lesions in apolipoprotein−/− (ApoE−/−) mice via suppression of proinflammatory gene expression. This observation may therefore provide a possible explanation for ineffectiveness of antioxidant therapies and suggests that selective targeting of cRel activation may provide a novel approach for the treatment of stress-related inflammatory vascular disease. PMID:23114885

  16. Identification of Homeotic Target Genes in Drosophila Melanogaster Including Nervy, a Proto-Oncogene Homologue

    PubMed Central

    Feinstein, P. G.; Kornfeld, K.; Hogness, D. S.; Mann, R. S.

    1995-01-01

    In Drosophila, the specific morphological characteristics of each segment are determined by the homeotic genes that regulate the expression of downstream target genes. We used a subtractive hybridization procedure to isolate activated target genes of the homeotic gene Ultrabithorax (Ubx). In addition, we constructed a set of mutant genotypes that measures the regulatory contribution of individual homeotic genes to a complex target gene expression pattern. Using these mutants, we demonstrate that homeotic genes can regulate target gene expression at the start of gastrulation, suggesting a previously unknown role for the homeotic genes at this early stage. We also show that, in abdominal segments, the levels of expression for two target genes increase in response to high levels of Ubx, demonstrating that the normal down-regulation of Ubx in these segments is functional. Finally, the DNA sequence of cDNAs for one of these genes predicts a protein that is similar to a human proto-oncogene involved in acute myeloid leukemias. These results illustrate potentially general rules about the homeotic control of target gene expression and suggest that subtractive hybridization can be used to isolate interesting homeotic target genes. PMID:7498738

  17. miR-182 attenuates atrophy-related gene expression by targeting FoxO3 in skeletal muscle

    PubMed Central

    Rahnert, Jill A.; Zheng, Bin; Woodworth-Hobbs, Myra E.; Franch, Harold A.; Russ Price, S.

    2014-01-01

    Skeletal muscle atrophy occurs in response to a variety of conditions including chronic kidney disease, diabetes, cancer, and elevated glucocorticoids. MicroRNAs (miR) may play a role in the wasting process. Activation of the forkhead box O3 (FoxO3) transcription factor causes skeletal muscle atrophy in patients, animals, and cultured cells by increasing the expression of components of the ubiquitin-proteasome and autophagy-lysosome proteolytic systems. To identify microRNAs that potentially modulate the atrophy process, an in silico target analysis was performed and miR-182 was predicted to target FoxO3 mRNA. Using a combination of immunoblot analysis, quantitative real-time RT-PCR, and FoxO3 3′-UTR luciferase reporter genes, miR-182 was confirmed to regulate FoxO3 expression in C2C12 myotubes. Transfection of miR-182 into muscle cells decreased FoxO3 mRNA 30% and FoxO3 protein 67% (P < 0.05) and also prevented a glucocorticoid-induced upregulation of multiple FoxO3 gene targets including MAFbx/atrogin-1, autophagy-related protein 12 (ATG12), cathepsin L, and microtubule-associated protein light chain 3 (LC3). Treatment of C2C12 myotubes with dexamethasone (Dex) (1 μM, 6 h) to induce muscle atrophy decreased miR-182 expression by 63% (P < 0.05). Similarly, miR-182 was decreased 44% (P < 0.05) in the gastrocnemius muscle of rats injected with streptozotocin to induce diabetes compared with controls. Finally, miR-182 was present in exosomes isolated from the media of C2C12 myotubes and Dex increased its abundance. These data identify miR-182 as an important regulator of FoxO3 expression that participates in the control of atrophy-inducing genes during catabolic diseases. PMID:24871856

  18. RNAi targeting GPR4 influences HMEC-1 gene expression by microarray analysis

    PubMed Central

    Ren, Juan; Zhang, Yuelang; Cai, Hui; Ma, Hongbing; Zhao, Dongli; Zhang, Xiaozhi; Li, Zongfang; Wang, Shufeng; Wang, Jiangsheng; Liu, Rui; Li, Yi; Qian, Jiansheng; Wei, Hongxia; Niu, Liying; Liu, Yan; Xiao, Lisha; Ding, Muyang; Jiang, Shiwen

    2014-01-01

    G-protein coupled receptor 4 (GPR4) belongs to a protein family comprised of 3 closely related G protein-coupled receptors. Recent studies have shown that GPR4 plays important roles in angiogenesis, proton sensing, and regulating tumor cells as an oncogenic gene. How GPR4 conducts its functions? Rare has been known. In order to detect the genes related to GPR4, microarray technology was employed. GPR4 is highly expressed in human vascular endothelial cell HMEC-1. Small interfering RNA against GPR4 was used to knockdown GPR4 expression in HMEC-1. Then RNA from the GPR4 knockdown cells and control cells were analyzed through genome microarray. Microarray results shown that among the whole genes and expressed sequence tags, 447 differentially expressed genes were identified, containing 318 up-regulated genes and 129 down-regulated genes. These genes whose expression dramatically changed may be involved in the GPR4 functions. These genes were related to cell apoptosis, cytoskeleton and signal transduction, cell proliferation, differentiation and cell-cycle regulation, gene transcription and translation and cell material and energy metabolism. PMID:24753754

  19. Expression of microRNA-26b and identification of its target gene EphA2 in pituitary tissues in Yanbian cattle

    PubMed Central

    YUAN, BAO; YU, WANG-YANG; DAI, LI-SHENG; GAO, YAN; DING, YU; YU, XIAN-FENG; CHEN, JIAN; ZHANG, JIA-BAO

    2015-01-01

    microRNAs (miRNAs/miRs) are a class of single-stranded non-coding RNA molecules of 19–24 nucleotides (nt) in length. They are widely expressed in animals, plants, bacteria and viruses. Via specific mRNA complementary pairing of target genes, miRNAs are able to regulate the expression of mRNA levels or inhibit protein translation following transcription. miRNA expression has a time- and space specificity, and it is involved in cell proliferation and differentiation, apoptosis, development, tumor metastasis occurrence and other biological processes. miR-26b is an miRNA of 22 nt and is important in the regulation of cellular processes. With the advancement of molecular biology techniques in recent years, there have been extensive investigations into miR-26b. Numerous studies have observed that miR-26b is involved in early embryonic development, cell proliferation regulation, pituitary hormone secretion and other physiological activities. miRNAs are associated with the function of propagation. The present study used reverse transcription quantitative polymerase chain reaction to detect the relative expression levels of miR-26b in the pituitary tissue of Yanbian cattle at different developmental stages. The 2−ΔΔCt method was used to calculate the relative gene expression levels. The miRNA target gene database TargetScan and RNA22 were used for prediction of the miR-26b target gene and selective recognition was also performed. The results demonstrated that miR-26b is expressed in the pituitary tissues of Yanbian cattle at 6 and 24 months of age. The relative expression levels of miR-26b in the pituitary tissues of 24-month-old Yanbian cattle were 2.41 times that of those in the six-month-old Yanbian cattle, demonstrating significant differences in the relative expression (P<0.01). The relative expression of the candidate target genes, EphA2 and miR-26b, exhibited the opposite expression pattern. The relative expression levels in the pituitary tissues of six

  20. Expression of microRNA‑26b and identification of its target gene EphA2 in pituitary tissues in Yanbian cattle.

    PubMed

    Yuan, Bao; Yu, Wang-Yang; Dai, Li-Sheng; Gao, Yan; Ding, Yu; Yu, Xian-Feng; Chen, Jian; Zhang, Jia-Bao

    2015-10-01

    microRNAs (miRNAs/miRs) are a class of single‑stranded non‑coding RNA molecules of 19‑24 nucleotides (nt) in length. They are widely expressed in animals, plants, bacteria and viruses. Via specific mRNA complementary pairing of target genes, miRNAs are able to regulate the expression of mRNA levels or inhibit protein translation following transcription. miRNA expression has a time‑ and space specificity, and it is involved in cell proliferation and differentiation, apoptosis, development, tumor metastasis occurrence and other biological processes. miR‑26b is an miRNA of 22 nt and is important in the regulation of cellular processes. With the advancement of molecular biology techniques in recent years, there have been extensive investigations into miR‑26b. Numerous studies have observed that miR‑26b is involved in early embryonic development, cell proliferation regulation, pituitary hormone secretion and other physiological activities. miRNAs are associated with the function of propagation. The present study used reverse transcription quantitative polymerase chain reaction to detect the relative expression levels of miR‑26b in the pituitary tissue of Yanbian cattle at different developmental stages. The 2‑∆∆Ct method was used to calculate the relative gene expression levels. The miRNA target gene database TargetScan and RNA22 were used for prediction of the miR‑26b target gene and selective recognition was also performed. The results demonstrated that miR‑26b is expressed in the pituitary tissues of Yanbian cattle at 6 and 24 months of age. The relative expression levels of miR‑26b in the pituitary tissues of 24‑month‑old Yanbian cattle were 2.41 times that of those in the six‑month‑old Yanbian cattle, demonstrating significant differences in the relative expression (P<0.01). The relative expression of the candidate target genes, EphA2 and miR‑26b, exhibited the opposite expression pattern. The relative expression levels in the

  1. Multi-focal control of mitochondrial gene expression by oncogenic MYC provides potential therapeutic targets in cancer

    PubMed Central

    Oran, Amanda R.; Adams, Clare M.; Zhang, Xiao-yong; Gennaro, Victoria J.; Pfeiffer, Harla K.; Mellert, Hestia S.; Seidel, Hans E.; Mascioli, Kirsten; Kaplan, Jordan; Gaballa, Mahmoud R.; Shen, Chen; Rigoutsos, Isidore; King, Michael P.; Cotney, Justin L.; Arnold, Jamie J.; Sharma, Suresh D.; Martinez, Ubaldo E.; Vakoc, Christopher R.; Chodosh, Lewis A.; Thompson, James E.; Bradner, James E.; Cameron, Craig E.; Shadel, Gerald S.; Eischen, Christine M.; McMahon, Steven B.

    2016-01-01

    Despite ubiquitous activation in human cancer, essential downstream effector pathways of the MYC transcription factor have been difficult to define and target. Using a structure/function-based approach, we identified the mitochondrial RNA polymerase (POLRMT) locus as a critical downstream target of MYC. The multifunctional POLRMT enzyme controls mitochondrial gene expression, a process required both for mitochondrial function and mitochondrial biogenesis. We further demonstrate that inhibition of this newly defined MYC effector pathway causes robust and selective tumor cell apoptosis, via an acute, checkpoint-like mechanism linked to aberrant electron transport chain complex assembly and mitochondrial reactive oxygen species (ROS) production. Fortuitously, MYC-dependent tumor cell death can be induced by inhibiting the mitochondrial gene expression pathway using a variety of strategies, including treatment with FDA-approved antibiotics. In vivo studies using a mouse model of Burkitt's Lymphoma provide pre-clinical evidence that these antibiotics can successfully block progression of MYC-dependent tumors. PMID:27590350

  2. Multi-focal control of mitochondrial gene expression by oncogenic MYC provides potential therapeutic targets in cancer.

    PubMed

    Oran, Amanda R; Adams, Clare M; Zhang, Xiao-Yong; Gennaro, Victoria J; Pfeiffer, Harla K; Mellert, Hestia S; Seidel, Hans E; Mascioli, Kirsten; Kaplan, Jordan; Gaballa, Mahmoud R; Shen, Chen; Rigoutsos, Isidore; King, Michael P; Cotney, Justin L; Arnold, Jamie J; Sharma, Suresh D; Martinez-Outschoorn, Ubaldo E; Vakoc, Christopher R; Chodosh, Lewis A; Thompson, James E; Bradner, James E; Cameron, Craig E; Shadel, Gerald S; Eischen, Christine M; McMahon, Steven B

    2016-11-08

    Despite ubiquitous activation in human cancer, essential downstream effector pathways of the MYC transcription factor have been difficult to define and target. Using a structure/function-based approach, we identified the mitochondrial RNA polymerase (POLRMT) locus as a critical downstream target of MYC. The multifunctional POLRMT enzyme controls mitochondrial gene expression, a process required both for mitochondrial function and mitochondrial biogenesis. We further demonstrate that inhibition of this newly defined MYC effector pathway causes robust and selective tumor cell apoptosis, via an acute, checkpoint-like mechanism linked to aberrant electron transport chain complex assembly and mitochondrial reactive oxygen species (ROS) production. Fortuitously, MYC-dependent tumor cell death can be induced by inhibiting the mitochondrial gene expression pathway using a variety of strategies, including treatment with FDA-approved antibiotics. In vivo studies using a mouse model of Burkitt's Lymphoma provide pre-clinical evidence that these antibiotics can successfully block progression of MYC-dependent tumors.

  3. Targeted gene insertion for molecular medicine.

    PubMed

    Voigt, Katrin; Izsvák, Zsuzsanna; Ivics, Zoltán

    2008-11-01

    Genomic insertion of a functional gene together with suitable transcriptional regulatory elements is often required for long-term therapeutical benefit in gene therapy for several genetic diseases. A variety of integrating vectors for gene delivery exist. Some of them exhibit random genomic integration, whereas others have integration preferences based on attributes of the targeted site, such as primary DNA sequence and physical structure of the DNA, or through tethering to certain DNA sequences by host-encoded cellular factors. Uncontrolled genomic insertion bears the risk of the transgene being silenced due to chromosomal position effects, and can lead to genotoxic effects due to mutagenesis of cellular genes. None of the vector systems currently used in either preclinical experiments or clinical trials displays sufficient preferences for target DNA sequences that would ensure appropriate and reliable expression of the transgene and simultaneously prevent hazardous side effects. We review in this paper the advantages and disadvantages of both viral and non-viral gene delivery technologies, discuss mechanisms of target site selection of integrating genetic elements (viruses and transposons), and suggest distinct molecular strategies for targeted gene delivery.

  4. Stochastic model of transcription factor-regulated gene expression

    NASA Astrophysics Data System (ADS)

    Karmakar, Rajesh; Bose, Indrani

    2006-09-01

    We consider a stochastic model of transcription factor (TF)-regulated gene expression. The model describes two genes, gene A and gene B, which synthesize the TFs and the target gene proteins, respectively. We show through analytic calculations that the TF fluctuations have a significant effect on the distribution of the target gene protein levels when the mean TF level falls in the highest sensitive region of the dose-response curve. We further study the effect of reducing the copy number of gene A from two to one. The enhanced TF fluctuations yield results different from those in the deterministic case. The probability that the target gene protein level exceeds a threshold value is calculated with the knowledge of the probability density functions associated with the TF and target gene protein levels. Numerical simulation results for a more detailed stochastic model are shown to be in agreement with those obtained through analytic calculations. The relevance of these results in the context of the genetic disorder haploinsufficiency is pointed out. Some experimental observations on the haploinsufficiency of the tumour suppressor gene, Nkx 3.1, are explained with the help of the stochastic model of TF-regulated gene expression.

  5. Vitamin D Impacts the Expression of Runx2 Target Genes and Modulates Inflammation, Oxidative Stress and Membrane Vesicle Biogenesis Gene Networks in 143B Osteosarcoma Cells.

    PubMed

    Garimella, Rama; Tadikonda, Priyanka; Tawfik, Ossama; Gunewardena, Sumedha; Rowe, Peter; Van Veldhuizen, Peter

    2017-03-16

    Osteosarcoma (OS) is an aggressive malignancy of bone affecting children, adolescents and young adults. Understanding vitamin D metabolism and vitamin D regulated genes in OS is an important aspect of vitamin D/cancer paradigm, and in evaluating vitamin D as adjuvant therapy for human OS. Vitamin D treatment of 143B OS cells induced significant and novel changes in the expression of genes that regulate: (a) inflammation and immunity; (b) formation of reactive oxygen species, metabolism of cyclic nucleotides, sterols, vitamins and mineral (calcium), quantity of gap junctions and skeletogenesis; (c) bone mineral density; and (d) cell viability of skeletal cells, aggregation of bone cancer cells and exocytosis of secretory vesicles. Ingenuity pathway analysis revealed significant reduction in Runx2 target genes such as fibroblast growth factor -1, -12 ( FGF1 and FGF12 ), bone morphogenetic factor-1 ( BMP1 ), SWI/SNF related, matrix associated actin dependent regulator of chromatin subfamily a, member 4 ( SMARCA4 ), Matrix extracellular phosphoglycoprotein ( MEPE ), Integrin, β4 ( ITGBP4 ), Matrix Metalloproteinase -1, -28 ( MMP1 and MMP28 ), and signal transducer and activator of transcription-4 ( STAT4 ) in vitamin D treated 143B OS cells. These genes interact with the inflammation, oxidative stress and membrane vesicle biogenesis gene networks. Vitamin D not only inhibited the expression of Runx2 target genes MMP1 , MMP28 and kallikrein related peptidase-7 ( KLK7 ), but also migration and invasion of 143B OS cells. Vitamin D regulated Runx2 target genes or their products represent potential therapeutic targets and laboratory biomarkers for applications in translational oncology.

  6. Vitamin D Impacts the Expression of Runx2 Target Genes and Modulates Inflammation, Oxidative Stress and Membrane Vesicle Biogenesis Gene Networks in 143B Osteosarcoma Cells

    PubMed Central

    Garimella, Rama; Tadikonda, Priyanka; Tawfik, Ossama; Gunewardena, Sumedha; Rowe, Peter; Van Veldhuizen, Peter

    2017-01-01

    Osteosarcoma (OS) is an aggressive malignancy of bone affecting children, adolescents and young adults. Understanding vitamin D metabolism and vitamin D regulated genes in OS is an important aspect of vitamin D/cancer paradigm, and in evaluating vitamin D as adjuvant therapy for human OS. Vitamin D treatment of 143B OS cells induced significant and novel changes in the expression of genes that regulate: (a) inflammation and immunity; (b) formation of reactive oxygen species, metabolism of cyclic nucleotides, sterols, vitamins and mineral (calcium), quantity of gap junctions and skeletogenesis; (c) bone mineral density; and (d) cell viability of skeletal cells, aggregation of bone cancer cells and exocytosis of secretory vesicles. Ingenuity pathway analysis revealed significant reduction in Runx2 target genes such as fibroblast growth factor -1, -12 (FGF1 and FGF12), bone morphogenetic factor-1 (BMP1), SWI/SNF related, matrix associated actin dependent regulator of chromatin subfamily a, member 4 (SMARCA4), Matrix extracellular phosphoglycoprotein (MEPE), Integrin, β4 (ITGBP4), Matrix Metalloproteinase -1, -28 (MMP1 and MMP28), and signal transducer and activator of transcription-4 (STAT4) in vitamin D treated 143B OS cells. These genes interact with the inflammation, oxidative stress and membrane vesicle biogenesis gene networks. Vitamin D not only inhibited the expression of Runx2 target genes MMP1, MMP28 and kallikrein related peptidase-7 (KLK7), but also migration and invasion of 143B OS cells. Vitamin D regulated Runx2 target genes or their products represent potential therapeutic targets and laboratory biomarkers for applications in translational oncology. PMID:28300755

  7. Gravity-regulated gene expression in Arabidopsis thaliana

    NASA Astrophysics Data System (ADS)

    Sederoff, Heike; Brown, Christopher S.; Heber, Steffen; Kajla, Jyoti D.; Kumar, Sandeep; Lomax, Terri L.; Wheeler, Benjamin; Yalamanchili, Roopa

    Plant growth and development is regulated by changes in environmental signals. Plants sense environmental changes and respond to them by modifying gene expression programs to ad-just cell growth, differentiation, and metabolism. Functional expression of genes comprises many different processes including transcription, translation, post-transcriptional and post-translational modifications, as well as the degradation of RNA and proteins. Recently, it was discovered that small RNAs (sRNA, 18-24 nucleotides long), which are heritable and systemic, are key elements in regulating gene expression in response to biotic and abiotic changes. Sev-eral different classes of sRNAs have been identified that are part of a non-cell autonomous and phloem-mobile network of regulators affecting transcript stability, translational kinetics, and DNA methylation patterns responsible for heritable transcriptional silencing (epigenetics). Our research has focused on gene expression changes in response to gravistimulation of Arabidopsis roots. Using high-throughput technologies including microarrays and 454 sequencing, we iden-tified rapid changes in transcript abundance of genes as well as differential expression of small RNA in Arabidopsis root apices after minutes of reorientation. Some of the differentially regu-lated transcripts are encoded by genes that are important for the bending response. Functional mutants of those genes respond faster to reorientation than the respective wild type plants, indicating that these proteins are repressors of differential cell elongation. We compared the gravity responsive sRNAs to the changes in transcript abundances of their putative targets and identified several potential miRNA: target pairs. Currently, we are using mutant and transgenic Arabidopsis plants to characterize the function of those miRNAs and their putative targets in gravitropic and phototropic responses in Arabidopsis.

  8. About miRNAs, miRNA seeds, target genes and target pathways.

    PubMed

    Kehl, Tim; Backes, Christina; Kern, Fabian; Fehlmann, Tobias; Ludwig, Nicole; Meese, Eckart; Lenhof, Hans-Peter; Keller, Andreas

    2017-12-05

    miRNAs are typically repressing gene expression by binding to the 3' UTR, leading to degradation of the mRNA. This process is dominated by the eight-base seed region of the miRNA. Further, miRNAs are known not only to target genes but also to target significant parts of pathways. A logical line of thoughts is: miRNAs with similar (seed) sequence target similar sets of genes and thus similar sets of pathways. By calculating similarity scores for all 3.25 million pairs of 2,550 human miRNAs, we found that this pattern frequently holds, while we also observed exceptions. Respective results were obtained for both, predicted target genes as well as experimentally validated targets. We note that miRNAs target gene set similarity follows a bimodal distribution, pointing at a set of 282 miRNAs that seems to target genes with very high specificity. Further, we discuss miRNAs with different (seed) sequences that nonetheless regulate similar gene sets or pathways. Most intriguingly, we found miRNA pairs that regulate different gene sets but similar pathways such as miR-6886-5p and miR-3529-5p. These are jointly targeting different parts of the MAPK signaling cascade. The main goal of this study is to provide a general overview on the results, to highlight a selection of relevant results on miRNAs, miRNA seeds, target genes and target pathways and to raise awareness for artifacts in respective comparisons. The full set of information that allows to infer detailed results on each miRNA has been included in miRPathDB, the miRNA target pathway database (https://mpd.bioinf.uni-sb.de).

  9. The targeting expression of the vascular endothelial growth factor gene in endothelial cells regulated by HRE.ppET-1.

    PubMed

    Zheng, Xiangrong; Zhang, Shangshang; Yang, Yujia; Wang, Xia; Zhong, Le; Yu, Xiaohe

    2008-11-01

    The success of gene therapy depends largely on the efficacy of gene delivery vector systems that can deliver genes to target organs or cells selectively and efficiently with minimal toxicity. Here, we show that by using the HRE.ppET-1 regulatory element, we were able to restrict expression of the transgene of vascular endothelial growth factor (VEGF) to endothelial cells exclusively in hypoxic conditions. Eukaryotic expression vectors such as pEGFP-HRE.ppET-1, pcDNA3.1-VEGF+Pa, pcDNA3.1-ppET-1+ EGF+Pa, and pcDNA3.1-HRE.ppET-1+VEGF+Pa were constructed by using a series of nuclear molecule handling methods like PCR, enzyme digestion. The recombinant vectors were transfected into HUVEC cells and HL7702 cells by the lipofectin method. GFP expression was observed with a fluorescence microscope to validate the specificity of expression in endothelial cells under the regulation of HRE.ppET-1 element. Cobalt chloride (final concentration 100 mumol/L) was added to the medium to mimic hypoxia in vitro. After transfection of vectors, the expression of VEGF mRNA was detected by RT-PCR, and the expression of VEGF was detected by Western blotting and ELISA methods under normoxia and hypoxia, respectively. The cell proliferation rate was detected by the MTT test. The expression of GFP revealed that the exterior gene was transcripted effectively in endothelial cells regulated by the HRE.ppET-1 element, while the expression of GFP was very weak in nonendothelial cells. The results of RT-PCR, Western blotting and ELISA showed that VEGF gene expression in the pcDNA3.1-HRE.ppET-1+VEGF+Pa group and in the pcDNA3.1-ppET-1+VEGF+Pa group was higher in hypoxia than it was in normoxia (P<0.05). The MTT test showed that the proliferation rate of HUVEC transfected with HPVA under hypoxia exceeded that of the control group. We conclude that the HRE.ppET-1 element was expressed specifically in endothelial cells, and can increase the expression of VEGF in hypoxia and stimulate proliferation of

  10. Systemic injection of AAV9 carrying a periostin promoter targets gene expression to a myofibroblast-like lineage in mouse hearts after reperfused myocardial infarction.

    PubMed

    Piras, B A; Tian, Y; Xu, Y; Thomas, N A; O'Connor, D M; French, B A

    2016-05-01

    Adeno-associated virus (AAV) has been used to direct gene transfer to a variety of tissues, including heart, liver, skeletal muscle, brain, kidney and lung, but it has not previously been shown to effectively target fibroblasts in vivo, including cardiac fibroblasts. We constructed expression cassettes using a modified periostin promoter to drive gene expression in a cardiac myofibroblast-like lineage, with only occasional spillover into cardiomyocyte-like cells. We compared AAV serotypes 6 and 9 and found robust gene expression when the vectors were delivered by systemic injection after myocardial infarction (MI), with little expression in healthy, non-infarcted mice. AAV9 provided expression in a greater number of cells than AAV6, with reporter gene expression visible in the cardiac infarct and border zones from 5 to 62 days post MI, as assessed by luciferase and Cre-activated green fluorescent protein expression. Although common myofibroblast markers were expressed in low abundance, most of the targeted cells expressed myosin IIb, an embryonic form of smooth muscle myosin heavy chain that has previously been associated with myofibroblasts after reperfused MI. This study is the first to demonstrate AAV-mediated expression in a potentially novel myofibroblast-like lineage in mouse hearts post MI and may open new avenues of gene therapy to treat patients surviving MI.

  11. Targeted polymeric nanoparticles for cancer gene therapy

    PubMed Central

    Kim, Jayoung; Wilson, David R.; Zamboni, Camila G.; Green, Jordan J.

    2015-01-01

    In this article, advances in designing polymeric nanoparticles for targeted cancer gene therapy are reviewed. Characterization and evaluation of biomaterials, targeting ligands, and transcriptional elements are each discussed. Advances in biomaterials have driven improvements to nanoparticle stability and tissue targeting, conjugation of ligands to the surface of polymeric nanoparticles enable binding to specific cancer cells, and the design of transcriptional elements has enabled selective DNA expression specific to the cancer cells. Together, these features have improved the performance of polymeric nanoparticles as targeted non-viral gene delivery vectors to treat cancer. As polymeric nanoparticles can be designed to be biodegradable, non-toxic, and to have reduced immunogenicity and tumorigenicity compared to viral platforms, they have significant potential for clinical use. Results of polymeric gene therapy in clinical trials and future directions for the engineering of nanoparticle systems for targeted cancer gene therapy are also presented. PMID:26061296

  12. Transposon Variants and Their Effects on Gene Expression in Arabidopsis

    PubMed Central

    Wang, Xi; Weigel, Detlef; Smith, Lisa M.

    2013-01-01

    Transposable elements (TEs) make up the majority of many plant genomes. Their transcription and transposition is controlled through siRNAs and epigenetic marks including DNA methylation. To dissect the interplay of siRNA–mediated regulation and TE evolution, and to examine how TE differences affect nearby gene expression, we investigated genome-wide differences in TEs, siRNAs, and gene expression among three Arabidopsis thaliana accessions. Both TE sequence polymorphisms and presence of linked TEs are positively correlated with intraspecific variation in gene expression. The expression of genes within 2 kb of conserved TEs is more stable than that of genes next to variant TEs harboring sequence polymorphisms. Polymorphism levels of TEs and closely linked adjacent genes are positively correlated as well. We also investigated the distribution of 24-nt-long siRNAs, which mediate TE repression. TEs targeted by uniquely mapping siRNAs are on average farther from coding genes, apparently because they more strongly suppress expression of adjacent genes. Furthermore, siRNAs, and especially uniquely mapping siRNAs, are enriched in TE regions missing in other accessions. Thus, targeting by uniquely mapping siRNAs appears to promote sequence deletions in TEs. Overall, our work indicates that siRNA–targeting of TEs may influence removal of sequences from the genome and hence evolution of gene expression in plants. PMID:23408902

  13. Ndrg2 is a PGC-1α/ERRα target gene that controls protein synthesis and expression of contractile-type genes in C2C12 myotubes.

    PubMed

    Foletta, Victoria C; Brown, Erin L; Cho, Yoshitake; Snow, Rod J; Kralli, Anastasia; Russell, Aaron P

    2013-12-01

    The stress-responsive, tumor suppressor N-myc downstream-regulated gene 2 (Ndrg2) is highly expressed in striated muscle. In response to anabolic and catabolic signals, Ndrg2 is suppressed and induced, respectively, in mouse C2C12 myotubes. However, little is known about the mechanisms regulating Ndrg2 expression in muscle, as well as the biological role for Ndrg2 in differentiated myotubes. Here, we show that Ndrg2 is a target of a peroxisome proliferator-activated receptor-gamma coactivator-1α (PGC-1α) and estrogen-related receptor alpha (ERRα) transcriptional program and is induced in response to endurance exercise, a physiological stress known also to increase PGC-1α/ERRα activity. Analyses of global gene and protein expression profiles in C2C12 myotubes with reduced levels of NDRG2, suggest that NDRG2 affects muscle growth, contractile properties, MAPK signaling, ion and vesicle transport and oxidative phosphorylation. Indeed, suppression of NDRG2 in myotubes increased protein synthesis and the expression of fast glycolytic myosin heavy chain isoforms, while reducing the expression of embryonic myosin Myh3, other contractile-associated genes and the MAPK p90 RSK1. Conversely, enhanced expression of NDRG2 reduced protein synthesis, and furthermore, partially blocked the increased protein synthesis rates elicited by a constitutively active form of ERRα. In contrast, suppressing or increasing levels of NDRG2 did not affect mRNA expression of genes involved in mitochondrial biogenesis that are regulated by PGC-1α or ERRα. This study shows that in C2C12 myotubes Ndrg2 is a novel PGC-1α/ERRα transcriptional target, which influences protein turnover and the regulation of genes involved in muscle contraction and function. © 2013 Elsevier B.V. All rights reserved.

  14. Identification and expression analysis of leptin-regulated immediate early response and late target genes.

    PubMed

    Waelput, W; Verhee, A; Broekaert, D; Eyckerman, S; Vandekerckhove, J; Beattie, J H; Tavernier, J

    2000-05-15

    Using PC12 cells as an in vitro model system, we have identified a series of transcripts induced through activation of the leptin receptor. On the basis of kinetic studies, two distinct gene sets could be discerned: signal transducer and activator of transciption-3 (STAT-3), suppressor of cytokine signalling-3 (SOCS-3), MT-II (metallothionein-II), the serine/threonine kinase fibroblast-growth-factor-inducible kinase (Fnk) and modulator recognition factor (MRF-1), which are immediate early response genes, and pancreatitis-associated protein I (PAP I), squalene epoxidase, uridine diphosphate glucuronosyltransferase and annexin VIII, which are late induced target genes. At late time points a strong co-stimulation with beta-nerve growth factor or with the adenylate cyclase activator forskolin was observed. To assess the validity of the PC12-cell model system, we examined the effect of leptin administration on the gene transcription of STAT-3, MT-II, Fnk and PAP I in vivo. Leptin treatment of leptin-deficient ob/ob mice increased the STAT-3, SOCS-3, MT-II and Fnk mRNA, and MT-I protein levels in liver, whereas, in jejunum, expression of PAP I mRNA was down-regulated. Furthermore, administration of leptin to starved wild-type mice enhanced the expression of MT-II and Fnk mRNA in liver, but decreased MT-II and PAP I mRNA expression in jejunum. These findings may help to explain the obese phenotype observed in some colonies of MT-I- and MT-II-null mice and/or the observation that leptin protects against tumour-necrosis-factor toxicity in vivo.

  15. RNA-guided genome editing for target gene mutations in wheat.

    PubMed

    Upadhyay, Santosh Kumar; Kumar, Jitesh; Alok, Anshu; Tuli, Rakesh

    2013-12-09

    The clustered, regularly interspaced, short palindromic repeats (CRISPR) and CRISPR-associated protein (Cas) system has been used as an efficient tool for genome editing. We report the application of CRISPR-Cas-mediated genome editing to wheat (Triticum aestivum), the most important food crop plant with a very large and complex genome. The mutations were targeted in the inositol oxygenase (inox) and phytoene desaturase (pds) genes using cell suspension culture of wheat and in the pds gene in leaves of Nicotiana benthamiana. The expression of chimeric guide RNAs (cgRNA) targeting single and multiple sites resulted in indel mutations in all the tested samples. The expression of Cas9 or sgRNA alone did not cause any mutation. The expression of duplex cgRNA with Cas9 targeting two sites in the same gene resulted in deletion of DNA fragment between the targeted sequences. Multiplexing the cgRNA could target two genes at one time. Target specificity analysis of cgRNA showed that mismatches at the 3' end of the target site abolished the cleavage activity completely. The mismatches at the 5' end reduced cleavage, suggesting that the off target effects can be abolished in vivo by selecting target sites with unique sequences at 3' end. This approach provides a powerful method for genome engineering in plants.

  16. p53 targets chromatin structure alteration to repress alpha-fetoprotein gene expression.

    PubMed

    Ogden, S K; Lee, K C; Wernke-Dollries, K; Stratton, S A; Aronow, B; Barton, M C

    2001-11-09

    Many of the functions ascribed to p53 tumor suppressor protein are mediated through transcription regulation. We have shown that p53 represses hepatic-specific alpha-fetoprotein (AFP) gene expression by direct interaction with a composite HNF-3/p53 DNA binding element. Using solid-phase, chromatin-assembled AFP DNA templates and analysis of chromatin structure and transcription in vitro, we find that p53 binds DNA and alters chromatin structure at the AFP core promoter to regulate transcription. Chromatin assembled in the presence of hepatoma extracts is activated for AFP transcription with an open, accessible core promoter structure. Distal (-850) binding of p53 during chromatin assembly, but not post-assembly, reverses transcription activation concomitant with promoter inaccessibility to restriction enzyme digestion. Inhibition of histone deacetylase activity by trichostatin-A (TSA) addition, prior to and during chromatin assembly, activated chromatin transcription in parallel with increased core promoter accessibility. Chromatin immunoprecipitation analyses showed increased H3 and H4 acetylated histones at the core promoter in the presence of TSA, while histone acetylation remained unchanged at the site of distal p53 binding. Our data reveal that p53 targets chromatin structure alteration at the core promoter, independently of effects on histone acetylation, to establish repressed AFP gene expression.

  17. Comparative gene expression analysis of Dtg, a novel target gene of Dpp signaling pathway in the early Drosophila melanogaster embryo.

    PubMed

    Hodar, Christian; Zuñiga, Alejandro; Pulgar, Rodrigo; Travisany, Dante; Chacon, Carlos; Pino, Michael; Maass, Alejandro; Cambiazo, Verónica

    2014-02-10

    In the early Drosophila melanogaster embryo, Dpp, a secreted molecule that belongs to the TGF-β superfamily of growth factors, activates a set of downstream genes to subdivide the dorsal region into amnioserosa and dorsal epidermis. Here, we examined the expression pattern and transcriptional regulation of Dtg, a new target gene of Dpp signaling pathway that is required for proper amnioserosa differentiation. We showed that the expression of Dtg was controlled by Dpp and characterized a 524-bp enhancer that mediated expression in the dorsal midline, as well as, in the differentiated amnioserosa in transgenic reporter embryos. This enhancer contained a highly conserved region of 48-bp in which bioinformatic predictions and in vitro assays identified three Mad binding motifs. Mutational analysis revealed that these three motifs were necessary for proper expression of a reporter gene in transgenic embryos, suggesting that short and highly conserved genomic sequences may be indicative of functional regulatory regions in D. melanogaster genes. Dtg orthologs were not detected in basal lineages of Dipterans, which unlike D. melanogaster develop two extra-embryonic membranes, amnion and serosa, nevertheless Dtg orthologs were identified in the transcriptome of Musca domestica, in which dorsal ectoderm patterning leads to the formation of a single extra-embryonic membrane. These results suggest that Dtg was recruited as a new component of the network that controls dorsal ectoderm patterning in the lineage leading to higher Cyclorrhaphan flies, such as D. melanogaster and M. domestica. Copyright © 2013 Elsevier B.V. All rights reserved.

  18. GENE EXPRESSION PROFILING OF ACCESSIBLE SURROGATE TISSUES TO MONITOR MOLECULAR CHANGES IN INACCESSIBLE TARGET TISSUES FOLLOWING TOXICANT EXPOSURE

    EPA Science Inventory

    Gene Expression Profiling Of Accessible Surrogate Tissues To Monitor Molecular Changes In Inaccessible Target Tissues Following Toxicant Exposure
    John C. Rockett, Chad R. Blystone, Amber K. Goetz, Rachel N. Murrell, Judith E. Schmid and David J. Dix
    Reproductive Toxicology ...

  19. NF-kB activation and its downstream target genes expression after heavy ions exposure

    NASA Astrophysics Data System (ADS)

    Chishti, Arif Ali; Baumstark-Khan, Christa; Hellweg, Christine; Schmitz, Claudia; Koch, Kristina; Feles, Sebastian

    2016-07-01

    To enable long-term human space flight cellular radiation response to densely ionizing radiation needs to be better understood for developing appropriate countermeasures to mitigate acute effects and late radiation risks for the astronaut. The biological effectiveness of accelerated heavy ions (which constitute the most important radiation type in space) with high linear energy transfer (LET) for effecting DNA damage response pathways as a gateway to cell death or survival is of major concern not only for space missions but also for new regimes of tumor radiotherapy. In the current research study, the contribution of NF-κB in response to space-relevant radiation qualities was determined by a NF-κB reporter cell line (HEK-pNF-κB-d2EGFP/Neo L2). The NF-κB dependent reporter gene expression (d2EGFP) after ionizing radiation (X-rays and heavy ions) exposure was evaluated by flow cytometry. Because of differences in the extent of NF-κB activation after X-irradiation and heavy ions exposure, it was expected that radiation quality (LET) might play an important role in the cellular radiation response. In addition, the biological effectiveness (RBE) of NF-κB activation and reduction of cellular survival was examined for heavy ions having a broad range of LET (˜0.3 - 9674 keV/µm). Furthermore, the effect of LET on NF-κB target gene expression was analyzed by real time reverse transcriptase quantitative PCR (RT-qPCR). In this study it was proven that NF-κB activation and NF-κB dependent gene expression comprises an early step in cellular radiation response. Taken together, this study clearly demonstrates that NF-κB activation and NF-κB-dependent gene expression by heavy ions are highest in the LET range of ˜50-200 keV/μupm. The up-regulated chemokines and cytokines (CXCL1, CXCL2, CXCL10, IL-8 and TNF) might be important for cell-cell communication among hit as well as unhit cells (bystander effect). The results obtained suggest the NF-κB pathway to be a

  20. Multiple microRNAs regulate human FOXP2 gene expression by targeting sequences in its 3' untranslated region.

    PubMed

    Fu, Lijuan; Shi, Zhimin; Luo, Guanzheng; Tu, Weihong; Wang, XiuJie; Fang, Zhide; Li, XiaoChing

    2014-10-01

    Mutations in the human FOXP2 gene cause speech and language impairments. The FOXP2 protein is a transcription factor that regulates the expression of many downstream genes, which may have important roles in nervous system development and function. An adequate amount of functional FOXP2 protein is thought to be critical for the proper development of the neural circuitry underlying speech and language. However, how FOXP2 gene expression is regulated is not clearly understood. The FOXP2 mRNA has an approximately 4-kb-long 3' untranslated region (3' UTR), twice as long as its protein coding region, indicating that FOXP2 can be regulated by microRNAs (miRNAs). We identified multiple miRNAs that regulate the expression of the human FOXP2 gene using sequence analysis and in vitro cell systems. Focusing on let-7a, miR-9, and miR-129-5p, three brain-enriched miRNAs, we show that these miRNAs regulate human FOXP2 expression in a dosage-dependent manner and target specific sequences in the FOXP2 3' UTR. We further show that these three miRNAs are expressed in the cerebellum of the human fetal brain, where FOXP2 is known to be expressed. Our results reveal novel regulatory functions of the human FOXP2 3' UTR sequence and regulatory interactions between multiple miRNAs and the human FOXP2 gene. The expression of let-7a, miR-9, and miR-129-5p in the human fetal cerebellum is consistent with their roles in regulating FOXP2 expression during early cerebellum development. These results suggest that various genetic and environmental factors may contribute to speech and language development and related neural developmental disorders via the miRNA-FOXP2 regulatory network.

  1. In silico identification of miRNAs and their target genes and analysis of gene co-expression network in saffron (Crocus sativus L.) stigma

    PubMed Central

    Zinati, Zahra; Shamloo-Dashtpagerdi, Roohollah; Behpouri, Ali

    2016-01-01

    As an aromatic and colorful plant of substantive taste, saffron (Crocus sativus L.) owes such properties of matter to growing class of the secondary metabolites derived from the carotenoids, apocarotenoids. Regarding the critical role of microRNAs in secondary metabolic synthesis and the limited number of identified miRNAs in C. sativus, on the other hand, one may see the point how the characterization of miRNAs along with the corresponding target genes in C. sativus might expand our perspectives on the roles of miRNAs in carotenoid/apocarotenoid biosynthetic pathway. A computational analysis was used to identify miRNAs and their targets using EST (Expressed Sequence Tag) library from mature saffron stigmas. Then, a gene co- expression network was constructed to identify genes which are potentially involved in carotenoid/apocarotenoid biosynthetic pathways. EST analysis led to the identification of two putative miRNAs (miR414 and miR837-5p) along with the corresponding stem- looped precursors. To our knowledge, this is the first report on miR414 and miR837-5p in C. sativus. Co-expression network analysis indicated that miR414 and miR837-5p may play roles in C. sativus metabolic pathways and led to identification of candidate genes including six transcription factors and one protein kinase probably involved in carotenoid/apocarotenoid biosynthetic pathway. Presence of transcription factors, miRNAs and protein kinase in the network indicated multiple layers of regulation in saffron stigma. The candidate genes from this study may help unraveling regulatory networks underlying the carotenoid/apocarotenoid biosynthesis in saffron and designing metabolic engineering for enhanced secondary metabolites. PMID:28261627

  2. The drug target genes show higher evolutionary conservation than non-target genes.

    PubMed

    Lv, Wenhua; Xu, Yongdeng; Guo, Yiying; Yu, Ziqi; Feng, Guanglong; Liu, Panpan; Luan, Meiwei; Zhu, Hongjie; Liu, Guiyou; Zhang, Mingming; Lv, Hongchao; Duan, Lian; Shang, Zhenwei; Li, Jin; Jiang, Yongshuai; Zhang, Ruijie

    2016-01-26

    Although evidence indicates that drug target genes share some common evolutionary features, there have been few studies analyzing evolutionary features of drug targets from an overall level. Therefore, we conducted an analysis which aimed to investigate the evolutionary characteristics of drug target genes. We compared the evolutionary conservation between human drug target genes and non-target genes by combining both the evolutionary features and network topological properties in human protein-protein interaction network. The evolution rate, conservation score and the percentage of orthologous genes of 21 species were included in our study. Meanwhile, four topological features including the average shortest path length, betweenness centrality, clustering coefficient and degree were considered for comparison analysis. Then we got four results as following: compared with non-drug target genes, 1) drug target genes had lower evolutionary rates; 2) drug target genes had higher conservation scores; 3) drug target genes had higher percentages of orthologous genes and 4) drug target genes had a tighter network structure including higher degrees, betweenness centrality, clustering coefficients and lower average shortest path lengths. These results demonstrate that drug target genes are more evolutionarily conserved than non-drug target genes. We hope that our study will provide valuable information for other researchers who are interested in evolutionary conservation of drug targets.

  3. Identification of apoptosis-related PLZF target genes

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Bernardo, Maria Victoria; Yelo, Estefania; Gimeno, Lourdes

    2007-07-27

    The PLZF gene encodes a BTB/POZ-zinc finger-type transcription factor, involved in physiological development, proliferation, differentiation, and apoptosis. In this paper, we investigate proliferation, survival, and gene expression regulation in stable clones from the human haematopoietic K562, DG75, and Jurkat cell lines with inducible expression of PLZF. In Jurkat cells, but not in K562 and DG75 cells, PLZF induced growth suppression and apoptosis in a cell density-dependent manner. Deletion of the BTB/POZ domain of PLZF abrogated growth suppression and apoptosis. PLZF was expressed with a nuclear speckled pattern distinctively in the full-length PLZF-expressing Jurkat clones, suggesting that the nuclear speckled localizationmore » is required for PLZF-induced apoptosis. By microarray analysis, we identified that the apoptosis-inducer TP53INP1, ID1, and ID3 genes were upregulated, and the apoptosis-inhibitor TERT gene was downregulated. The identification of apoptosis-related PLZF target genes may have biological and clinical relevance in cancer typified by altered PLZF expression.« less

  4. Gene expression profile of mouse prostate tumors reveals dysregulations in major biological processes and identifies potential murine targets for preclinical development of human prostate cancer therapy.

    PubMed

    Haram, Kerstyn M; Peltier, Heidi J; Lu, Bin; Bhasin, Manoj; Otu, Hasan H; Choy, Bob; Regan, Meredith; Libermann, Towia A; Latham, Gary J; Sanda, Martin G; Arredouani, Mohamed S

    2008-10-01

    Translation of preclinical studies into effective human cancer therapy is hampered by the lack of defined molecular expression patterns in mouse models that correspond to the human counterpart. We sought to generate an open source TRAMP mouse microarray dataset and to use this array to identify differentially expressed genes from human prostate cancer (PCa) that have concordant expression in TRAMP tumors, and thereby represent lead targets for preclinical therapy development. We performed microarrays on total RNA extracted and amplified from eight TRAMP tumors and nine normal prostates. A subset of differentially expressed genes was validated by QRT-PCR. Differentially expressed TRAMP genes were analyzed for concordant expression in publicly available human prostate array datasets and a subset of resulting genes was analyzed by QRT-PCR. Cross-referencing differentially expressed TRAMP genes to public human prostate array datasets revealed 66 genes with concordant expression in mouse and human PCa; 56 between metastases and normal and 10 between primary tumor and normal tissues. Of these 10 genes, two, Sox4 and Tubb2a, were validated by QRT-PCR. Our analysis also revealed various dysregulations in major biologic pathways in the TRAMP prostates. We report a TRAMP microarray dataset of which a gene subset was validated by QRT-PCR with expression patterns consistent with previous gene-specific TRAMP studies. Concordance analysis between TRAMP and human PCa associated genes supports the utility of the model and suggests several novel molecular targets for preclinical therapy.

  5. PAI-1, a target gene of miR-143, regulates invasion and metastasis by upregulating MMP-13 expression of human osteosarcoma.

    PubMed

    Hirahata, Mio; Osaki, Mitsuhiko; Kanda, Yusuke; Sugimoto, Yui; Yoshioka, Yusuke; Kosaka, Nobuyoshi; Takeshita, Fumitaka; Fujiwara, Tomohiro; Kawai, Akira; Ito, Hisao; Ochiya, Takahiro; Okada, Futoshi

    2016-05-01

    Despite recent improvements in the therapy for osteosarcoma, 30-40% of osteosarcoma patients die of this disease, mainly due to its lung metastasis. We have previously reported that intravenous injection of miR-143 significantly suppresses lung metastasis of human osteosarcoma cells (143B) in a mouse model. In this study, we examined the biological role and mechanism of miR-143 in the metastasis of human osteosarcoma cells. We identified plasminogen activator inhibitor-1 (PAI-1) as a direct target gene of miR-143. To determine the role of PAI-1 in human osteosarcoma cells, siRNA was transfected into 143B cells for knockdown of PAI-1 expression. An in vitro study showed that downregulation of PAI-1 suppressed cell invasion activity, but not proliferation. Moreover, injection of PAI-1 siRNA into a primary lesion in the osteosarcoma mouse model inhibited lung metastasis compared to control siRNA-injected mice, without influencing the proliferative activity of the tumor cells. Subsequent examination using 143B cells revealed that knockdown of PAI-1 expression resulted in downregulation of the expression and secretion of matrix metalloproteinase-13 (MMP-13), which is also a target gene of miR-143 and a proteolytic enzyme that regulates tumor-induced osteolysis. Immunohistochemical analysis using clinical samples showed that higher miR-143 expressing cases showed poor expression of PAI-1 in the primary tumor cells. All such cases belonged to the lung metastasis-negative group. Moreover, the frequency of lung metastasis-positive cases was significantly higher in PAI-1 and MMP-13 double-positive cases than in PAI-1 or MMP-13 single-positive or double-negative cases (P < 0.05). These results indicated that PAI-1, a target gene of miR-143, regulates invasion and lung metastasis via enhancement of MMP-13 expression and secretion in human osteosarcoma cells, suggesting that these molecules could be potential therapeutic target genes for preventing lung metastasis in

  6. Generation of novel resistance genes using mutation and targeted gene editing.

    PubMed

    Gal-On, Amit; Fuchs, Marc; Gray, Stewart

    2017-10-01

    Classical breeding for virus resistance is a lengthy process and is restricted by the availability of resistance genes. Precise genome editing is a 'dream technology' to improve plants for virus resistance and these tools have opened new and very promising ways to generate virus resistant plants by disrupting host susceptibility genes, or by increasing the expression of viral resistance genes. However, precise targets must be identified and their roles understood to minimize potential negative effects on the plant. Nonetheless, the opportunities for genome editing are expanding, as are the technologies to generate effective and broad-spectrum resistance against plant viruses. Here we provide insights into recent progress related to gene targets and gene editing technologies. Published by Elsevier B.V.

  7. Polycistronic gene expression in Aspergillus niger.

    PubMed

    Schuetze, Tabea; Meyer, Vera

    2017-09-25

    Genome mining approaches predict dozens of biosynthetic gene clusters in each of the filamentous fungal genomes sequenced so far. However, the majority of these gene clusters still remain cryptic because they are not expressed in their natural host. Simultaneous expression of all genes belonging to a biosynthetic pathway in a heterologous host is one approach to activate biosynthetic gene clusters and to screen the metabolites produced for bioactivities. Polycistronic expression of all pathway genes under control of a single and tunable promoter would be the method of choice, as this does not only simplify cloning procedures, but also offers control on timing and strength of expression. However, polycistronic gene expression is a feature not commonly found in eukaryotic host systems, such as Aspergillus niger. In this study, we tested the suitability of the viral P2A peptide for co-expression of three genes in A. niger. Two genes descend from Fusarium oxysporum and are essential to produce the secondary metabolite enniatin (esyn1, ekivR). The third gene (luc) encodes the reporter luciferase which was included to study position effects. Expression of the polycistronic gene cassette was put under control of the Tet-On system to ensure tunable gene expression in A. niger. In total, three polycistronic expression cassettes which differed in the position of luc were constructed and targeted to the pyrG locus in A. niger. This allowed direct comparison of the luciferase activity based on the position of the luciferase gene. Doxycycline-mediated induction of the Tet-On expression cassettes resulted in the production of one long polycistronic mRNA as proven by Northern analyses, and ensured comparable production of enniatin in all three strains. Notably, gene position within the polycistronic expression cassette matters, as, luciferase activity was lowest at position one and had a comparable activity at positions two and three. The P2A peptide can be used to express at

  8. Specific c-Jun target genes in malignant melanoma.

    PubMed

    Schummer, Patrick; Kuphal, Silke; Vardimon, Lily; Bosserhoff, Anja K; Kappelmann, Melanie

    2016-05-03

    A fundamental event in the development and progression of malignant melanoma is the de-regulation of cancer-relevant transcription factors. We recently showed that c-Jun is a main regulator of melanoma progression and, thus, is the most important member of the AP-1 transcription factor family in this disease. Surprisingly, no cancer-related specific c-Jun target genes in melanoma were described in the literature, so far. Therefore, we focused on pre-existing ChIP-Seq data (Encyclopedia of DNA Elements) of 3 different non-melanoma cell lines to screen direct c-Jun target genes. Here, a specific c-Jun antibody to immunoprecipitate the associated promoter DNA was used. Consequently, we identified 44 direct c-Jun targets and a detailed analysis of 6 selected genes confirmed their deregulation in malignant melanoma. The identified genes were differentially regulated comparing 4 melanoma cell lines and normal human melanocytes and we confirmed their c-Jun dependency. Direct interaction between c-Jun and the promoter/enhancer regions of the identified genes was confirmed by us via ChIP experiments. Interestingly, we revealed that the direct regulation of target gene expression via c-Jun can be independent of the existence of the classical AP-1 (5´-TGA(C/G)TCA-3´) consensus sequence allowing for the subsequent down- or up-regulation of the expression of these cancer-relevant genes. In summary, the results of this study indicate that c-Jun plays a crucial role in the development and progression of malignant melanoma via direct regulation of cancer-relevant target genes and that inhibition of direct c-Jun targets through inhibition of c-Jun is a potential novel therapeutic option for treatment of malignant melanoma.

  9. HOXB homeobox gene expression in cervical carcinoma.

    PubMed

    López, R; Garrido, E; Piña, P; Hidalgo, A; Lazos, M; Ochoa, R; Salcedo, M

    2006-01-01

    The homeobox (HOX) genes are a family of transcription factors that bind to specific DNA sequences in target genes regulating gene expression. Thirty-nine HOX genes have been mapped in four conserved clusters: A, B, C, and D; they act as master genes regulating the identity of body segments along the anteroposterior axis of the embryo. The role played by HOX genes in adult cell differentiation is unclear to date, but growing evidence suggests that they may play an important role in the development of cancer. To study the role played by HOX genes in cervical cancer, in the present work, we analyzed the expression of HOXB genes and the localization of their transcripts in human cervical tissues. Reverse transcription-polymerase chain reaction analysis and nonradioactive RNA in situ hybridization were used to detect HOXB expression in 11 normal cervical tissues and 17 cervical carcinomas. It was determined that HOXB1, B3, B5, B6, B7, B8, and B9 genes are expressed in normal adult cervical epithelium and squamous cervical carcinomas. Interestingly, HOXB2, HOXB4, and HOXB13 gene expression was found only in tumor tissues. Our findings suggest that the new expression of HOXB2, HOXB4, and B13 genes is involved in cervical cancer.

  10. Reconstitution of the ERG Gene Expression Network Reveals New Biomarkers and Therapeutic Targets in ERG Positive Prostate Tumors

    PubMed Central

    Dubovenko, Alexey; Serebryiskaya, Tatiana; Nikolsky, Yuri; Nikolskaya, Tatiana; Perlina, Ally; JeBailey, Lellean; Bureeva, Svetlana; Katta, Shilpa; Srivastava, Shiv; Dobi, Albert; Khasanova, Tatiana

    2015-01-01

    Background: Despite a growing number of studies evaluating cancer of prostate (CaP) specific gene alterations, oncogenic activation of the ETS Related Gene (ERG) by gene fusions remains the most validated cancer gene alteration in CaP. Prevalent gene fusions have been described between the ERG gene and promoter upstream sequences of androgen-inducible genes, predominantly TMPRSS2 (transmembrane protease serine 2). Despite the extensive evaluations of ERG genomic rearrangements, fusion transcripts and the ERG oncoprotein, the prognostic value of ERG remains to be better understood. Using gene expression dataset from matched prostate tumor and normal epithelial cells from an 80 GeneChip experiment examining 40 tumors and their matching normal pairs in 40 patients with known ERG status, we conducted a cancer signaling-focused functional analysis of prostatic carcinoma representing moderate and aggressive cancers stratified by ERG expression. Results: In the present study of matched pairs of laser capture microdissected normal epithelial cells and well-to-moderately differentiated tumor epithelial cells with known ERG gene expression status from 20 patients with localized prostate cancer, we have discovered novel ERG associated biochemical networks. Conclusions: Using causal network reconstruction methods, we have identified three major signaling pathways related to MAPK/PI3K cascade that may indeed contribute synergistically to the ERG dependent tumor development. Moreover, the key components of these pathways have potential as biomarkers and therapeutic target for ERG positive prostate tumors. PMID:26000039

  11. Progress in gene targeting and gene therapy for retinitis pigmentosa

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Farrar, G.J.; Humphries, M.M.; Erven, A.

    1994-09-01

    Previously, we localized disease genes involved in retinitis pigmentosa (RP), an inherited retinal degeneration, close to the rhodopsin and peripherin genes on 3q and 6p. Subsequently, we and others identified mutations in these genes in RP patients. Currently animal models for human retinopathies are being generated using gene targeting by homologous recombination in embryonic stem (ES) cells. Genomic clones for retinal genes including rhodopsin and peripherin have been obtained from a phage library carrying mouse DNA isogenic with the ES cell line (CC1.2). The peripherin clone has been sequenced to establish the genomic structure of the mouse gene. Targeting vectorsmore » for rhodopsin and peripherin including a neomycin cassette for positive selection and thymidine kinase genes enabling selection against random intergrants are under construction. Progress in vector construction will be presented. Simultaneously we are developing systems for delivery of gene therapies to retinal tissues utilizing replication-deficient adenovirus (Ad5). Efficacy of infection subsequent to various methods of intraocular injection and with varying viral titers is being assayed using an adenovirus construct containing a CMV promoter LacZ fusion as reporter and the range of tissues infected and the level of duration of LacZ expression monitored. Viral constructs with the LacZ reporter gene under the control of retinal specific promoters such as rhodopsin and IRBP cloned into pXCJL.1 are under construction. An update on developments in photoreceptor cell-directed expression of virally delivered genes will be presented.« less

  12. The novel cyst nematode effector protein 30D08 targets host nuclear functions to alter gene expression in feeding sites.

    PubMed

    Verma, Anju; Lee, Chris; Morriss, Stephanie; Odu, Fiona; Kenning, Charlotte; Rizzo, Nancy; Spollen, William G; Lin, Marriam; McRae, Amanda G; Givan, Scott A; Hewezi, Tarek; Hussey, Richard; Davis, Eric L; Baum, Thomas J; Mitchum, Melissa G

    2018-05-04

    Cyst nematodes deliver effector proteins into host cells to manipulate cellular processes and establish a metabolically hyperactive feeding site. The novel 30D08 effector protein is produced in the dorsal gland of parasitic juveniles, but its function has remained unknown. We demonstrate that expression of 30D08 contributes to nematode parasitism, the protein is packaged into secretory granules and it is targeted to the plant nucleus where it interacts with SMU2 (homolog of suppressor of mec-8 and unc-52 2), an auxiliary spliceosomal protein. We show that SMU2 is expressed in feeding sites and an smu2 mutant is less susceptible to nematode infection. In Arabidopsis expressing 30D08 under the SMU2 promoter, several genes were found to be alternatively spliced and the most abundant functional classes represented among differentially expressed genes were involved in RNA processing, transcription and binding, as well as in development, and hormone and secondary metabolism, representing key cellular processes known to be important for feeding site formation. In conclusion, we demonstrated that the 30D08 effector is secreted from the nematode and targeted to the plant nucleus where its interaction with a host auxiliary spliceosomal protein may alter the pre-mRNA splicing and expression of a subset of genes important for feeding site formation. © 2018 The Authors. New Phytologist © 2018 New Phytologist Trust.

  13. Multilevel regulation of gene expression by microRNAs.

    PubMed

    Makeyev, Eugene V; Maniatis, Tom

    2008-03-28

    MicroRNAs (miRNAs) are approximately 22-nucleotide-long noncoding RNAs that normally function by suppressing translation and destabilizing messenger RNAs bearing complementary target sequences. Some miRNAs are expressed in a cell- or tissue-specific manner and may contribute to the establishment and/or maintenance of cellular identity. Recent studies indicate that tissue-specific miRNAs may function at multiple hierarchical levels of gene regulatory networks, from targeting hundreds of effector genes incompatible with the differentiated state to controlling the levels of global regulators of transcription and alternative pre-mRNA splicing. This multilevel regulation may allow individual miRNAs to profoundly affect the gene expression program of differentiated cells.

  14. DNMT3B modulates the expression of cancer-related genes and downregulates the expression of the gene VAV3 via methylation

    PubMed Central

    Peralta-Arrieta, Irlanda; Hernández-Sotelo, Daniel; Castro-Coronel, Yaneth; Leyva-Vázquez, Marco Antonio; Illades-Aguiar, Berenice

    2017-01-01

    Altered promoter DNA methylation is one of the most important epigenetic abnormalities in human cancer. DNMT3B, de novo methyltransferase, is clearly related to abnormal methylation of tumour suppressor genes, DNA repair genes and its overexpression contributes to oncogenic processes and tumorigenesis in vivo. The purpose of this study was to assess the effect of the overexpression of DNMT3B in HaCaT cells on global gene expression and on the methylation of selected genes to the identification of genes that can be target of DNMT3B. We found that the overexpression of DNMT3B in HaCaT cells, modulate the expression of genes related to cancer, downregulated the expression of 151 genes with CpG islands and downregulated the expression of the VAV3 gene via methylation of its promoter. These results highlight the importance of DNMT3B in gene expression and human cancer. PMID:28123849

  15. DNMT3B modulates the expression of cancer-related genes and downregulates the expression of the gene VAV3 via methylation.

    PubMed

    Peralta-Arrieta, Irlanda; Hernández-Sotelo, Daniel; Castro-Coronel, Yaneth; Leyva-Vázquez, Marco Antonio; Illades-Aguiar, Berenice

    2017-01-01

    Altered promoter DNA methylation is one of the most important epigenetic abnormalities in human cancer. DNMT3B, de novo methyltransferase, is clearly related to abnormal methylation of tumour suppressor genes, DNA repair genes and its overexpression contributes to oncogenic processes and tumorigenesis in vivo . The purpose of this study was to assess the effect of the overexpression of DNMT3B in HaCaT cells on global gene expression and on the methylation of selected genes to the identification of genes that can be target of DNMT3B. We found that the overexpression of DNMT3B in HaCaT cells, modulate the expression of genes related to cancer, downregulated the expression of 151 genes with CpG islands and downregulated the expression of the VAV3 gene via methylation of its promoter. These results highlight the importance of DNMT3B in gene expression and human cancer.

  16. Systemic bioinformatics analysis of skeletal muscle gene expression profiles of sepsis

    PubMed Central

    Yang, Fang; Wang, Yumei

    2018-01-01

    Sepsis is a type of systemic inflammatory response syndrome with high morbidity and mortality. Skeletal muscle dysfunction is one of the major complications of sepsis that may also influence the outcome of sepsis. The aim of the present study was to explore and identify potential mechanisms and therapeutic targets of sepsis. Systemic bioinformatics analysis of skeletal muscle gene expression profiles from the Gene Expression Omnibus was performed. Differentially expressed genes (DEGs) in samples from patients with sepsis and control samples were screened out using the limma package. Differential co-expression and coregulation (DCE and DCR, respectively) analysis was performed based on the Differential Co-expression Analysis package to identify differences in gene co-expression and coregulation patterns between the control and sepsis groups. Gene Ontology terms and Kyoto Encyclopedia of Genes and Genomes pathways of DEGs were identified using the Database for Annotation, Visualization and Integrated Discovery, and inflammatory, cancer and skeletal muscle development-associated biological processes and pathways were identified. DCE and DCR analysis revealed several potential therapeutic targets for sepsis, including genes and transcription factors. The results of the present study may provide a basis for the development of novel therapeutic targets and treatment methods for sepsis. PMID:29805480

  17. Genomic identification of direct target genes of LEAFY

    PubMed Central

    William, Dilusha A.; Su, Yanhui; Smith, Michael R.; Lu, Meina; Baldwin, Don A.; Wagner, Doris

    2004-01-01

    The switch from vegetative to reproductive development in plants necessitates a switch in the developmental program of the descendents of the stem cells in the shoot apical meristem. Genetic and molecular investigations have demonstrated that the plant-specific transcription factor and meristem identity regulator LEAFY (LFY) controls this developmental transition by inducing expression of a second transcription factor, APETALA1, and by regulating the expression of additional, as yet unknown, genes. Here we show that the additional LFY targets include the APETALA1-related factor, CAULI-FLOWER, as well as three transcription factors and two putative signal transduction pathway components. These genes are up-regulated by LFY even when protein synthesis is inhibited and, hence, appear to be direct targets of LFY. Supporting this conclusion, cis-regulatory regions upstream of these genes are bound by LFY in vivo. The newly identified LFY targets likely initiate the transcriptional changes that are required for the switch from vegetative to reproductive development in Arabidopsis. PMID:14736918

  18. Comprehensive evaluation of AmpliSeq transcriptome, a novel targeted whole transcriptome RNA sequencing methodology for global gene expression analysis.

    PubMed

    Li, Wenli; Turner, Amy; Aggarwal, Praful; Matter, Andrea; Storvick, Erin; Arnett, Donna K; Broeckel, Ulrich

    2015-12-16

    Whole transcriptome sequencing (RNA-seq) represents a powerful approach for whole transcriptome gene expression analysis. However, RNA-seq carries a few limitations, e.g., the requirement of a significant amount of input RNA and complications led by non-specific mapping of short reads. The Ion AmpliSeq Transcriptome Human Gene Expression Kit (AmpliSeq) was recently introduced by Life Technologies as a whole-transcriptome, targeted gene quantification kit to overcome these limitations of RNA-seq. To assess the performance of this new methodology, we performed a comprehensive comparison of AmpliSeq with RNA-seq using two well-established next-generation sequencing platforms (Illumina HiSeq and Ion Torrent Proton). We analyzed standard reference RNA samples and RNA samples obtained from human induced pluripotent stem cell derived cardiomyocytes (hiPSC-CMs). Using published data from two standard RNA reference samples, we observed a strong concordance of log2 fold change for all genes when comparing AmpliSeq to Illumina HiSeq (Pearson's r = 0.92) and Ion Torrent Proton (Pearson's r = 0.92). We used ROC, Matthew's correlation coefficient and RMSD to determine the overall performance characteristics. All three statistical methods demonstrate AmpliSeq as a highly accurate method for differential gene expression analysis. Additionally, for genes with high abundance, AmpliSeq outperforms the two RNA-seq methods. When analyzing four closely related hiPSC-CM lines, we show that both AmpliSeq and RNA-seq capture similar global gene expression patterns consistent with known sources of variations. Our study indicates that AmpliSeq excels in the limiting areas of RNA-seq for gene expression quantification analysis. Thus, AmpliSeq stands as a very sensitive and cost-effective approach for very large scale gene expression analysis and mRNA marker screening with high accuracy.

  19. Differentially Expressed Genes Associated with Low-Dose Gamma Radiation

    NASA Astrophysics Data System (ADS)

    Hegyesi, Hargita; Sándor, Nikolett; Schilling, Boglárka; Kis, Enikő; Lumniczky, Katalin; Sáfrány, Géza

    We have studied low dose radiation induced gene expression alterations in a primary human fibroblast cell line using Agilent's whole human genome microarray. Cells were irradiated with 60Co γ-rays (0; 0.1; 0.5 Gy) and 2 hours later total cellular RNA was isolated. We observed differential regulation of approximately 300-500 genes represented on the microarray. Of these, 126 were differentially expressed at both doses, among them significant elevation of GDF-15 and KITLG was confirmed by qRT-PCR. Based on the transcriptional studies we selected GDF-15 to assess its role in radiation response, since GDF-15 is one of the p53 gene targets and is believed to participate in mediating p53 activities. First we confirmed gamma-radiation induced dose-dependent changes in GDF-15 expression by qRT-PCR. Next we determined the effect of GDF-15 silencing on radiosensitivity. Four GDF-15 targeting shRNA expressing lentiviral vectors were transfected into immortalized human fibroblast cells. We obtained efficient GDF-15 silencing in one of the four constructs. RNA interference inhibited GDF-15 gene expression and enhanced the radiosensitivity of the cells. Our studies proved that GDF-15 plays an essential role in radiation response and may serve as a promising target in radiation therapy.

  20. Id-1 gene and gene products as therapeutic targets for treatment of breast cancer and other types of carcinoma

    DOEpatents

    Desprez, Pierre-Yves; Campisi, Judith

    2014-08-19

    A method for treatment of breast cancer and other types of cancer. The method comprises targeting and modulating Id-1 gene expression, if any, for the Id-1 gene, or gene products in breast or other epithelial cancers in a patient by delivering products that modulate Id-1 gene expression. When expressed, Id-1 gene is a prognostic indicator that cancer cells are invasive and metastatic.

  1. Combined serial analysis of gene expression and transcription factor binding site prediction identifies novel-candidate-target genes of Nr2e1 in neocortex development.

    PubMed

    Schmouth, Jean-François; Arenillas, David; Corso-Díaz, Ximena; Xie, Yuan-Yun; Bohacec, Slavita; Banks, Kathleen G; Bonaguro, Russell J; Wong, Siaw H; Jones, Steven J M; Marra, Marco A; Simpson, Elizabeth M; Wasserman, Wyeth W

    2015-07-24

    Nr2e1 (nuclear receptor subfamily 2, group e, member 1) encodes a transcription factor important in neocortex development. Previous work has shown that nuclear receptors can have hundreds of target genes, and bind more than 300 co-interacting proteins. However, recognition of the critical role of Nr2e1 in neural stem cells and neocortex development is relatively recent, thus the molecular mechanisms involved for this nuclear receptor are only beginning to be understood. Serial analysis of gene expression (SAGE), has given researchers both qualitative and quantitative information pertaining to biological processes. Thus, in this work, six LongSAGE mouse libraries were generated from laser microdissected tissue samples of dorsal VZ/SVZ (ventricular zone and subventricular zone) from the telencephalon of wild-type (Wt) and Nr2e1-null embryos at the critical development ages E13.5, E15.5, and E17.5. We then used a novel approach, implementing multiple computational methods followed by biological validation to further our understanding of Nr2e1 in neocortex development. In this work, we have generated a list of 1279 genes that are differentially expressed in response to altered Nr2e1 expression during in vivo neocortex development. We have refined this list to 64 candidate direct-targets of NR2E1. Our data suggested distinct roles for Nr2e1 during different neocortex developmental stages. Most importantly, our results suggest a possible novel pathway by which Nr2e1 regulates neurogenesis, which includes Lhx2 as one of the candidate direct-target genes, and SOX9 as a co-interactor. In conclusion, we have provided new candidate interacting partners and numerous well-developed testable hypotheses for understanding the pathways by which Nr2e1 functions to regulate neocortex development.

  2. Gene targeting in mosquito cells: a demonstration of 'knockout' technology in extrachromosomal gene arrays

    PubMed Central

    Eggleston, Paul; Zhao, Yuguang

    2001-01-01

    Background Gene targeting would offer a number of advantages over current transposon-based strategies for insect transformation. These include freedom from both position effects associated with quasi-random integration and concerns over transgene instability mediated by endogenous transposases, independence from phylogenetic restrictions on transposon mobility and the ability to generate gene knockouts. Results We describe here our initial investigations of gene targeting in the mosquito. The target site was a hygromycin resistance gene, stably maintained as part of an extrachromosomal array. Using a promoter-trap strategy to enrich for targeted events, a neomycin resistance gene was integrated into the target site. This resulted in knockout of hygromycin resistance concurrent with the expression of high levels of neomycin resistance from the resident promoter. PCR amplification of the targeted site generated a product that was specific to the targeted cell line and consistent with precise integration of the neomycin resistance gene into the 5' end of the hygromycin resistance gene. Sequencing of the PCR product and Southern analysis of cellular DNA subsequently confirmed this molecular structure. Conclusions These experiments provide the first demonstration of gene targeting in mosquito tissue and show that mosquito cells possess the necessary machinery to bring about precise integration of exogenous sequences through homologous recombination. Further development of these procedures and their extension to chromosomally located targets hold much promise for the exploitation of gene targeting in a wide range of medically and economically important insect species. PMID:11513755

  3. Identification of HMX1 target genes: A predictive promoter model approach

    PubMed Central

    Boulling, Arnaud; Wicht, Linda

    2013-01-01

    Purpose A homozygous mutation in the H6 family homeobox 1 (HMX1) gene is responsible for a new oculoauricular defect leading to eye and auricular developmental abnormalities as well as early retinal degeneration (MIM 612109). However, the HMX1 pathway remains poorly understood, and in the first approach to better understand the pathway’s function, we sought to identify the target genes. Methods We developed a predictive promoter model (PPM) approach using a comparative transcriptomic analysis in the retina at P15 of a mouse model lacking functional Hmx1 (dmbo mouse) and its respective wild-type. This PPM was based on the hypothesis that HMX1 binding site (HMX1-BS) clusters should be more represented in promoters of HMX1 target genes. The most differentially expressed genes in the microarray experiment that contained HMX1-BS clusters were used to generate the PPM, which was then statistically validated. Finally, we developed two genome-wide target prediction methods: one that focused on conserving PPM features in human and mouse and one that was based on the co-occurrence of HMX1-BS pairs fitting the PPM, in human or in mouse, independently. Results The PPM construction revealed that sarcoglycan, gamma (35kDa dystrophin-associated glycoprotein) (Sgcg), teashirt zinc finger homeobox 2 (Tshz2), and solute carrier family 6 (neurotransmitter transporter, glycine) (Slc6a9) genes represented Hmx1 targets in the mouse retina at P15. Moreover, the genome-wide target prediction revealed that mouse genes belonging to the retinal axon guidance pathway were targeted by Hmx1. Expression of these three genes was experimentally validated using a quantitative reverse transcription PCR approach. The inhibitory activity of Hmx1 on Sgcg, as well as protein tyrosine phosphatase, receptor type, O (Ptpro) and Sema3f, two targets identified by the PPM, were validated with luciferase assay. Conclusions Gene expression analysis between wild-type and dmbo mice allowed us to develop a PPM

  4. Conservation of transcription factor binding events predicts gene expression across species

    PubMed Central

    Hemberg, Martin; Kreiman, Gabriel

    2011-01-01

    Recent technological advances have made it possible to determine the genome-wide binding sites of transcription factors (TFs). Comparisons across species have suggested a relatively low degree of evolutionary conservation of experimentally defined TF binding events (TFBEs). Using binding data for six different TFs in hepatocytes and embryonic stem cells from human and mouse, we demonstrate that evolutionary conservation of TFBEs within orthologous proximal promoters is closely linked to function, defined as expression of the target genes. We show that (i) there is a significantly higher degree of conservation of TFBEs when the target gene is expressed in both species; (ii) there is increased conservation of binding events for groups of TFs compared to individual TFs; and (iii) conserved TFBEs have a greater impact on the expression of their target genes than non-conserved ones. These results link conservation of structural elements (TFBEs) to conservation of function (gene expression) and suggest a higher degree of functional conservation than implied by previous studies. PMID:21622661

  5. Identification of differentially expressed small RNAs and prediction of target genes in Italian Large White pigs with divergent backfat deposition.

    PubMed

    Davoli, R; Gaffo, E; Zappaterra, M; Bortoluzzi, S; Zambonelli, P

    2018-06-01

    The identification of the molecular mechanisms regulating pathways associated with the potential for fat deposition in pigs can lead to the detection of key genes and markers for the genetic improvement of fat traits. Interactions of microRNAs (miRNAs) with target RNAs regulate gene expression and modulate pathway activation in cells and tissues. In pigs, miRNA discovery is far from saturation, and the knowledge of miRNA expression in backfat tissue and particularly of the impact of miRNA variations is still fragmentary. Using RNA-seq, we characterized the small RNA (sRNA) expression profiles in Italian Large White pig backfat tissue. Comparing two groups of pigs divergent for backfat deposition, we detected 31 significant differentially expressed (DE) sRNAs: 14 up-regulated (including ssc-miR-132, ssc-miR-146b, ssc-miR-221-5p, ssc-miR-365-5p and the moRNA ssc-moR-21-5p) and 17 down-regulated (including ssc-miR-136, ssc-miR-195, ssc-miR-199a-5p and ssc-miR-335). To understand the biological impact of the observed miRNA expression variations, we used the expression correlation of DE miRNA target transcripts expressed in the same samples to define a regulatory network of 193 interactions between DE miRNAs and 40 DE target transcripts showing opposite expression profiles and being involved in specific pathways. Several miRNAs and mRNAs in the network were found to be expressed from backfat-related pig QTL. These results are informative for the complex mechanisms influencing fat traits, shed light on a new aspect of the genetic regulation of fat deposition in pigs and facilitate the prospective implementation of innovative strategies of pig genetic improvement based on genomic markers. © 2018 Stichting International Foundation for Animal Genetics.

  6. Gene expression promoted by the SV40 DNA targeting sequence and the hypoxia-responsive element under normoxia and hypoxia.

    PubMed

    Sacramento, C B; Moraes, J Z; Denapolis, P M A; Han, S W

    2010-08-01

    The main objective of the present study was to find suitable DNA-targeting sequences (DTS) for the construction of plasmid vectors to be used to treat ischemic diseases. The well-known Simian virus 40 nuclear DTS (SV40-DTS) and hypoxia-responsive element (HRE) sequences were used to construct plasmid vectors to express the human vascular endothelial growth factor gene (hVEGF). The rate of plasmid nuclear transport and consequent gene expression under normoxia (20% O2) and hypoxia (less than 5% O2) were determined. Plasmids containing the SV40-DTS or HRE sequences were constructed and used to transfect the A293T cell line (a human embryonic kidney cell line) in vitro and mouse skeletal muscle cells in vivo. Plasmid transport to the nucleus was monitored by real-time PCR, and the expression level of the hVEGF gene was measured by ELISA. The in vitro nuclear transport efficiency of the SV40-DTS plasmid was about 50% lower under hypoxia, while the HRE plasmid was about 50% higher under hypoxia. Quantitation of reporter gene expression in vitro and in vivo, under hypoxia and normoxia, confirmed that the SV40-DTS plasmid functioned better under normoxia, while the HRE plasmid was superior under hypoxia. These results indicate that the efficiency of gene expression by plasmids containing DNA binding sequences is affected by the concentration of oxygen in the medium.

  7. Promising Nanocarriers for PEDF Gene Targeting Delivery to Cervical Cancer Cells Mediated by the Over-expressing FRα.

    PubMed

    Yang, Yuhan; He, Lili; Liu, Yongmei; Xia, Shan; Fang, Aiping; Xie, Yafei; Gan, Li; He, Zhiyao; Tan, Xiaoyue; Jiang, Chunling; Tong, Aiping; Song, Xiangrong

    2016-08-31

    Cervical cancer presents extremely low PEDF expression which is associated with tumor progression and poor prognosis. In this study, folate receptor α (FRα)-targeted nano-liposomes (FLP) were designed to enhance the anti-tumor effect by targeting delivery of exogenous PEDF gene to cervical cancer cells. The targeting molecule F-PEG-Chol was firstly synthesized by a novel simpler method. FLP encapsulating PEDF gene (FLP/PEDF) with a typical lipid-membrane structure were prepared by a film dispersion method. The transfection experiment found FLP could effectively transfect human cervical cancer cells (HeLa cells). FLP/PEDF significantly inhibited the growth of HeLa cells and human umbilical vein endothelial cells (HUVEC cells) and suppressed adhension, invasion and migration of HeLa cells in vitro. In the abdominal metastatic tumor model of cervical cancer, FLP/PEDF administered by intraperitoneal injection exhibited a superior anti-tumor effect probably due to the up-regulated PEDF. FLP/PEDF could not only sharply reduce the microvessel density but also dramatically inhibit proliferation and markedly induce apoptosis of tumor cells in vivo. Moreover, the preliminary safety investigation revealed that FLP/PEDF had no obvious toxicity. These results clearly showed that FLP were desired carriers for PEDF gene and FLP/PEDF might represent a potential novel strategy for gene therapy of cervical cancer.

  8. Promising Nanocarriers for PEDF Gene Targeting Delivery to Cervical Cancer Cells Mediated by the Over-expressing FRα

    PubMed Central

    Yang, Yuhan; He, Lili; Liu, Yongmei; Xia, Shan; Fang, Aiping; Xie, Yafei; Gan, Li; He, Zhiyao; Tan, Xiaoyue; Jiang, Chunling; Tong, Aiping; Song, Xiangrong

    2016-01-01

    Cervical cancer presents extremely low PEDF expression which is associated with tumor progression and poor prognosis. In this study, folate receptor α (FRα)-targeted nano-liposomes (FLP) were designed to enhance the anti-tumor effect by targeting delivery of exogenous PEDF gene to cervical cancer cells. The targeting molecule F-PEG-Chol was firstly synthesized by a novel simpler method. FLP encapsulating PEDF gene (FLP/PEDF) with a typical lipid-membrane structure were prepared by a film dispersion method. The transfection experiment found FLP could effectively transfect human cervical cancer cells (HeLa cells). FLP/PEDF significantly inhibited the growth of HeLa cells and human umbilical vein endothelial cells (HUVEC cells) and suppressed adhension, invasion and migration of HeLa cells in vitro. In the abdominal metastatic tumor model of cervical cancer, FLP/PEDF administered by intraperitoneal injection exhibited a superior anti-tumor effect probably due to the up-regulated PEDF. FLP/PEDF could not only sharply reduce the microvessel density but also dramatically inhibit proliferation and markedly induce apoptosis of tumor cells in vivo. Moreover, the preliminary safety investigation revealed that FLP/PEDF had no obvious toxicity. These results clearly showed that FLP were desired carriers for PEDF gene and FLP/PEDF might represent a potential novel strategy for gene therapy of cervical cancer. PMID:27576898

  9. Multifaceted Targeting of the Chromatin Mediates Gonadotropin-Releasing Hormone Effects on Gene Expression in the Gonadotrope.

    PubMed

    Melamed, Philippa; Haj, Majd; Yosefzon, Yahav; Rudnizky, Sergei; Wijeweera, Andrea; Pnueli, Lilach; Kaplan, Ariel

    2018-01-01

    Gonadotropin-releasing hormone (GnRH) stimulates the expression of multiple genes in the pituitary gonadotropes, most notably to induce synthesis of the gonadotropins, luteinizing hormone (LH), and follicle-stimulating hormone (FSH), but also to ensure the appropriate functioning of these cells at the center of the mammalian reproductive endocrine axis. Aside from the activation of gene-specific transcription factors, GnRH stimulates through its membrane-bound receptor, alterations in the chromatin that facilitate transcription of its target genes. These include changes in the histone and DNA modifications, nucleosome positioning, and chromatin packaging at the regulatory regions of each gene. The requirements for each of these events vary according to the DNA sequence which determines the basal chromatin packaging at the regulatory regions. Despite considerable progress in this field in recent years, we are only beginning to understand some of the complexities involved in the role and regulation of this chromatin structure, including new modifications, extensive cross talk, histone variants, and the actions of distal enhancers and non-coding RNAs. This short review aims to integrate the latest findings on GnRH-induced alterations in the chromatin of its target genes, which indicate multiple and diverse actions. Understanding these processes is illuminating not only in the context of the activation of these hormones during the reproductive life span but may also reveal how aberrant epigenetic regulation of these genes leads to sub-fertility.

  10. Multifaceted Targeting of the Chromatin Mediates Gonadotropin-Releasing Hormone Effects on Gene Expression in the Gonadotrope

    PubMed Central

    Melamed, Philippa; Haj, Majd; Yosefzon, Yahav; Rudnizky, Sergei; Wijeweera, Andrea; Pnueli, Lilach; Kaplan, Ariel

    2018-01-01

    Gonadotropin-releasing hormone (GnRH) stimulates the expression of multiple genes in the pituitary gonadotropes, most notably to induce synthesis of the gonadotropins, luteinizing hormone (LH), and follicle-stimulating hormone (FSH), but also to ensure the appropriate functioning of these cells at the center of the mammalian reproductive endocrine axis. Aside from the activation of gene-specific transcription factors, GnRH stimulates through its membrane-bound receptor, alterations in the chromatin that facilitate transcription of its target genes. These include changes in the histone and DNA modifications, nucleosome positioning, and chromatin packaging at the regulatory regions of each gene. The requirements for each of these events vary according to the DNA sequence which determines the basal chromatin packaging at the regulatory regions. Despite considerable progress in this field in recent years, we are only beginning to understand some of the complexities involved in the role and regulation of this chromatin structure, including new modifications, extensive cross talk, histone variants, and the actions of distal enhancers and non-coding RNAs. This short review aims to integrate the latest findings on GnRH-induced alterations in the chromatin of its target genes, which indicate multiple and diverse actions. Understanding these processes is illuminating not only in the context of the activation of these hormones during the reproductive life span but may also reveal how aberrant epigenetic regulation of these genes leads to sub-fertility. PMID:29535683

  11. [Regulation of the β-globin gene family expression, useful in the search for new therapeutic targets for hemoglobinopathies].

    PubMed

    Scheps, Karen G; Varela, Viviana

    Different hemoglobin isoforms are expressed during the embryonic, fetal and postnatal stages. They are formed by combination of polypeptide chains synthesized from the α- and β-globin gene clusters. Based on the fact that the presence of high hemoglobin F levels is beneficial in both sickle cell disease and severe thalassemic syndromes, a revision of the regulation of the β-globin cluster expression is proposed, especially regarding the genes encoding the y-globin chains (HBG1 and HBG2). In this review we describe the current knowledge about transcription factors and epigenetic regulators involved in the switches of the β-globin cluster. It is expected that the consolidation of knowledge in this field will allow finding new therapeutic targets for the treatment of hemoglobinopathies.

  12. Identification of downstream metastasis-associated target genes regulated by LSD1 in colon cancer cells.

    PubMed

    Chen, Jiang; Ding, Jie; Wang, Ziwei; Zhu, Jian; Wang, Xuejian; Du, Jiyi

    2017-03-21

    This study aims to identify downstream target genes regulated by lysine-specific demethylase 1 (LSD1) in colon cancer cells and investigate the molecular mechanisms of LSD1 influencing invasion and metastasis of colon cancer. We obtained the expression changes of downstream target genes regulated by small-interfering RNA-LSD1 and LSD1-overexpression via gene expression profiling in two human colon cancer cell lines. An Affymetrix Human Transcriptome Array 2.0 was used to identify differentially expressed genes (DEGs). We screened out LSD1-target gene associated with proliferation, metastasis, and invasion from DEGs via Gene Ontology and Pathway Studio. Subsequently, four key genes (CABYR, FOXF2, TLE4, and CDH1) were computationally predicted as metastasis-related LSD1-target genes. ChIp-PCR was applied after RT-PCR and Western blot validations to detect the occupancy of LSD1-target gene promoter-bound LSD1. A total of 3633 DEGs were significantly upregulated, and 4642 DEGs were downregulated in LSD1-silenced SW620 cells. A total of 4047 DEGs and 4240 DEGs were upregulated and downregulated in LSD1-overexpressed HT-29 cells, respectively. RT-PCR and Western blot validated the microarray analysis results. ChIP assay results demonstrated that LSD1 might be negative regulators for target genes CABYR and CDH1. The expression level of LSD1 is negatively correlated with mono- and dimethylation of histone H3 lysine4(H3K4) at LSD1- target gene promoter region. No significant mono-methylation and dimethylation of H3 lysine9 methylation was detected at the promoter region of CABYR and CDH1. LSD1- depletion contributed to the upregulation of CABYR and CDH1 through enhancing the dimethylation of H3K4 at the LSD1-target genes promoter. LSD1- overexpression mediated the downregulation of CABYR and CDH1expression through decreasing the mono- and dimethylation of H3K4 at LSD1-target gene promoter in colon cancer cells. CABYR and CDH1 might be potential LSD1-target genes in colon

  13. Msn2 Coordinates a Stoichiometric Gene Expression Program

    PubMed Central

    Stewart-Ornstein, Jacob; Nelson, Christopher; DeRisi, Joe; Weissman, Jonathan S.; El-Samad, Hana

    2014-01-01

    Summary Background Many cellular processes operate in an “analog” regime in which the magnitude of the response is precisely tailored to the intensity of the stimulus. In order to maintain the coherence of such responses, the cell must provide for proportional expression of multiple target genes across a wide dynamic range of induction states. Our understanding of the strategies used to achieve graded gene regulation is limited. Results In this work, we document a relationship between stress responsive gene expression and the transcription factor Msn2 that is graded over a large range of Msn2 cocnentrations. We use computational modeling, in vivo, and in vitro analysis to dissect the roots of this relationship. Our studies reveal a simple and general strategy based on non-cooperative low-affinity interactions between Msn2 and its cognate binding sites, as well as competition over a large number of Msn2 binding sites in the genome relative to the number of Msn2 molecules. Conclusions In addition to enabling precise tuning of gene expression to the state of the environment, this strategy ensures co-linear activation of target genes, allowing for stoichiometric expression of large groups of genes without extensive promoter tuning. Furthermore, such a strategy enables precise modulation of the activity of any given promoter by addition of binding sites without altering the qualitative relationship between different genes in a regulon. This feature renders a given regulon highly ‘evolvable’. PMID:24210615

  14. Gene therapy for human ovarian cancer cells using efficient expression of Fas gene combined with γδT cells.

    PubMed

    Lin, Jiajing; Zeng, Dingyuan; He, Hongying; Tan, Guangping; Lan, Ying; Jiang, Fuyan; Sheng, Shuting

    2017-10-01

    Low tissue specificity and efficiency of exogenous gene expression are the two major obstacles in tumor‑targeted gene therapy. The Fas cell surface death receptor (Fas)/Fas ligand pathway is one of the primary pathways responsible for the regulation of cell apoptosis. The aim of the present study was to explore whether the regulation of tumor specific promoters and a two‑step transcriptional amplification system (TSTA) assured efficient, targeted expression of their downstream Fas gene in human ovarian cancer cells, and to assess the killing effect of γδT cells on these cells with high Fas expression. Three shuttle plasmids containing different control elements of the human telomerase reverse transcriptase (hTERT) promoter and/or TSTA were constructed and packaged into adenovirus 5 (Ad5) vectors for the expression of exogenous Fas gene. The human ovarian cancer cell line SKOV3 and a control human embryonic lung fibroblast cell line were transfected with Ad5‑hTERT‑Fas or Ad5‑hTERT‑TSTA‑Fas. Fas mRNA and protein expression were examined by reverse transcription‑quantitative polymerase chain reaction and western blot analysis. γδT lymphocytes were isolated, cultured and mixed at different ratios with SKOV3 cells with Fas expression in order to assess the killing effect of γδT cells. hTERT promoter induced the specific expression of FAS gene in SKOV3 cells, and the TSTA strategy increased FAS expression by 14.2‑fold. The killing effect of γδT cells increased with the expression level of Fas and the effector‑target cell ratio. The killing rate for SKOV3 cells with high FAS expression was 72.5% at an effector‑target cell ratio of 40:1. The regulators of hTERT promoter and TSTA assure the efficient and targeted expression of their downstream Fas gene in SKOV3 cells. The killing effect of γδT cells for ovarian cancer cells with relatively high Fas expression was improved.

  15. A resource for characterizing genome-wide binding and putative target genes of transcription factors expressed during secondary growth and wood formation in Populus.

    PubMed

    Liu, Lijun; Ramsay, Trevor; Zinkgraf, Matthew; Sundell, David; Street, Nathaniel Robert; Filkov, Vladimir; Groover, Andrew

    2015-06-01

    Identifying transcription factor target genes is essential for modeling the transcriptional networks underlying developmental processes. Here we report a chromatin immunoprecipitation sequencing (ChIP-seq) resource consisting of genome-wide binding regions and associated putative target genes for four Populus homeodomain transcription factors expressed during secondary growth and wood formation. Software code (programs and scripts) for processing the Populus ChIP-seq data are provided within a publically available iPlant image, including tools for ChIP-seq data quality control and evaluation adapted from the human Encyclopedia of DNA Elements (ENCODE) project. Basic information for each transcription factor (including members of Class I KNOX, Class III HD ZIP, BEL1-like families) binding are summarized, including the number and location of binding regions, distribution of binding regions relative to gene features, associated putative target genes, and enriched functional categories of putative target genes. These ChIP-seq data have been integrated within the Populus Genome Integrative Explorer (PopGenIE) where they can be analyzed using a variety of web-based tools. We present an example analysis that shows preferential binding of transcription factor ARBORKNOX1 to the nearest neighbor genes in a pre-calculated co-expression network module, and enrichment for meristem-related genes within this module including multiple orthologs of Arabidopsis KNOTTED-like Arabidopsis 2/6. © 2015 Society for Experimental Biology and John Wiley & Sons Ltd This article has been contributed to by US Government employees and their work is in the public domain in the USA.

  16. An Assessment of Database-Validated microRNA Target Genes in Normal Colonic Mucosa: Implications for Pathway Analysis.

    PubMed

    Slattery, Martha L; Herrick, Jennifer S; Stevens, John R; Wolff, Roger K; Mullany, Lila E

    2017-01-01

    Determination of functional pathways regulated by microRNAs (miRNAs), while an essential step in developing therapeutics, is challenging. Some miRNAs have been studied extensively; others have limited information. In this study, we focus on 254 miRNAs previously identified as being associated with colorectal cancer and their database-identified validated target genes. We use RNA-Seq data to evaluate messenger RNA (mRNA) expression for 157 subjects who also had miRNA expression data. In the replication phase of the study, we replicated associations between 254 miRNAs associated with colorectal cancer and mRNA expression of database-identified target genes in normal colonic mucosa. In the discovery phase of the study, we evaluated expression of 18 miR-NAs (those with 20 or fewer database-identified target genes along with miR-21-5p, miR-215-5p, and miR-124-3p which have more than 500 database-identified target genes) with expression of 17 434 mRNAs to identify new targets in colon tissue. Seed region matches between miRNA and newly identified targeted mRNA were used to help determine direct miRNA-mRNA associations. From the replication of the 121 miRNAs that had at least 1 database-identified target gene using mRNA expression methods, 97.9% were expressed in normal colonic mucosa. Of the 8622 target miRNA-mRNA associations identified in the database, 2658 (30.2%) were associated with gene expression in normal colonic mucosa after adjusting for multiple comparisons. Of the 133 miRNAs with database-identified target genes by non-mRNA expression methods, 97.2% were expressed in normal colonic mucosa. After adjustment for multiple comparisons, 2416 miRNA-mRNA associations remained significant (19.8%). Results from the discovery phase based on detailed examination of 18 miRNAs identified more than 80 000 miRNA-mRNA associations that had not previously linked to the miRNA. Of these miRNA-mRNA associations, 15.6% and 14.8% had seed matches for CRCh38 and CRCh37

  17. CRISPR/Cas9-mediated gene targeting in Arabidopsis using sequential transformation.

    PubMed

    Miki, Daisuke; Zhang, Wenxin; Zeng, Wenjie; Feng, Zhengyan; Zhu, Jian-Kang

    2018-05-17

    Homologous recombination-based gene targeting is a powerful tool for precise genome modification and has been widely used in organisms ranging from yeast to higher organisms such as Drosophila and mouse. However, gene targeting in higher plants, including the most widely used model plant Arabidopsis thaliana, remains challenging. Here we report a sequential transformation method for gene targeting in Arabidopsis. We find that parental lines expressing the bacterial endonuclease Cas9 from the egg cell- and early embryo-specific DD45 gene promoter can improve the frequency of single-guide RNA-targeted gene knock-ins and sequence replacements via homologous recombination at several endogenous sites in the Arabidopsis genome. These heritable gene targeting can be identified by regular PCR. Our approach enables routine and fine manipulation of the Arabidopsis genome.

  18. Gene expression patterns combined with bioinformatics analysis identify genes associated with cholangiocarcinoma.

    PubMed

    Li, Chen; Shen, Weixing; Shen, Sheng; Ai, Zhilong

    2013-12-01

    To explore the molecular mechanisms of cholangiocarcinoma (CC), microarray technology was used to find biomarkers for early detection and diagnosis. The gene expression profiles from 6 patients with CC and 5 normal controls were downloaded from Gene Expression Omnibus and compared. As a result, 204 differentially co-expressed genes (DCGs) in CC patients compared to normal controls were identified using a computational bioinformatics analysis. These genes were mainly involved in coenzyme metabolic process, peptidase activity and oxidation reduction. A regulatory network was constructed by mapping the DCGs to known regulation data. Four transcription factors, FOXC1, ZIC2, NKX2-2 and GCGR, were hub nodes in the network. In conclusion, this study provides a set of targets useful for future investigations into molecular biomarker studies. Copyright © 2013 Elsevier Ltd. All rights reserved.

  19. Applications of Gene Targeting Technology to Mental Retardation and Developmental Disability Research

    ERIC Educational Resources Information Center

    Pimenta, Aurea F.; Levitt, Pat

    2005-01-01

    The human and mouse genome projects elucidated the sequence and position map of innumerous genes expressed in the central nervous system (CNS), advancing our ability to manipulate these sequences and create models to investigate regulation of gene expression and function. In this article, we reviewed gene targeting methodologies with emphasis on…

  20. Identifying osteosarcoma metastasis associated genes by weighted gene co-expression network analysis (WGCNA).

    PubMed

    Tian, Honglai; Guan, Donghui; Li, Jianmin

    2018-06-01

    Osteosarcoma (OS), the most common malignant bone tumor, accounts for the heavy healthy threat in the period of children and adolescents. OS occurrence usually correlates with early metastasis and high death rate. This study aimed to better understand the mechanism of OS metastasis.Based on Gene Expression Omnibus (GEO) database, we downloaded 4 expression profile data sets associated with OS metastasis, and selected differential expressed genes. Weighted gene co-expression network analysis (WGCNA) approach allowed us to investigate the most OS metastasis-correlated module. Gene Ontology functional and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses were used to give annotation of selected OS metastasis-associated genes.We select 897 differential expressed genes from OS metastasis and OS non-metastasis groups. Based on these selected genes, WGCNA further explored 142 genes included in the most OS metastasis-correlated module. Gene Ontology functional and KEGG pathway enrichment analyses showed that significantly OS metastasis-associated genes were involved in pathway correlated with insulin-like growth factor binding.Our research figured out several potential molecules participating in metastasis process and factors acting as biomarker. With this study, we could better explore the mechanism of OS metastasis and further discover more therapy targets.

  1. Multiscale Embedded Gene Co-expression Network Analysis

    PubMed Central

    Song, Won-Min; Zhang, Bin

    2015-01-01

    Gene co-expression network analysis has been shown effective in identifying functional co-expressed gene modules associated with complex human diseases. However, existing techniques to construct co-expression networks require some critical prior information such as predefined number of clusters, numerical thresholds for defining co-expression/interaction, or do not naturally reproduce the hallmarks of complex systems such as the scale-free degree distribution of small-worldness. Previously, a graph filtering technique called Planar Maximally Filtered Graph (PMFG) has been applied to many real-world data sets such as financial stock prices and gene expression to extract meaningful and relevant interactions. However, PMFG is not suitable for large-scale genomic data due to several drawbacks, such as the high computation complexity O(|V|3), the presence of false-positives due to the maximal planarity constraint, and the inadequacy of the clustering framework. Here, we developed a new co-expression network analysis framework called Multiscale Embedded Gene Co-expression Network Analysis (MEGENA) by: i) introducing quality control of co-expression similarities, ii) parallelizing embedded network construction, and iii) developing a novel clustering technique to identify multi-scale clustering structures in Planar Filtered Networks (PFNs). We applied MEGENA to a series of simulated data and the gene expression data in breast carcinoma and lung adenocarcinoma from The Cancer Genome Atlas (TCGA). MEGENA showed improved performance over well-established clustering methods and co-expression network construction approaches. MEGENA revealed not only meaningful multi-scale organizations of co-expressed gene clusters but also novel targets in breast carcinoma and lung adenocarcinoma. PMID:26618778

  2. Multiscale Embedded Gene Co-expression Network Analysis.

    PubMed

    Song, Won-Min; Zhang, Bin

    2015-11-01

    Gene co-expression network analysis has been shown effective in identifying functional co-expressed gene modules associated with complex human diseases. However, existing techniques to construct co-expression networks require some critical prior information such as predefined number of clusters, numerical thresholds for defining co-expression/interaction, or do not naturally reproduce the hallmarks of complex systems such as the scale-free degree distribution of small-worldness. Previously, a graph filtering technique called Planar Maximally Filtered Graph (PMFG) has been applied to many real-world data sets such as financial stock prices and gene expression to extract meaningful and relevant interactions. However, PMFG is not suitable for large-scale genomic data due to several drawbacks, such as the high computation complexity O(|V|3), the presence of false-positives due to the maximal planarity constraint, and the inadequacy of the clustering framework. Here, we developed a new co-expression network analysis framework called Multiscale Embedded Gene Co-expression Network Analysis (MEGENA) by: i) introducing quality control of co-expression similarities, ii) parallelizing embedded network construction, and iii) developing a novel clustering technique to identify multi-scale clustering structures in Planar Filtered Networks (PFNs). We applied MEGENA to a series of simulated data and the gene expression data in breast carcinoma and lung adenocarcinoma from The Cancer Genome Atlas (TCGA). MEGENA showed improved performance over well-established clustering methods and co-expression network construction approaches. MEGENA revealed not only meaningful multi-scale organizations of co-expressed gene clusters but also novel targets in breast carcinoma and lung adenocarcinoma.

  3. The expression of Hedgehog genes (Ihh, Dhh) and Hedgehog target genes (Ptc1, Gli1, Coup-TfII) is affected by estrogenic stimuli in the uterus of immature female rats

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Katayama, Seiichi; Ashizawa, Koji; Gohma, Hiroshi

    2006-12-15

    The objective of this study was to investigate the effects of estrogen receptor (ER) agonists and an ER antagonist on the expression of Hedgehog genes (Indian hedgehog: Ihh; Desert hedgehog: Dhh) and Hedgehog target genes (Patched 1: Ptc1; glioma-associated oncogene homolog 1: Gli1; chicken ovalbumin upstream promoter transcription factor II: Coup-TfII) in the rat uterus. Immature female rats were administered once with 17{alpha}-ethynyl estradiol (EE, an ER agonist), propyl pyrazole triole (PPT, an ER{alpha}-selective agonist), diarylpropionitrile (DPN, an ER{beta}-selective agonist), or ICI 182,780 (an ER antagonist). Expression of mRNA for Ihh, Dhh, and Ptc1 was dose-dependently downregulated by EE inmore » the uterus of immature rats, mediated by ER as confirmed by coadministration of ICI 182,780. The mRNA expression levels of Ptc1, Gli1, and Coup-TfII were simultaneously downregulated during the period in which the mRNA expression levels of Ihh and Dhh were downregulated in the uterus after administration of EE. PPT downregulated the transcription of Ihh, Dhh, Ptc1, Gli1, and Coup-TfII, indicating that expression of these genes was regulated by the ER{alpha}-dependent pathway. DPN also downregulated the transcription of Ihh and Dhh, although the effect was weaker than that of PPT, indicating that the regulation of uterine Ihh and Dhh transcription was also affected by the ER{beta}-dependent pathway. These results suggest that the expression of Hedgehog genes (Ihh, Dhh) and Hedgehog target genes (Ptc1, Gli1, Coup-TfII) is affected by estrogenic stimuli in the uterus of immature female rats.« less

  4. Origins of extrinsic variability in eukaryotic gene expression

    NASA Astrophysics Data System (ADS)

    Volfson, Dmitri; Marciniak, Jennifer; Blake, William J.; Ostroff, Natalie; Tsimring, Lev S.; Hasty, Jeff

    2006-02-01

    Variable gene expression within a clonal population of cells has been implicated in a number of important processes including mutation and evolution, determination of cell fates and the development of genetic disease. Recent studies have demonstrated that a significant component of expression variability arises from extrinsic factors thought to influence multiple genes simultaneously, yet the biological origins of this extrinsic variability have received little attention. Here we combine computational modelling with fluorescence data generated from multiple promoter-gene inserts in Saccharomyces cerevisiae to identify two major sources of extrinsic variability. One unavoidable source arising from the coupling of gene expression with population dynamics leads to a ubiquitous lower limit for expression variability. A second source, which is modelled as originating from a common upstream transcription factor, exemplifies how regulatory networks can convert noise in upstream regulator expression into extrinsic noise at the output of a target gene. Our results highlight the importance of the interplay of gene regulatory networks with population heterogeneity for understanding the origins of cellular diversity.

  5. Origins of extrinsic variability in eukaryotic gene expression

    NASA Astrophysics Data System (ADS)

    Volfson, Dmitri; Marciniak, Jennifer; Blake, William J.; Ostroff, Natalie; Tsimring, Lev S.; Hasty, Jeff

    2006-03-01

    Variable gene expression within a clonal population of cells has been implicated in a number of important processes including mutation and evolution, determination of cell fates and the development of genetic disease. Recent studies have demonstrated that a significant component of expression variability arises from extrinsic factors thought to influence multiple genes in concert, yet the biological origins of this extrinsic variability have received little attention. Here we combine computational modeling with fluorescence data generated from multiple promoter-gene inserts in Saccharomyces cerevisiae to identify two major sources of extrinsic variability. One unavoidable source arising from the coupling of gene expression with population dynamics leads to a ubiquitous noise floor in expression variability. A second source which is modeled as originating from a common upstream transcription factor exemplifies how regulatory networks can convert noise in upstream regulator expression into extrinsic noise at the output of a target gene. Our results highlight the importance of the interplay of gene regulatory networks with population heterogeneity for understanding the origins of cellular diversity.

  6. STAT3 Target Genes Relevant to Human Cancers

    PubMed Central

    Carpenter, Richard L.; Lo, Hui-Wen

    2014-01-01

    Since its discovery, the STAT3 transcription factor has been extensively studied for its function as a transcriptional regulator and its role as a mediator of development, normal physiology, and pathology of many diseases, including cancers. These efforts have uncovered an array of genes that can be positively and negatively regulated by STAT3, alone and in cooperation with other transcription factors. Through regulating gene expression, STAT3 has been demonstrated to play a pivotal role in many cellular processes including oncogenesis, tumor growth and progression, and stemness. Interestingly, recent studies suggest that STAT3 may behave as a tumor suppressor by activating expression of genes known to inhibit tumorigenesis. Additional evidence suggested that STAT3 may elicit opposing effects depending on cellular context and tumor types. These mixed results signify the need for a deeper understanding of STAT3, including its upstream regulators, parallel transcription co-regulators, and downstream target genes. To help facilitate fulfilling this unmet need, this review will be primarily focused on STAT3 downstream target genes that have been validated to associate with tumorigenesis and/or malignant biology of human cancers. PMID:24743777

  7. Organ-specific gene expression: the bHLH protein Sage provides tissue specificity to Drosophila FoxA.

    PubMed

    Fox, Rebecca M; Vaishnavi, Aria; Maruyama, Rika; Andrew, Deborah J

    2013-05-01

    FoxA transcription factors play major roles in organ-specific gene expression, regulating, for example, glucagon expression in the pancreas, GLUT2 expression in the liver, and tyrosine hydroxylase expression in dopaminergic neurons. Organ-specific gene regulation by FoxA proteins is achieved through cooperative regulation with a broad array of transcription factors with more limited expression domains. Fork head (Fkh), the sole Drosophila FoxA family member, is required for the development of multiple distinct organs, yet little is known regarding how Fkh regulates tissue-specific gene expression. Here, we characterize Sage, a bHLH transcription factor expressed exclusively in the Drosophila salivary gland (SG). We show that Sage is required for late SG survival and normal tube morphology. We find that many Sage targets, identified by microarray analysis, encode SG-specific secreted cargo, transmembrane proteins, and the enzymes that modify these proteins. We show that both Sage and Fkh are required for the expression of Sage target genes, and that co-expression of Sage and Fkh is sufficient to drive target gene expression in multiple cell types. Sage and Fkh drive expression of the bZip transcription factor Senseless (Sens), which boosts expression of Sage-Fkh targets, and Sage, Fkh and Sens colocalize on SG chromosomes. Importantly, expression of Sage-Fkh target genes appears to simply add to the tissue-specific gene expression programs already established in other cell types, and Sage and Fkh cannot alter the fate of most embryonic cell types even when expressed early and continuously.

  8. Organ-specific gene expression: the bHLH protein Sage provides tissue specificity to Drosophila FoxA

    PubMed Central

    Fox, Rebecca M.; Vaishnavi, Aria; Maruyama, Rika; Andrew, Deborah J.

    2013-01-01

    FoxA transcription factors play major roles in organ-specific gene expression, regulating, for example, glucagon expression in the pancreas, GLUT2 expression in the liver, and tyrosine hydroxylase expression in dopaminergic neurons. Organ-specific gene regulation by FoxA proteins is achieved through cooperative regulation with a broad array of transcription factors with more limited expression domains. Fork head (Fkh), the sole Drosophila FoxA family member, is required for the development of multiple distinct organs, yet little is known regarding how Fkh regulates tissue-specific gene expression. Here, we characterize Sage, a bHLH transcription factor expressed exclusively in the Drosophila salivary gland (SG). We show that Sage is required for late SG survival and normal tube morphology. We find that many Sage targets, identified by microarray analysis, encode SG-specific secreted cargo, transmembrane proteins, and the enzymes that modify these proteins. We show that both Sage and Fkh are required for the expression of Sage target genes, and that co-expression of Sage and Fkh is sufficient to drive target gene expression in multiple cell types. Sage and Fkh drive expression of the bZip transcription factor Senseless (Sens), which boosts expression of Sage-Fkh targets, and Sage, Fkh and Sens colocalize on SG chromosomes. Importantly, expression of Sage-Fkh target genes appears to simply add to the tissue-specific gene expression programs already established in other cell types, and Sage and Fkh cannot alter the fate of most embryonic cell types even when expressed early and continuously. PMID:23578928

  9. Celastrol suppresses tumor cell growth through targeting an AR-ERG-NF-κB pathway in TMPRSS2/ERG fusion gene expressing prostate cancer.

    PubMed

    Shao, Longjiang; Zhou, Zhansong; Cai, Yi; Castro, Patricia; Dakhov, Olga; Shi, Ping; Bai, Yaoxia; Ji, Huixiang; Shen, Wenhao; Wang, Jianghua

    2013-01-01

    The TMPRSS2/ERG (T/E) fusion gene is present in the majority of all prostate cancers (PCa). We have shown previously that NF-kB signaling is highly activated in these T/E fusion expressing cells via phosphorylation of NF-kB p65 Ser536 (p536). We therefore hypothesize that targeting NF-kB signaling may be an efficacious approach for the subgroup of PCas that carry T/E fusions. Celastrol is a well known NF-kB inhibitor, and thus may inhibit T/E fusion expressing PCa cell growth. We therefore evaluated Celastrol's effects in vitro and in vivo in VCaP cells, which express the T/E fusion gene. VCaP cells were treated with different concentrations of Celastrol and growth inhibition and target expression were evaluated. To test its ability to inhibit growth in vivo, 0.5 mg/kg Celastrol was used to treat mice bearing subcutaneous VCaP xenograft tumors. Our results show Celastrol can significantly inhibit the growth of T/E fusion expressing PCa cells both in vitro and in vivo through targeting three critical signaling pathways: AR, ERG and NF-kB in these cells. When mice received 0.5 mg/kg Celastrol for 4 times/week, significant growth inhibition was seen with no obvious toxicity or significant weight loss. Therefore, Celastrol is a promising candidate drug for T/E fusion expressing PCa. Our findings provide a novel strategy for the targeted therapy which may benefit the more than half of PCa patients who have T/E fusion expressing PCas.

  10. Hypoxia-inducible tumour-specific promoters as a dual-targeting transcriptional regulation system for cancer gene therapy

    PubMed Central

    Javan, Bita; Shahbazi, Majid

    2017-01-01

    Transcriptional targeting is the best approach for specific gene therapy. Hypoxia is a common feature of the tumour microenvironment. Therefore, targeting gene expression in hypoxic cells by placing transgene under the control of a hypoxia-responsive promoter can be a good strategy for cancer-specific gene therapy. The hypoxia-inducible gene expression system has been investigated more in suicide gene therapy and it can also be of great help in knocking down cancer gene therapy with siRNAs. However, this system needs to be optimised to have maximum efficacy with minimum side effects in normal tissues. The combination of tissue-/tumour-specific promoters with HRE core sequences has been found to enhance the specificity and efficacy of this system. In this review, hypoxia-inducible gene expression system as well as gene therapy strategies targeting tumour hypoxia will be discussed. This review will also focus on hypoxia-inducible tumour-specific promoters as a dual-targeting transcriptional regulation systems developed for cancer-specific gene therapy. PMID:28798809

  11. Regulation of ecmF gene expression and genetic hierarchy among STATa, CudA, and MybC on several prestalk A-specific gene expressions in Dictyostelium.

    PubMed

    Saga, Yukika; Inamura, Tomoka; Shimada, Nao; Kawata, Takefumi

    2016-05-01

    STATa, a Dictyostelium homologue of metazoan signal transducer and activator of transcription, is important for the organizer function in the tip region of the migrating Dictyostelium slug. We previously showed that ecmF gene expression depends on STATa in prestalk A (pstA) cells, where STATa is activated. Deletion and site-directed mutagenesis analysis of the ecmF/lacZ fusion gene in wild-type and STATa null strains identified an imperfect inverted repeat sequence, ACAAATANTATTTGT, as a STATa-responsive element. An upstream sequence element was required for efficient expression in the rear region of pstA zone; an element downstream of the inverted repeat was necessary for sufficient prestalk expression during culmination. Band shift analyses using purified STATa protein detected no sequence-specific binding to those ecmF elements. The only verified upregulated target gene of STATa is cudA gene; CudA directly activates expL7 gene expression in prestalk cells. However, ecmF gene expression was almost unaffected in a cudA null mutant. Several previously reported putative STATa target genes were also expressed in cudA null mutant but were downregulated in STATa null mutant. Moreover, mybC, which encodes another transcription factor, belonged to this category, and ecmF expression was downregulated in a mybC null mutant. These findings demonstrate the existence of a genetic hierarchy for pstA-specific genes, which can be classified into two distinct STATa downstream pathways, CudA dependent and independent. The ecmF expression is indirectly upregulated by STATa in a CudA-independent activation manner but dependent on MybC, whose expression is positively regulated by STATa. © 2016 Japanese Society of Developmental Biologists.

  12. Differential co-expression analysis of a microarray gene expression profiles of pulmonary adenocarcinoma.

    PubMed

    Fu, Shijie; Pan, Xufeng; Fang, Wentao

    2014-08-01

    Lung cancer severely reduces the quality of life worldwide and causes high socioeconomic burdens. However, key genes leading to the generation of pulmonary adenocarcinoma remain elusive despite intensive research efforts. The present study aimed to identify the potential associations between transcription factors (TFs) and differentially co‑expressed genes (DCGs) in the regulation of transcription in pulmonary adenocarcinoma. Gene expression profiles of pulmonary adenocarcinoma were downloaded from the Gene Expression Omnibus, and gene expression was analyzed using a computational method. A total of 37,094 differentially co‑expressed links (DCLs) and 251 DCGs were identified, which were significantly enriched in 10 pathways. The construction of the regulatory network and the analysis of the regulatory impact factors revealed eight crucial TFs in the regulatory network. These TFs regulated the expression of DCGs by promoting or inhibiting their expression. In addition, certain TFs and target genes associated with DCGs did not appear in the DCLs, which indicated that those TFs could be synergistic with other factors. This is likely to provide novel insights for research into pulmonary adenocarcinoma. In conclusion, the present study may enhance the understanding of disease mechanisms and lead to an improved diagnosis of lung cancer. However, further studies are required to confirm these observations.

  13. Transgenic Cotton Plants Expressing Double-stranded RNAs Target HMG-CoA Reductase (HMGR) Gene Inhibits the Growth, Development and Survival of Cotton Bollworms

    PubMed Central

    Tian, Geng; Cheng, Linlin; Qi, Xuewei; Ge, Zonghe; Niu, Changying; Zhang, Xianlong; Jin, Shuangxia

    2015-01-01

    RNA interference (RNAi) has been developed as a powerful technique in the research of functional genomics as well as plant pest control. In this report, double-stranded RNAs (dsRNA) targeting 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR) gene, which catalyze a rate-limiting enzymatic reaction in the mevalonate pathway of juvenile hormone (JH) synthesis in cotton bollworm, was expressed in cotton plants via Agrobacterium tumefaciens-mediated transformation. PCR and Sothern analysis revealed the integration of HMGR gene into cotton genome. RT-PCR and qRT-PCR confirmed the high transcription level of dsHMGR in transgenic cotton lines. The HMGR expression both in transcription and translation level was significantly downregulated in cotton bollworms (helicoverpa armigera) larvae after feeding on the leaves of HMGR transgenic plants. The transcription level of HMGR gene in larvae reared on transgenic cotton leaves was as much as 80.68% lower than that of wild type. In addition, the relative expression level of vitellogenin (Vg, crucial source of nourishment for offspring embryo development) gene was also reduced by 76.86% when the insect larvae were fed with transgenic leaves. The result of insect bioassays showed that the transgenic plant harboring dsHMGR not only inhibited net weight gain but also delayed the growth of cotton bollworm larvae. Taken together, transgenic cotton plant expressing dsRNAs successfully downregulated HMGR gene and impaired the development and survival of target insect, which provided more option for plant pest control. PMID:26435695

  14. Ortho-aminoazotoluene activates mouse constitutive androstane receptor (mCAR) and increases expression of mCAR target genes

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Smetanina, Mariya A., E-mail: maria.smetanina@gmail.com; Laboratory of Gene Expression Control, Institute of Cytology and Genetics of the Siberian Branch of the Russian Academy of Sciences, prospekt Lavrentyeva 10, Novosibirsk 630090; Group of Pharmacogenomics, Institute of Chemical Biology and Fundamental Medicine of the Siberian Branch of the Russian Academy of Sciences, prospekt Lavrentyeva 8, Novosibirsk 630090

    2'-3-dimethyl-4-aminoazobenzene (ortho-aminoazotoluene, OAT) is an azo dye and a rodent carcinogen that has been evaluated by the International Agency for Research on Cancer (IARC) as a possible (class 2B) human carcinogen. Its mechanism of action remains unclear. We examined the role of the xenobiotic receptor Constitutive Androstane Receptor (CAR, NR1I3) as a mediator of the effects of OAT. We found that OAT increases mouse CAR (mCAR) transactivation in a dose-dependent manner. This effect is specific because another closely related azo dye, 3'-methyl-4-dimethyl-aminoazobenzene (3'MeDAB), did not activate mCAR. Real-time Q-PCR analysis in wild-type C57BL/6 mice revealed that OAT induces the hepaticmore » mRNA expression of the following CAR target genes: Cyp2b10, Cyp2c29, Cyp3a11, Ugt1a1, Mrp4, Mrp2 and c-Myc. CAR-null (Car{sup -/-}) mice showed no increased expression of these genes following OAT treatment, demonstrating that CAR is required for their OAT dependent induction. The OAT-induced CAR-dependent increase of Cyp2b10 and c-Myc expression was confirmed by Western blotting. Immunohistochemistry analysis of wild-type and Car{sup -/-} livers showed that OAT did not acutely induce hepatocyte proliferation, but at much later time points showed an unexpected CAR-dependent proliferative response. These studies demonstrate that mCAR is an OAT xenosensor, and indicate that at least some of the biological effects of this compound are mediated by this nuclear receptor. - Highlights: > The azo dye and mouse carcinogen OAT is a very effective mCAR activator. > OAT increases mCAR transactivation in a dose-dependent manner. > OAT CAR-dependently increases the expression of a specific subset of CAR target genes. > OAT induces an unexpectedly deferred, but CAR-dependent hepatocyte proliferation.« less

  15. Targeted adenovirus mediated inhibition of NF-κB-dependent inflammatory gene expression in endothelial cells in vitro and in vivo.

    PubMed

    Kułdo, J M; Ásgeirsdóttir, S A; Zwiers, P J; Bellu, A R; Rots, M G; Schalk, J A C; Ogawara, K I; Trautwein, C; Banas, B; Haisma, H J; Molema, G; Kamps, J A A M

    2013-02-28

    In chronic inflammatory diseases the endothelium expresses mediators responsible for harmful leukocyte infiltration. We investigated whether targeted delivery of a therapeutic transgene that inhibits nuclear factor κB signal transduction could silence the proinflammatory activation status of endothelial cells. For this, an adenovirus encoding dominant-negative IκB (dnIκB) as a therapeutic transgene was employed. Selectivity for the endothelial cells was achieved by introduction of antibodies specific for inflammatory endothelial adhesion molecules E-selectin or VCAM-1 chemically linked to the virus via polyethylene glycol. In vitro, the retargeted adenoviruses selectively infected cytokine-activated endothelial cells to express functional transgene. The comparison of transductional capacity of both retargeted viruses revealed that E-selectin based transgene delivery exerted superior pharmacological effects. Targeted delivery mediated dnIκB transgene expression in endothelial cells inhibited the induced expression of several inflammatory genes, including adhesion molecules, cytokines, and chemokines. In vivo, in mice suffering from glomerulonephritis, E-selectin-retargeted adenovirus selectively homed in the kidney to microvascular glomerular endothelium. Subsequent downregulation of endothelial adhesion molecule expression 2 days after induction of inflammation demonstrated the pharmacological potential of this gene therapy approach. The data justify further studies towards therapeutic virus design and optimization of treatment schedules to investigate their capacity to interfere with inflammatory disease progression. Copyright © 2012 Elsevier B.V. All rights reserved.

  16. C/EBPβ Mediates Growth Hormone-Regulated Expression of Multiple Target Genes

    PubMed Central

    Cui, Tracy X.; Lin, Grace; LaPensee, Christopher R.; Calinescu, Anda-Alexandra; Rathore, Maanjot; Streeter, Cale; Piwien-Pilipuk, Graciela; Lanning, Nathan; Jin, Hui; Carter-Su, Christin; Qin, Zhaohui S.

    2011-01-01

    Regulation of c-Fos transcription by GH is mediated by CCAAT/enhancer binding protein β (C/EBPβ). This study examines the role of C/EBPβ in mediating GH activation of other early response genes, including Cyr61, Btg2, Socs3, Zfp36, and Socs1. C/EBPβ depletion using short hairpin RNA impaired responsiveness of these genes to GH, as seen for c-Fos. Rescue with wild-type C/EBPβ led to GH-dependent recruitment of the coactivator p300 to the c-Fos promoter. In contrast, rescue with C/EBPβ mutated at the ERK phosphorylation site at T188 failed to induce GH-dependent recruitment of p300, indicating that ERK-mediated phosphorylation of C/EBPβ at T188 is required for GH-induced recruitment of p300 to c-Fos. GH also induced the occupancy of phosphorylated C/EBPβ and p300 on Cyr61, Btg2, and Socs3 at predicted C/EBP-cAMP response element-binding protein motifs in their promoters. Consistent with a role for ERKs in GH-induced expression of these genes, treatment with U0126 to block ERK phosphorylation inhibited their GH-induced expression. In contrast, GH-dependent expression of Zfp36 and Socs1 was not inhibited by U0126. Thus, induction of multiple early response genes by GH in 3T3-F442A cells is mediated by C/EBPβ. A subset of these genes is regulated similarly to c-Fos, through a mechanism involving GH-stimulated ERK 1/2 activation, phosphorylation of C/EBPβ, and recruitment of p300. Overall, these studies suggest that C/EBPβ, like the signal transducer and activator of transcription proteins, regulates multiple genes in response to GH. PMID:21292824

  17. Gene expression profiling of whole blood: Comparison of target preparation methods for accurate and reproducible microarray analysis

    PubMed Central

    Vartanian, Kristina; Slottke, Rachel; Johnstone, Timothy; Casale, Amanda; Planck, Stephen R; Choi, Dongseok; Smith, Justine R; Rosenbaum, James T; Harrington, Christina A

    2009-01-01

    Background Peripheral blood is an accessible and informative source of transcriptomal information for many human disease and pharmacogenomic studies. While there can be significant advantages to analyzing RNA isolated from whole blood, particularly in clinical studies, the preparation of samples for microarray analysis is complicated by the need to minimize artifacts associated with highly abundant globin RNA transcripts. The impact of globin RNA transcripts on expression profiling data can potentially be reduced by using RNA preparation and labeling methods that remove or block globin RNA during the microarray assay. We compared four different methods for preparing microarray hybridization targets from human whole blood collected in PAXGene tubes. Three of the methods utilized the Affymetrix one-cycle cDNA synthesis/in vitro transcription protocol but varied treatment of input RNA as follows: i. no treatment; ii. treatment with GLOBINclear; or iii. treatment with globin PNA oligos. In the fourth method cDNA targets were prepared with the Ovation amplification and labeling system. Results We find that microarray targets generated with labeling methods that reduce globin mRNA levels or minimize the impact of globin transcripts during hybridization detect more transcripts in the microarray assay compared with the standard Affymetrix method. Comparison of microarray results with quantitative PCR analysis of a panel of genes from the NF-kappa B pathway shows good correlation of transcript measurements produced with all four target preparation methods, although method-specific differences in overall correlation were observed. The impact of freezing blood collected in PAXGene tubes on data reproducibility was also examined. Expression profiles show little or no difference when RNA is extracted from either fresh or frozen blood samples. Conclusion RNA preparation and labeling methods designed to reduce the impact of globin mRNA transcripts can significantly improve the

  18. Histidine-rich stabilized polyplexes for cMet-directed tumor-targeted gene transfer

    NASA Astrophysics Data System (ADS)

    Kos, Petra; Lächelt, Ulrich; Herrmann, Annika; Mickler, Frauke Martina; Döblinger, Markus; He, Dongsheng; Krhač Levačić, Ana; Morys, Stephan; Bräuchle, Christoph; Wagner, Ernst

    2015-03-01

    Overexpression of the hepatocyte growth factor receptor/c-Met proto oncogene on the surface of a variety of tumor cells gives an opportunity to specifically target cancerous tissues. Herein, we report the first use of c-Met as receptor for non-viral tumor-targeted gene delivery. Sequence-defined oligomers comprising the c-Met binding peptide ligand cMBP2 for targeting, a monodisperse polyethylene glycol (PEG) for polyplex surface shielding, and various cationic (oligoethanamino) amide cores containing terminal cysteines for redox-sensitive polyplex stabilization, were assembled by solid-phase supported syntheses. The resulting oligomers exhibited a greatly enhanced cellular uptake and gene transfer over non-targeted control sequences, confirming the efficacy and target-specificity of the formed polyplexes. Implementation of endosomal escape-promoting histidines in the cationic core was required for gene expression without additional endosomolytic agent. The histidine-enriched polyplexes demonstrated stability in serum as well as receptor-specific gene transfer in vivo upon intratumoral injection. The co-formulation with an analogous PEG-free cationic oligomer led to a further compaction of pDNA polyplexes with an obvious change of shape as demonstrated by transmission electron microscopy. Such compaction was critically required for efficient intravenous gene delivery which resulted in greatly enhanced, cMBP2 ligand-dependent gene expression in the distant tumor.Overexpression of the hepatocyte growth factor receptor/c-Met proto oncogene on the surface of a variety of tumor cells gives an opportunity to specifically target cancerous tissues. Herein, we report the first use of c-Met as receptor for non-viral tumor-targeted gene delivery. Sequence-defined oligomers comprising the c-Met binding peptide ligand cMBP2 for targeting, a monodisperse polyethylene glycol (PEG) for polyplex surface shielding, and various cationic (oligoethanamino) amide cores containing

  19. Chemical Approaches to Control Gene Expression

    PubMed Central

    Gottesfeld, Joel M.; Turner, James M.; Dervan, Peter B.

    2000-01-01

    A current goal in molecular medicine is the development of new strategies to interfere with gene expression in living cells in the hope that novel therapies for human disease will result from these efforts. This review focuses on small-molecule or chemical approaches to manipulate gene expression by modulating either transcription of messenger RNA-coding genes or protein translation. The molecules under study include natural products, designed ligands, and compounds identified through functional screens of combinatorial libraries. The cellular targets for these molecules include DNA, messenger RNA, and the protein components of the transcription, RNA processing, and translational machinery. Studies with model systems have shown promise in the inhibition of both cellular and viral gene transcription and mRNA utilization. Moreover, strategies for both repression and activation of gene transcription have been described. These studies offer promise for treatment of diseases of pathogenic (viral, bacterial, etc.) and cellular origin (cancer, genetic diseases, etc.). PMID:11097426

  20. FARO server: Meta-analysis of gene expression by matching gene expression signatures to a compendium of public gene expression data.

    PubMed

    Manijak, Mieszko P; Nielsen, Henrik B

    2011-06-11

    Although, systematic analysis of gene annotation is a powerful tool for interpreting gene expression data, it sometimes is blurred by incomplete gene annotation, missing expression response of key genes and secondary gene expression responses. These shortcomings may be partially circumvented by instead matching gene expression signatures to signatures of other experiments. To facilitate this we present the Functional Association Response by Overlap (FARO) server, that match input signatures to a compendium of 242 gene expression signatures, extracted from more than 1700 Arabidopsis microarray experiments. Hereby we present a publicly available tool for robust characterization of Arabidopsis gene expression experiments which can point to similar experimental factors in other experiments. The server is available at http://www.cbs.dtu.dk/services/faro/.

  1. Haloperidol induces pharmacoepigenetic response by modulating miRNA expression, global DNA methylation and expression profiles of methylation maintenance genes and genes involved in neurotransmission in neuronal cells.

    PubMed

    Swathy, Babu; Banerjee, Moinak

    2017-01-01

    Haloperidol has been extensively used in various psychiatric conditions. It has also been reported to induce severe side effects. We aimed to evaluate whether haloperidol can influence host methylome, and if so what are the possible mechanisms for it in neuronal cells. Impact on host methylome and miRNAs can have wide spread alterations in gene expression, which might possibly help in understanding how haloperidol may impact treatment response or induce side effects. SK-N-SH, a neuroblasoma cell line was treated with haloperidol at 10μm concentration for 24 hours and global DNA methylation was evaluated. Methylation at global level is maintained by methylation maintenance machinery and certain miRNAs. Therefore, the expression of methylation maintenance genes and their putative miRNA expression profiles were assessed. These global methylation alterations could result in gene expression changes. Therefore genes expressions for neurotransmitter receptors, regulators, ion channels and transporters were determined. Subsequently, we were also keen to identify a strong candidate miRNA based on biological and in-silico approach which can reflect on the pharmacoepigenetic trait of haloperidol and can also target the altered neuroscience panel of genes used in the study. Haloperidol induced increase in global DNA methylation which was found to be associated with corresponding increase in expression of various epigenetic modifiers that include DNMT1, DNMT3A, DNMT3B and MBD2. The expression of miR-29b that is known to putatively regulate the global methylation by modulating the expression of epigenetic modifiers was observed to be down regulated by haloperidol. In addition to miR-29b, miR-22 was also found to be downregulated by haloperidol treatment. Both these miRNA are known to putatively target several genes associated with various epigenetic modifiers, pharmacogenes and neurotransmission. Interestingly some of these putative target genes involved in neurotransmission

  2. Haloperidol induces pharmacoepigenetic response by modulating miRNA expression, global DNA methylation and expression profiles of methylation maintenance genes and genes involved in neurotransmission in neuronal cells

    PubMed Central

    Swathy, Babu

    2017-01-01

    Introduction Haloperidol has been extensively used in various psychiatric conditions. It has also been reported to induce severe side effects. We aimed to evaluate whether haloperidol can influence host methylome, and if so what are the possible mechanisms for it in neuronal cells. Impact on host methylome and miRNAs can have wide spread alterations in gene expression, which might possibly help in understanding how haloperidol may impact treatment response or induce side effects. Methods SK-N-SH, a neuroblasoma cell line was treated with haloperidol at 10μm concentration for 24 hours and global DNA methylation was evaluated. Methylation at global level is maintained by methylation maintenance machinery and certain miRNAs. Therefore, the expression of methylation maintenance genes and their putative miRNA expression profiles were assessed. These global methylation alterations could result in gene expression changes. Therefore genes expressions for neurotransmitter receptors, regulators, ion channels and transporters were determined. Subsequently, we were also keen to identify a strong candidate miRNA based on biological and in-silico approach which can reflect on the pharmacoepigenetic trait of haloperidol and can also target the altered neuroscience panel of genes used in the study. Results Haloperidol induced increase in global DNA methylation which was found to be associated with corresponding increase in expression of various epigenetic modifiers that include DNMT1, DNMT3A, DNMT3B and MBD2. The expression of miR-29b that is known to putatively regulate the global methylation by modulating the expression of epigenetic modifiers was observed to be down regulated by haloperidol. In addition to miR-29b, miR-22 was also found to be downregulated by haloperidol treatment. Both these miRNA are known to putatively target several genes associated with various epigenetic modifiers, pharmacogenes and neurotransmission. Interestingly some of these putative target genes

  3. Genome-wide prediction and analysis of human tissue-selective genes using microarray expression data

    PubMed Central

    2013-01-01

    Background Understanding how genes are expressed specifically in particular tissues is a fundamental question in developmental biology. Many tissue-specific genes are involved in the pathogenesis of complex human diseases. However, experimental identification of tissue-specific genes is time consuming and difficult. The accurate predictions of tissue-specific gene targets could provide useful information for biomarker development and drug target identification. Results In this study, we have developed a machine learning approach for predicting the human tissue-specific genes using microarray expression data. The lists of known tissue-specific genes for different tissues were collected from UniProt database, and the expression data retrieved from the previously compiled dataset according to the lists were used for input vector encoding. Random Forests (RFs) and Support Vector Machines (SVMs) were used to construct accurate classifiers. The RF classifiers were found to outperform SVM models for tissue-specific gene prediction. The results suggest that the candidate genes for brain or liver specific expression can provide valuable information for further experimental studies. Our approach was also applied for identifying tissue-selective gene targets for different types of tissues. Conclusions A machine learning approach has been developed for accurately identifying the candidate genes for tissue specific/selective expression. The approach provides an efficient way to select some interesting genes for developing new biomedical markers and improve our knowledge of tissue-specific expression. PMID:23369200

  4. Divergent and nonuniform gene expression patterns in mouse brain

    PubMed Central

    Morris, John A.; Royall, Joshua J.; Bertagnolli, Darren; Boe, Andrew F.; Burnell, Josh J.; Byrnes, Emi J.; Copeland, Cathy; Desta, Tsega; Fischer, Shanna R.; Goldy, Jeff; Glattfelder, Katie J.; Kidney, Jolene M.; Lemon, Tracy; Orta, Geralyn J.; Parry, Sheana E.; Pathak, Sayan D.; Pearson, Owen C.; Reding, Melissa; Shapouri, Sheila; Smith, Kimberly A.; Soden, Chad; Solan, Beth M.; Weller, John; Takahashi, Joseph S.; Overly, Caroline C.; Lein, Ed S.; Hawrylycz, Michael J.; Hohmann, John G.; Jones, Allan R.

    2010-01-01

    Considerable progress has been made in understanding variations in gene sequence and expression level associated with phenotype, yet how genetic diversity translates into complex phenotypic differences remains poorly understood. Here, we examine the relationship between genetic background and spatial patterns of gene expression across seven strains of mice, providing the most extensive cellular-resolution comparative analysis of gene expression in the mammalian brain to date. Using comprehensive brainwide anatomic coverage (more than 200 brain regions), we applied in situ hybridization to analyze the spatial expression patterns of 49 genes encoding well-known pharmaceutical drug targets. Remarkably, over 50% of the genes examined showed interstrain expression variation. In addition, the variability was nonuniformly distributed across strain and neuroanatomic region, suggesting certain organizing principles. First, the degree of expression variance among strains mirrors genealogic relationships. Second, expression pattern differences were concentrated in higher-order brain regions such as the cortex and hippocampus. Divergence in gene expression patterns across the brain could contribute significantly to variations in behavior and responses to neuroactive drugs in laboratory mouse strains and may help to explain individual differences in human responsiveness to neuroactive drugs. PMID:20956311

  5. Evaluation of Reference Genes for Normalization of Gene Expression Using Quantitative RT-PCR under Aluminum, Cadmium, and Heat Stresses in Soybean.

    PubMed

    Gao, Mengmeng; Liu, Yaping; Ma, Xiao; Shuai, Qin; Gai, Junyi; Li, Yan

    2017-01-01

    Quantitative reverse transcription polymerase chain reaction (qRT-PCR) is widely used to analyze the relative gene expression level, however, the accuracy of qRT-PCR is greatly affected by the stability of reference genes, which is tissue- and environment- dependent. Therefore, choosing the most stable reference gene in a specific tissue and environment is critical to interpret gene expression patterns. Aluminum (Al), cadmium (Cd), and heat stresses are three important abiotic factors limiting soybean (Glycine max) production in southern China. To identify the suitable reference genes for normalizing the expression levels of target genes by qRT-PCR in soybean response to Al, Cd and heat stresses, we studied the expression stability of ten commonly used housekeeping genes in soybean roots and leaves under these three abiotic stresses, using five approaches, BestKeeper, Delta Ct, geNorm, NormFinder and RefFinder. We found TUA4 is the most stable reference gene in soybean root tips under Al stress. Under Cd stress, Fbox and UKN2 are the most stable reference genes in roots and leaves, respectively, while 60S is the most suitable reference gene when analyzing both roots and leaves together. For heat stress, TUA4 and UKN2 are the most stable housekeeping genes in roots and leaves, respectively, and UKN2 is the best reference gene for analysis of roots and leaves together. To validate the reference genes, we quantified the relative expression levels of six target genes that were involved in soybean response to Al, Cd or heat stresses, respectively. The expression patterns of these target genes differed between using the most and least stable reference genes, suggesting the selection of a suitable reference gene is critical for gene expression studies.

  6. Inheritable Silencing of Endogenous Genes by Hit-and-Run Targeted Epigenetic Editing.

    PubMed

    Amabile, Angelo; Migliara, Alessandro; Capasso, Paola; Biffi, Mauro; Cittaro, Davide; Naldini, Luigi; Lombardo, Angelo

    2016-09-22

    Gene silencing is instrumental to interrogate gene function and holds promise for therapeutic applications. Here, we repurpose the endogenous retroviruses' silencing machinery of embryonic stem cells to stably silence three highly expressed genes in somatic cells by epigenetics. This was achieved by transiently expressing combinations of engineered transcriptional repressors that bind to and synergize at the target locus to instruct repressive histone marks and de novo DNA methylation, thus ensuring long-term memory of the repressive epigenetic state. Silencing was highly specific, as shown by genome-wide analyses, sharply confined to the targeted locus without spreading to nearby genes, resistant to activation induced by cytokine stimulation, and relieved only by targeted DNA demethylation. We demonstrate the portability of this technology by multiplex gene silencing, adopting different DNA binding platforms and interrogating thousands of genomic loci in different cell types, including primary T lymphocytes. Targeted epigenome editing might have broad application in research and medicine. Copyright © 2016 The Author(s). Published by Elsevier Inc. All rights reserved.

  7. Gene Expression Profile Analysis is Directly Affected by the Selected Reference Gene: The Case of Leaf-Cutting Atta Sexdens

    PubMed Central

    Máximo, Wesley P. F.; Zanetti, Ronald; Paiva, Luciano V.

    2018-01-01

    Although several ant species are important targets for the development of molecular control strategies, only a few studies focus on identifying and validating reference genes for quantitative reverse transcription polymerase chain reaction (RT-qPCR) data normalization. We provide here an extensive study to identify and validate suitable reference genes for gene expression analysis in the ant Atta sexdens, a threatening agricultural pest in South America. The optimal number of reference genes varies according to each sample and the result generated by RefFinder differed about which is the most suitable reference gene. Results suggest that the RPS16, NADH and SDHB genes were the best reference genes in the sample pool according to stability values. The SNF7 gene expression pattern was stable in all evaluated sample set. In contrast, when using less stable reference genes for normalization a large variability in SNF7 gene expression was recorded. There is no universal reference gene suitable for all conditions under analysis, since these genes can also participate in different cellular functions, thus requiring a systematic validation of possible reference genes for each specific condition. The choice of reference genes on SNF7 gene normalization confirmed that unstable reference genes might drastically change the expression profile analysis of target candidate genes. PMID:29419794

  8. AtSIG6 and other members of the sigma gene family jointly but differentially determine plastid target gene expression in Arabidopsis thaliana

    PubMed Central

    Bock, Sylvia; Ortelt, Jennifer; Link, Gerhard

    2014-01-01

    Plants contain a nuclear gene family for plastid sigma factors, i.e., proteins that associate with the “bacterial-type” organellar RNA polymerase and confer the ability for correct promoter binding and transcription initiation. Questions that are still unresolved relate to the “division of labor” among members of the sigma family, both in terms of their range of target genes and their temporal and spatial activity during development. Clues to the in vivo role of individual sigma genes have mainly come from studies of sigma knockout lines. Despite its obvious strengths, however, this strategy does not necessarily trace-down causal relationships between mutant phenotype and a single sigma gene, if other family members act in a redundant and/or compensatory manner. We made efforts to reduce the complexity by genetic crosses of Arabidopsis single mutants (with focus on a chlorophyll-deficient sig6 line) to generate double knockout lines. The latter typically had a similar visible phenotype as the parental lines, but tended to be more strongly affected in the transcript patterns of both plastid and sigma genes. Because triple mutants were lethal under our growth conditions, we exploited a strategy of transformation of single and double mutants with RNAi constructs that contained sequences from the unconserved sigma region (UCR). These RNAi/knockout lines phenotypically resembled their parental lines, but were even more strongly affected in their plastid transcript patterns. Expression patterns of sigma genes revealed both similarities and differences compared to the parental lines, with transcripts at reduced or unchanged amounts and others that were found to be present in higher (perhaps compensatory) amounts. Together, our results reveal considerable flexibility of gene activity at the levels of both sigma and plastid gene expression. A (still viable) “basal state” seems to be reached, if 2–3 of the 6 Arabidopsis sigma genes are functionally compromised

  9. Aging and calorie restriction regulate the expression of miR-125a-5p and its target genes Stat3, Casp2 and Stard13.

    PubMed

    Makwana, Kuldeep; Patel, Sonal Arvind; Velingkaar, Nikkhil; Ebron, Jey Sabith; Shukla, Girish C; Kondratov, Roman V Kondratov V

    2017-07-31

    Calorie restriction (CR) is a dietary intervention known to delay aging. In order, to understand molecular mechanisms of CR, we analyzed the expression of 983 MicroRNAs (miRNAs) in the liver of female mice after 2 years of 30% CR using micro-array. 16 miRNAs demonstrated significant changes in their expression upon CR in comparison with age-matched control. mmu-miR-125a-5p (miR-125a-5p) was significantly upregulated upon CR, and in agreement with this, the expression of mRNAs for its three predicted target genes: Stat3, Casp2, and Stard13 was significantly downregulated in the liver of CR animals. The expression of precursor miRNA for miR-125a-5p was also upregulated upon CR, which suggests its regulation at the level of transcription. Upon aging miR-125a-5p expression was downregulated while the expression of its target genes was upregulated. Thus, CR prevented age-associated changes in the expression of miR-125a-5p and its targets. We propose that miR-125a-5p dependent downregulation of Stat3, Casp2, and Stard13 contributes to the calorie restriction-mediated delay of aging.

  10. Targeting factor VIII expression to platelets for hemophilia A gene therapy does not induce an apparent thrombotic risk in mice.

    PubMed

    Baumgartner, C K; Mattson, J G; Weiler, H; Shi, Q; Montgomery, R R

    2017-01-01

    Essentials Platelet-Factor (F) VIII gene therapy is a promising treatment in hemophilia A. This study aims to evaluate if platelet-FVIII expression would increase the risk for thrombosis. Targeting FVIII expression to platelets does not induce or elevate thrombosis risk. Platelets expressing FVIII are neither hyper-activated nor hyper-responsive. Background Targeting factor (F) VIII expression to platelets is a promising gene therapy approach for hemophilia A, and is successful even in the presence of inhibitors. It is well known that platelets play important roles not only in hemostasis, but also in thrombosis and inflammation. Objective To evaluate whether platelet-FVIII expression might increase thrombotic risk and thereby compromise the safety of this approach. Methods In this study, platelet-FVIII-expressing transgenic mice were examined either in steady-state conditions or under prothrombotic conditions induced by inflammation or the FV Leiden mutation. Native whole blood thrombin generation assay, rotational thromboelastometry analysis and ferric chloride-induced vessel injury were used to evaluate the hemostatic properties. Various parameters associated with thrombosis risk, including D-dimer, thrombin-antithrombin complexes, fibrinogen, tissue fibrin deposition, platelet activation status and activatability, and platelet-leukocyte aggregates, were assessed. Results We generated a new line of transgenic mice that expressed 30-fold higher levels of platelet-expressed FVIII than are therapeutically required to restore hemostasis in hemophilic mice. Under both steady-state conditions and prothrombotic conditions induced by lipopolysaccharide-mediated inflammation or the FV Leiden mutation, supratherapeutic levels of platelet-expressed FVIII did not appear to be thrombogenic. Furthermore, FVIII-expressing platelets were neither hyperactivated nor hyperactivatable upon agonist activation. Conclusion We conclude that, in mice, more than 30-fold higher levels of

  11. Deficiency in Aryl Hydrocarbon Receptor (AHR) Expression throughout Aging Alters Gene Expression Profiles in Murine Long-Term Hematopoietic Stem Cells

    PubMed Central

    Bennett, John A.; Singh, Kameshwar P.; Unnisa, Zeenath; Welle, Stephen L.; Gasiewicz, Thomas A.

    2015-01-01

    Dysregulation of hematopoietic stem cell (HSC) signaling can contribute to the development of diseases of the blood system. Lack of aryl hydrocarbon receptor (AhR) has been associated with alterations in gene expression related to HSC function and the subsequent development of a myeloproliferative disorder in aging female mice. We sorted the most primitive population of HSCs with the highest stem cell potential (Long-term, or LT-HSCs) from 18-month-old AhR-null-allele (AhR-KO) and WT mice and analyzed gene expression using microarray to determine alterations in gene expression and cell signaling networks in HSCs that could potentially contribute to the aging phenotype of AhR-KO mice. Comparisons with previous array data from 8-week old mice indicated that aging alone is sufficient to alter gene expression. In addition, a significant number of gene expression differences were observed in aged LT-HSCs that are dependent on both aging and lack of AhR. Pathway analysis of these genes revealed networks related to hematopoietic stem cell activity or function. qPCR was used to confirm the differential expression of a subset of these genes, focusing on genes that may represent novel AhR targets due to the presence of a putative AhR binding site in their upstream regulatory region. We verified differential expression of PDGF-D, Smo, Wdfy1, Zbtb37 and Zfp382. Pathway analysis of this subset of genes revealed overlap between cellular functions of the novel AhR targets and AhR itself. Lentiviral-mediated knockdown of AhR in lineage-negative hematopoietic cells was sufficient to induce changes in all five of the candidate AhR targets identified. Taken together, these data suggest a role for AhR in HSC functional regulation, and identify novel HSC AhR target genes that may contribute to the phenotypes observed in AhR-KO mice. PMID:26208102

  12. Highly specific targeting of the TMPRSS2/ERG fusion gene using liposomal nanovectors

    PubMed Central

    Shao, Longjiang; Tekedereli, Ibrahim; Wang, Jianghua; Yuca, Erkan; Tsang, Susan; Sood, Anil; Lopez-Berestein, Gabriel; Ozpolat, Bulent; Ittmann, Michael

    2012-01-01

    Purpose The TMPRSS2/ERG (T/E) fusion gene is present in half of all prostate cancer (PCa) tumors. Fusion of the oncogenic ERG gene with the androgen-regulated TMPRSS2 gene promoter results in expression of fusion mRNAs in PCa cells. The junction of theTMPRSS2 and ERG derived portions of the fusion mRNA constitutes a cancer specific target in cells containing the T/E fusion gene. Targeting the most common alternatively spliced fusion gene mRNA junctional isoforms in vivo using siRNAs in liposomal nanovectors may potentially be a novel, low toxicity treatment for PCa. Experimental Design We designed and optimized siRNAs targeting the two most common T/E fusion gene mRNA junctional isoforms (Type III or Type VI). Specificity of siRNAs was assessed by transient co-transfection in vitro. To test their ability to inhibit growth of PCa cells expressing these fusion gene isoforms in vivo, specific siRNAs in liposomal nanovectors were used to treat mice bearing orthotopic or subcutaneous xenograft tumors expressing the targeted fusion isoforms. Results The targeting siRNAs were both potent and highly specific in vitro. In vivo they significantly inhibited tumor growth. The degree of growth inhibition was variable and was correlated with the extent of fusion gene knockdown. The growth inhibition was associated with marked inhibition of angiogenesis and, to a lesser degree, proliferation and a marked increase in apoptosis of tumor cells. No toxicity was observed. Conclusions Targeting the T/E fusion junction in vivo with specific siRNAs delivered via liposomal nanovectors is a promising therapy for men with PCa. PMID:23052253

  13. Highly specific targeting of the TMPRSS2/ERG fusion gene using liposomal nanovectors.

    PubMed

    Shao, Longjiang; Tekedereli, Ibrahim; Wang, Jianghua; Yuca, Erkan; Tsang, Susan; Sood, Anil; Lopez-Berestein, Gabriel; Ozpolat, Bulent; Ittmann, Michael

    2012-12-15

    The TMPRSS2/ERG (T/E) fusion gene is present in half of all prostate cancer tumors. Fusion of the oncogenic ERG gene with the androgen-regulated TMPRSS2 gene promoter results in expression of fusion mRNAs in prostate cancer cells. The junction of theTMPRSS2- and ERG-derived portions of the fusion mRNA constitutes a cancer-specific target in cells containing the T/E fusion gene. Targeting the most common alternatively spliced fusion gene mRNA junctional isoforms in vivo using siRNAs in liposomal nanovectors may potentially be a novel, low-toxicity treatment for prostate cancer. We designed and optimized siRNAs targeting the two most common T/E fusion gene mRNA junctional isoforms (type III or type VI). Specificity of siRNAs was assessed by transient co-transfection in vitro. To test their ability to inhibit growth of prostate cancer cells expressing these fusion gene isoforms in vivo, specific siRNAs in liposomal nanovectors were used to treat mice bearing orthotopic or subcutaneous xenograft tumors expressing the targeted fusion isoforms. The targeting siRNAs were both potent and highly specific in vitro. In vivo they significantly inhibited tumor growth. The degree of growth inhibition was variable and was correlated with the extent of fusion gene knockdown. The growth inhibition was associated with marked inhibition of angiogenesis and, to a lesser degree, proliferation and a marked increase in apoptosis of tumor cells. No toxicity was observed. Targeting the T/E fusion junction in vivo with specific siRNAs delivered via liposomal nanovectors is a promising therapy for men with prostate cancer. ©2012 AACR.

  14. Neighboring Genes Show Correlated Evolution in Gene Expression

    PubMed Central

    Ghanbarian, Avazeh T.; Hurst, Laurence D.

    2015-01-01

    When considering the evolution of a gene’s expression profile, we commonly assume that this is unaffected by its genomic neighborhood. This is, however, in contrast to what we know about the lack of autonomy between neighboring genes in gene expression profiles in extant taxa. Indeed, in all eukaryotic genomes genes of similar expression-profile tend to cluster, reflecting chromatin level dynamics. Does it follow that if a gene increases expression in a particular lineage then the genomic neighbors will also increase in their expression or is gene expression evolution autonomous? To address this here we consider evolution of human gene expression since the human-chimp common ancestor, allowing for both variation in estimation of current expression level and error in Bayesian estimation of the ancestral state. We find that in all tissues and both sexes, the change in gene expression of a focal gene on average predicts the change in gene expression of neighbors. The effect is highly pronounced in the immediate vicinity (<100 kb) but extends much further. Sex-specific expression change is also genomically clustered. As genes increasing their expression in humans tend to avoid nuclear lamina domains and be enriched for the gene activator 5-hydroxymethylcytosine, we conclude that, most probably owing to chromatin level control of gene expression, a change in gene expression of one gene likely affects the expression evolution of neighbors, what we term expression piggybacking, an analog of hitchhiking. PMID:25743543

  15. In vitro modulation of inflammatory target gene expression by a polyphenol-enriched fraction of rose oil distillation waste water.

    PubMed

    Wedler, Jonas; Weston, Anna; Rausenberger, Julia; Butterweck, Veronika

    2016-10-01

    Classical production of rose oil is based on water steam distillation from the flowers of Rosa damascena. During this process, large quantities of waste water accrue which are discharged to the environment, causing severe pollution of both, groundwater and surface water due to a high content of polyphenols. We recently developed a strategy to purify the waste water into a polyphenol-depleted and a polyphenol-enriched fraction RF20-(SP-207). RF20-(SP-207) and sub-fraction F(IV) significantly inhibited cell proliferation and migration of HaCaT cells. Since there is a close interplay between these actions and inflammatory processes, here we focused on the fractions' influence on pro-inflammatory biomarkers. HaCaT keratinocytes were treated with RF20-(SP-207), F(IV) (both at 50μg/mL) and ellagic acid (10μM) for 24h under TNF-α (20ng/mL) stimulated and non-stimulated conditions. Gene expression of IL-1β, IL-6, IL-8, RANTES and MCP-1 was analyzed by reverse transcriptase polymerase chain reaction (RT-PCR) and cellular protein secretion of IL-8, RANTES and MCP-1 was determined by ELISA based assays. RF20-(SP-207) and F(IV) significantly decreased the expression and cellular protein secretion of IL-1β, IL-6, IL-8, RANTES and MCP-1. The diminishing effects on inflammatory target gene expression were slightly less pronounced under TNF-α stimulated conditions. In conclusion, the recovered polyphenol fraction RF20-(SP-207) from rose oil distillation waste water markedly modified inflammatory target gene expression in vitro, and, therefore, could be further developed as alternative treatment of acute and chronic inflammation. Copyright © 2016 Elsevier B.V. All rights reserved.

  16. New TFII-I family target genes involved in embryonic development.

    PubMed

    Makeyev, Aleksandr V; Bayarsaihan, Dashzeveg

    2009-09-04

    Two members of the TFII-I family transcription factor genes, GTF2I and GTF2IRD1, are the prime candidates responsible for the craniofacial and cognitive abnormalities of Williams syndrome patients. We have previously generated mouse lines with targeted disruption of Gtf2i and Gtf2ird1. Microarray analysis revealed significant changes in the expression profile of mutant embryos. Here we described three unknown genes that were dramatically down-regulated in mutants. The 2410018M08Rik/Scand3 gene encodes a protein of unknown function with CHCH and hATC domains. Scand3 is down-regulated during mouse embryonic stem cell (ES) differentiation. 4933436H12Rik is a testis-specific gene, which encodes a protein with no known domains. It is expressed in mouse ES cells. 1110008P08Rik/Kbtbd7 encodes an adapter protein with BTB/POZ, BACK, and Kelch motifs, previously shown to recruit substrates to the enzymatic complexes of the histone modifying or E3 ubiquitin ligase activities. Based on its expression pattern Kbtbd7 may have a specific role in brain development and function. All three genes possess well-conserved TFII-I-binding consensus sites within proximal promoters. Therefore our analysis suggests that these genes can be direct targets of TFII-I proteins and their impaired expression, as a result of the GTF2I and GTF2IRD1 haploinsufficiency, could contribute to the etiology of Williams syndrome.

  17. Neighboring Genes Show Correlated Evolution in Gene Expression.

    PubMed

    Ghanbarian, Avazeh T; Hurst, Laurence D

    2015-07-01

    When considering the evolution of a gene's expression profile, we commonly assume that this is unaffected by its genomic neighborhood. This is, however, in contrast to what we know about the lack of autonomy between neighboring genes in gene expression profiles in extant taxa. Indeed, in all eukaryotic genomes genes of similar expression-profile tend to cluster, reflecting chromatin level dynamics. Does it follow that if a gene increases expression in a particular lineage then the genomic neighbors will also increase in their expression or is gene expression evolution autonomous? To address this here we consider evolution of human gene expression since the human-chimp common ancestor, allowing for both variation in estimation of current expression level and error in Bayesian estimation of the ancestral state. We find that in all tissues and both sexes, the change in gene expression of a focal gene on average predicts the change in gene expression of neighbors. The effect is highly pronounced in the immediate vicinity (<100 kb) but extends much further. Sex-specific expression change is also genomically clustered. As genes increasing their expression in humans tend to avoid nuclear lamina domains and be enriched for the gene activator 5-hydroxymethylcytosine, we conclude that, most probably owing to chromatin level control of gene expression, a change in gene expression of one gene likely affects the expression evolution of neighbors, what we term expression piggybacking, an analog of hitchhiking. © The Author 2015. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.

  18. NORMAL NASAL GENE EXPRESSION LEVELS USING CDNA ARRAY TECHNOLOGY

    EPA Science Inventory

    Normal Nasal Gene Expression Levels Using cDNA Array Technology.

    The nasal epithelium is a target site for chemically-induced toxicity and carcinogenicity. To detect and analyze genetic events which contribute to nasal tumor development, we first defined the gene expressi...

  19. Integrative analysis of RUNX1 downstream pathways and target genes

    PubMed Central

    Michaud, Joëlle; Simpson, Ken M; Escher, Robert; Buchet-Poyau, Karine; Beissbarth, Tim; Carmichael, Catherine; Ritchie, Matthew E; Schütz, Frédéric; Cannon, Ping; Liu, Marjorie; Shen, Xiaofeng; Ito, Yoshiaki; Raskind, Wendy H; Horwitz, Marshall S; Osato, Motomi; Turner, David R; Speed, Terence P; Kavallaris, Maria; Smyth, Gordon K; Scott, Hamish S

    2008-01-01

    Background The RUNX1 transcription factor gene is frequently mutated in sporadic myeloid and lymphoid leukemia through translocation, point mutation or amplification. It is also responsible for a familial platelet disorder with predisposition to acute myeloid leukemia (FPD-AML). The disruption of the largely unknown biological pathways controlled by RUNX1 is likely to be responsible for the development of leukemia. We have used multiple microarray platforms and bioinformatic techniques to help identify these biological pathways to aid in the understanding of why RUNX1 mutations lead to leukemia. Results Here we report genes regulated either directly or indirectly by RUNX1 based on the study of gene expression profiles generated from 3 different human and mouse platforms. The platforms used were global gene expression profiling of: 1) cell lines with RUNX1 mutations from FPD-AML patients, 2) over-expression of RUNX1 and CBFβ, and 3) Runx1 knockout mouse embryos using either cDNA or Affymetrix microarrays. We observe that our datasets (lists of differentially expressed genes) significantly correlate with published microarray data from sporadic AML patients with mutations in either RUNX1 or its cofactor, CBFβ. A number of biological processes were identified among the differentially expressed genes and functional assays suggest that heterozygous RUNX1 point mutations in patients with FPD-AML impair cell proliferation, microtubule dynamics and possibly genetic stability. In addition, analysis of the regulatory regions of the differentially expressed genes has for the first time systematically identified numerous potential novel RUNX1 target genes. Conclusion This work is the first large-scale study attempting to identify the genetic networks regulated by RUNX1, a master regulator in the development of the hematopoietic system and leukemia. The biological pathways and target genes controlled by RUNX1 will have considerable importance in disease progression in both

  20. Using RNA-seq data to select reference genes for normalizing gene expression in apple roots.

    PubMed

    Zhou, Zhe; Cong, Peihua; Tian, Yi; Zhu, Yanmin

    2017-01-01

    Gene expression in apple roots in response to various stress conditions is a less-explored research subject. Reliable reference genes for normalizing quantitative gene expression data have not been carefully investigated. In this study, the suitability of a set of 15 apple genes were evaluated for their potential use as reliable reference genes. These genes were selected based on their low variance of gene expression in apple root tissues from a recent RNA-seq data set, and a few previously reported apple reference genes for other tissue types. Four methods, Delta Ct, geNorm, NormFinder and BestKeeper, were used to evaluate their stability in apple root tissues of various genotypes and under different experimental conditions. A small panel of stably expressed genes, MDP0000095375, MDP0000147424, MDP0000233640, MDP0000326399 and MDP0000173025 were recommended for normalizing quantitative gene expression data in apple roots under various abiotic or biotic stresses. When the most stable and least stable reference genes were used for data normalization, significant differences were observed on the expression patterns of two target genes, MdLecRLK5 (MDP0000228426, a gene encoding a lectin receptor like kinase) and MdMAPK3 (MDP0000187103, a gene encoding a mitogen-activated protein kinase). Our data also indicated that for those carefully validated reference genes, a single reference gene is sufficient for reliable normalization of the quantitative gene expression. Depending on the experimental conditions, the most suitable reference genes can be specific to the sample of interest for more reliable RT-qPCR data normalization.

  1. Using RNA-seq data to select reference genes for normalizing gene expression in apple roots

    PubMed Central

    Zhou, Zhe; Cong, Peihua; Tian, Yi

    2017-01-01

    Gene expression in apple roots in response to various stress conditions is a less-explored research subject. Reliable reference genes for normalizing quantitative gene expression data have not been carefully investigated. In this study, the suitability of a set of 15 apple genes were evaluated for their potential use as reliable reference genes. These genes were selected based on their low variance of gene expression in apple root tissues from a recent RNA-seq data set, and a few previously reported apple reference genes for other tissue types. Four methods, Delta Ct, geNorm, NormFinder and BestKeeper, were used to evaluate their stability in apple root tissues of various genotypes and under different experimental conditions. A small panel of stably expressed genes, MDP0000095375, MDP0000147424, MDP0000233640, MDP0000326399 and MDP0000173025 were recommended for normalizing quantitative gene expression data in apple roots under various abiotic or biotic stresses. When the most stable and least stable reference genes were used for data normalization, significant differences were observed on the expression patterns of two target genes, MdLecRLK5 (MDP0000228426, a gene encoding a lectin receptor like kinase) and MdMAPK3 (MDP0000187103, a gene encoding a mitogen-activated protein kinase). Our data also indicated that for those carefully validated reference genes, a single reference gene is sufficient for reliable normalization of the quantitative gene expression. Depending on the experimental conditions, the most suitable reference genes can be specific to the sample of interest for more reliable RT-qPCR data normalization. PMID:28934340

  2. Analysis of multiplex gene expression maps obtained by voxelation.

    PubMed

    An, Li; Xie, Hongbo; Chin, Mark H; Obradovic, Zoran; Smith, Desmond J; Megalooikonomou, Vasileios

    2009-04-29

    Gene expression signatures in the mammalian brain hold the key to understanding neural development and neurological disease. Researchers have previously used voxelation in combination with microarrays for acquisition of genome-wide atlases of expression patterns in the mouse brain. On the other hand, some work has been performed on studying gene functions, without taking into account the location information of a gene's expression in a mouse brain. In this paper, we present an approach for identifying the relation between gene expression maps obtained by voxelation and gene functions. To analyze the dataset, we chose typical genes as queries and aimed at discovering similar gene groups. Gene similarity was determined by using the wavelet features extracted from the left and right hemispheres averaged gene expression maps, and by the Euclidean distance between each pair of feature vectors. We also performed a multiple clustering approach on the gene expression maps, combined with hierarchical clustering. Among each group of similar genes and clusters, the gene function similarity was measured by calculating the average gene function distances in the gene ontology structure. By applying our methodology to find similar genes to certain target genes we were able to improve our understanding of gene expression patterns and gene functions. By applying the clustering analysis method, we obtained significant clusters, which have both very similar gene expression maps and very similar gene functions respectively to their corresponding gene ontologies. The cellular component ontology resulted in prominent clusters expressed in cortex and corpus callosum. The molecular function ontology gave prominent clusters in cortex, corpus callosum and hypothalamus. The biological process ontology resulted in clusters in cortex, hypothalamus and choroid plexus. Clusters from all three ontologies combined were most prominently expressed in cortex and corpus callosum. The experimental

  3. Targeting pancreatic expressed PAX genes for the treatment of diabetes mellitus and pancreatic neuroendocrine tumors.

    PubMed

    Martin-Montalvo, Alejandro; Lorenzo, Petra I; López-Noriega, Livia; Gauthier, Benoit R

    2017-01-01

    Four members of the PAX family, PAX2, PAX4, PAX6 and PAX8 are known to be expressed in the pancreas. Accumulated evidences indicate that several pancreatic expressed PAX genes play a significant role in pancreatic development/functionality and alterations in these genes are involved in the pathogenesis of pancreatic diseases. Areas covered: In this review, we summarize the ongoing research related to pancreatic PAX genes in diabetes mellitus and pancreatic neuroendocrine tumors. We dissect the current knowledge at different levels; from mechanistic studies in cell lines performed to understand the molecular processes controlled by pancreatic PAX genes, to in vivo studies using rodent models that over-express or lack specific PAX genes. Finally, we describe human studies associating variants on pancreatic-expressed PAX genes with pancreatic diseases. Expert opinion: Based on the current literature, we propose that future interventions to treat pancreatic neuroendocrine tumors and diabetes mellitus could be developed via the modulation of PAX4 and/or PAX6 regulated pathways.

  4. Gene expression profiling in whole blood of patients with coronary artery disease

    PubMed Central

    Taurino, Chiara; Miller, William H.; McBride, Martin W.; McClure, John D.; Khanin, Raya; Moreno, María U.; Dymott, Jane A.; Delles, Christian; Dominiczak, Anna F.

    2010-01-01

    Owing to the dynamic nature of the transcriptome, gene expression profiling is a promising tool for discovery of disease-related genes and biological pathways. In the present study, we examined gene expression in whole blood of 12 patients with CAD (coronary artery disease) and 12 healthy control subjects. Furthermore, ten patients with CAD underwent whole-blood gene expression analysis before and after the completion of a cardiac rehabilitation programme following surgical coronary revascularization. mRNA and miRNA (microRNA) were isolated for expression profiling. Gene expression analysis identified 365 differentially expressed genes in patients with CAD compared with healthy controls (175 up- and 190 down-regulated in CAD), and 645 in CAD rehabilitation patients (196 up- and 449 down-regulated post-rehabilitation). Biological pathway analysis identified a number of canonical pathways, including oxidative phosphorylation and mitochondrial function, as being significantly and consistently modulated across the groups. Analysis of miRNA expression revealed a number of differentially expressed miRNAs, including hsa-miR-140-3p (control compared with CAD, P=0.017), hsa-miR-182 (control compared with CAD, P=0.093), hsa-miR-92a and hsa-miR-92b (post- compared with pre-exercise, P<0.01). Global analysis of predicted miRNA targets found significantly reduced expression of genes with target regions compared with those without: hsa-miR-140-3p (P=0.002), hsa-miR-182 (P=0.001), hsa-miR-92a and hsa-miR-92b (P=2.2×10−16). In conclusion, using whole blood as a ‘surrogate tissue’ in patients with CAD, we have identified differentially expressed miRNAs, differentially regulated genes and modulated pathways which warrant further investigation in the setting of cardiovascular function. This approach may represent a novel non-invasive strategy to unravel potentially modifiable pathways and possible therapeutic targets in cardiovascular disease. PMID:20528768

  5. Microgravity and Immunity: Changes in Lymphocyte Gene Expression

    NASA Technical Reports Server (NTRS)

    Risin, D.; Pellis, N. R.; Ward, N. E.; Risin, S. A.

    2006-01-01

    Earlier studies had shown that modeled and true microgravity (MG) cause multiple direct effects on human lymphocytes. MG inhibits lymphocyte locomotion, suppresses polyclonal and antigen-specific activation, affects signal transduction mechanisms, as well as activation-induced apoptosis. In this study we assessed changes in gene expression associated with lymphocyte exposure to microgravity in an attempt to identify microgravity-sensitive genes (MGSG) in general and specifically those genes that might be responsible for the functional and structural changes observed earlier. Two sets of experiments targeting different goals were conducted. In the first set, T-lymphocytes from normal donors were activated with antiCD3 and IL2 and then cultured in 1g (static) and modeled MG (MMG) conditions (Rotating Wall Vessel bioreactor) for 24 hours. This setting allowed searching for MGSG by comparison of gene expression patterns in zero and 1 g gravity. In the second set - activated T-cells after culturing for 24 hours in 1g and MMG were exposed three hours before harvesting to a secondary activation stimulus (PHA) thus triggering the apoptotic pathway. Total RNA was extracted using the RNeasy isolation kit (Qiagen, Valencia, CA). Affymetrix Gene Chips (U133A), allowing testing for 18,400 human genes, were used for microarray analysis. In the first set of experiments MMG exposure resulted in altered expression of 89 genes, 10 of them were up-regulated and 79 down-regulated. In the second set, changes in expression were revealed in 85 genes, 20 were up-regulated and 65 were down-regulated. The analysis revealed that significant numbers of MGS genes are associated with signal transduction and apoptotic pathways. Interestingly, the majority of genes that responded by up- or down-regulation in the alternative sets of experiments were not the same, possibly reflecting different functional states of the examined T-lymphocyte populations. The responder genes (MGSG) might play an

  6. Transposon integration enhances expression of stress response genes.

    PubMed

    Feng, Gang; Leem, Young-Eun; Levin, Henry L

    2013-01-01

    Transposable elements possess specific patterns of integration. The biological impact of these integration profiles is not well understood. Tf1, a long-terminal repeat retrotransposon in Schizosaccharomyces pombe, integrates into promoters with a preference for the promoters of stress response genes. To determine the biological significance of Tf1 integration, we took advantage of saturated maps of insertion activity and studied how integration at hot spots affected the expression of the adjacent genes. Our study revealed that Tf1 integration did not reduce gene expression. Importantly, the insertions activated the expression of 6 of 32 genes tested. We found that Tf1 increased gene expression by inserting enhancer activity. Interestingly, the enhancer activity of Tf1 could be limited by Abp1, a host surveillance factor that sequesters transposon sequences into structures containing histone deacetylases. We found the Tf1 promoter was activated by heat treatment and, remarkably, only genes that themselves were induced by heat could be activated by Tf1 integration, suggesting a synergy of Tf1 enhancer sequence with the stress response elements of target promoters. We propose that the integration preference of Tf1 for the promoters of stress response genes and the ability of Tf1 to enhance the expression of these genes co-evolved to promote the survival of cells under stress.

  7. Transposon integration enhances expression of stress response genes

    PubMed Central

    Feng, Gang; Leem, Young-Eun; Levin, Henry L.

    2013-01-01

    Transposable elements possess specific patterns of integration. The biological impact of these integration profiles is not well understood. Tf1, a long-terminal repeat retrotransposon in Schizosaccharomyces pombe, integrates into promoters with a preference for the promoters of stress response genes. To determine the biological significance of Tf1 integration, we took advantage of saturated maps of insertion activity and studied how integration at hot spots affected the expression of the adjacent genes. Our study revealed that Tf1 integration did not reduce gene expression. Importantly, the insertions activated the expression of 6 of 32 genes tested. We found that Tf1 increased gene expression by inserting enhancer activity. Interestingly, the enhancer activity of Tf1 could be limited by Abp1, a host surveillance factor that sequesters transposon sequences into structures containing histone deacetylases. We found the Tf1 promoter was activated by heat treatment and, remarkably, only genes that themselves were induced by heat could be activated by Tf1 integration, suggesting a synergy of Tf1 enhancer sequence with the stress response elements of target promoters. We propose that the integration preference of Tf1 for the promoters of stress response genes and the ability of Tf1 to enhance the expression of these genes co-evolved to promote the survival of cells under stress. PMID:23193295

  8. Identification of regulatory targets of tissue-specific transcription factors: application to retina-specific gene regulation

    PubMed Central

    Qian, Jiang; Esumi, Noriko; Chen, Yangjian; Wang, Qingliang; Chowers, Itay; Zack, Donald J.

    2005-01-01

    Identification of tissue-specific gene regulatory networks can yield insights into the molecular basis of a tissue's development, function and pathology. Here, we present a computational approach designed to identify potential regulatory target genes of photoreceptor cell-specific transcription factors (TFs). The approach is based on the hypothesis that genes related to the retina in terms of expression, disease and/or function are more likely to be the targets of retina-specific TFs than other genes. A list of genes that are preferentially expressed in retina was obtained by integrating expressed sequence tag, SAGE and microarray datasets. The regulatory targets of retina-specific TFs are enriched in this set of retina-related genes. A Bayesian approach was employed to integrate information about binding site location relative to a gene's transcription start site. Our method was applied to three retina-specific TFs, CRX, NRL and NR2E3, and a number of potential targets were predicted. To experimentally assess the validity of the bioinformatic predictions, mobility shift, transient transfection and chromatin immunoprecipitation assays were performed with five predicted CRX targets, and the results were suggestive of CRX regulation in 5/5, 3/5 and 4/5 cases, respectively. Together, these experiments strongly suggest that RP1, GUCY2D, ABCA4 are novel targets of CRX. PMID:15967807

  9. A multiplex branched DNA assay for parallel quantitative gene expression profiling.

    PubMed

    Flagella, Michael; Bui, Son; Zheng, Zhi; Nguyen, Cung Tuong; Zhang, Aiguo; Pastor, Larry; Ma, Yunqing; Yang, Wen; Crawford, Kimberly L; McMaster, Gary K; Witney, Frank; Luo, Yuling

    2006-05-01

    We describe a novel method to quantitatively measure messenger RNA (mRNA) expression of multiple genes directly from crude cell lysates and tissue homogenates without the need for RNA purification or target amplification. The multiplex branched DNA (bDNA) assay adapts the bDNA technology to the Luminex fluorescent bead-based platform through the use of cooperative hybridization, which ensures an exceptionally high degree of assay specificity. Using in vitro transcribed RNA as reference standards, we demonstrated that the assay is highly specific, with cross-reactivity less than 0.2%. We also determined that the assay detection sensitivity is 25,000 RNA transcripts with intra- and interplate coefficients of variance of less than 10% and less than 15%, respectively. Using three 10-gene panels designed to measure proinflammatory and apoptosis responses, we demonstrated sensitive and specific multiplex gene expression profiling directly from cell lysates. The gene expression change data demonstrate a high correlation coefficient (R(2)=0.94) compared with measurements obtained using the single-plex bDNA assay. Thus, the multiplex bDNA assay provides a powerful means to quantify the gene expression profile of a defined set of target genes in large sample populations.

  10. Blue Light Modulates Murine Microglial Gene Expression in the Absence of Optogenetic Protein Expression.

    PubMed

    Cheng, Kevin P; Kiernan, Elizabeth A; Eliceiri, Kevin W; Williams, Justin C; Watters, Jyoti J

    2016-02-17

    Neural optogenetic applications over the past decade have steadily increased; however the effects of commonly used blue light paradigms on surrounding, non-optogenetic protein-expressing CNS cells are rarely considered, despite their simultaneous exposure. Here we report that blue light (450 nm) repetitively delivered in both long-duration boluses and rapid optogenetic bursts gene-specifically altered basal expression of inflammatory and neurotrophic genes in immortalized and primary murine wild type microglial cultures. In addition, blue light reduced pro-inflammatory gene expression in microglia activated with lipopolysaccharide. These results demonstrate previously unreported, off-target effects of blue light in cells not expressing optogenetic constructs. The unexpected gene modulatory effects of blue light on wild type CNS resident immune cells have novel and important implications for the neuro-optogenetic field. Further studies are needed to elucidate the molecular mechanisms and potential therapeutic utility of blue light modulation of the wild type CNS.

  11. Identification of genes associated with renal cell carcinoma using gene expression profiling analysis.

    PubMed

    Yao, Ting; Wang, Qinfu; Zhang, Wenyong; Bian, Aihong; Zhang, Jinping

    2016-07-01

    Renal cell carcinoma (RCC) is the most common type of kidney cancer in adults and accounts for ~80% of all kidney cancer cases. However, the pathogenesis of RCC has not yet been fully elucidated. To interpret the pathogenesis of RCC at the molecular level, gene expression data and bio-informatics methods were used to identify RCC associated genes. Gene expression data was downloaded from Gene Expression Omnibus (GEO) database and identified differentially coexpressed genes (DCGs) and dysfunctional pathways in RCC patients compared with controls. In addition, a regulatory network was constructed using the known regulatory data between transcription factors (TFs) and target genes in the University of California Santa Cruz (UCSC) Genome Browser (http://genome.ucsc.edu) and the regulatory impact factor of each TF was calculated. A total of 258,0427 pairs of DCGs were identified. The regulatory network contained 1,525 pairs of regulatory associations between 126 TFs and 1,259 target genes and these genes were mainly enriched in cancer pathways, ErbB and MAPK. In the regulatory network, the 10 most strongly associated TFs were FOXC1, GATA3, ESR1, FOXL1, PATZ1, MYB, STAT5A, EGR2, EGR3 and PELP1. GATA3, ERG and MYB serve important roles in RCC while FOXC1, ESR1, FOXL1, PATZ1, STAT5A and PELP1 may be potential genes associated with RCC. In conclusion, the present study constructed a regulatory network and screened out several TFs that may be used as molecular biomarkers of RCC. However, future studies are needed to confirm the findings of the present study.

  12. Identification of genes associated with renal cell carcinoma using gene expression profiling analysis

    PubMed Central

    YAO, TING; WANG, QINFU; ZHANG, WENYONG; BIAN, AIHONG; ZHANG, JINPING

    2016-01-01

    Renal cell carcinoma (RCC) is the most common type of kidney cancer in adults and accounts for ~80% of all kidney cancer cases. However, the pathogenesis of RCC has not yet been fully elucidated. To interpret the pathogenesis of RCC at the molecular level, gene expression data and bio-informatics methods were used to identify RCC associated genes. Gene expression data was downloaded from Gene Expression Omnibus (GEO) database and identified differentially coexpressed genes (DCGs) and dysfunctional pathways in RCC patients compared with controls. In addition, a regulatory network was constructed using the known regulatory data between transcription factors (TFs) and target genes in the University of California Santa Cruz (UCSC) Genome Browser (http://genome.ucsc.edu) and the regulatory impact factor of each TF was calculated. A total of 258,0427 pairs of DCGs were identified. The regulatory network contained 1,525 pairs of regulatory associations between 126 TFs and 1,259 target genes and these genes were mainly enriched in cancer pathways, ErbB and MAPK. In the regulatory network, the 10 most strongly associated TFs were FOXC1, GATA3, ESR1, FOXL1, PATZ1, MYB, STAT5A, EGR2, EGR3 and PELP1. GATA3, ERG and MYB serve important roles in RCC while FOXC1, ESR1, FOXL1, PATZ1, STAT5A and PELP1 may be potential genes associated with RCC. In conclusion, the present study constructed a regulatory network and screened out several TFs that may be used as molecular biomarkers of RCC. However, future studies are needed to confirm the findings of the present study. PMID:27347102

  13. The Prx1 limb enhancers: targeted gene expression in developing zebrafish pectoral fins.

    PubMed

    Hernández-Vega, Amayra; Minguillón, Carolina

    2011-08-01

    Limbs represent an excellent model to study the induction, growth, and patterning of several organs. A breakthrough to study gene function in various tissues has been the characterization of regulatory elements that allow tissue-specific interference of gene function. The mouse Prx1 promoter has been used to generate limb-specific mutants and overexpress genes in tetrapod limbs. Although zebrafish possess advantages that favor their use to study limb morphogenesis, there is no driver described suitable for specifically interfering with gene function in developing fins. We report the generation of zebrafish lines that express enhanced green fluorescent protein (EGFP) driven by the mouse Prx1 enhancer in developing pectoral fins. We also describe the expression pattern of the zebrafish prrx1 genes and identify three conserved non-coding elements (CNEs) that we use to generate fin-specific EGFP reporter lines. Finally, we show that the mouse and zebrafish regulatory elements may be used to modify gene function in pectoral fins. Copyright © 2011 Wiley-Liss, Inc.

  14. Gene silencing in Tribolium castaneum as a tool for the targeted identification of candidate RNAi targets in crop pests.

    PubMed

    Knorr, Eileen; Fishilevich, Elane; Tenbusch, Linda; Frey, Meghan L F; Rangasamy, Murugesan; Billion, Andre; Worden, Sarah E; Gandra, Premchand; Arora, Kanika; Lo, Wendy; Schulenberg, Greg; Valverde-Garcia, Pablo; Vilcinskas, Andreas; Narva, Kenneth E

    2018-02-01

    RNAi shows potential as an agricultural technology for insect control, yet, a relatively low number of robust lethal RNAi targets have been demonstrated to control insects of agricultural interest. In the current study, a selection of lethal RNAi target genes from the iBeetle (Tribolium castaneum) screen were used to demonstrate efficacy of orthologous targets in the economically important coleopteran pests Diabrotica virgifera virgifera and Meligethes aeneus. Transcript orthologs of 50 selected genes were analyzed in D. v. virgifera diet-based RNAi bioassays; 21 of these RNAi targets showed mortality and 36 showed growth inhibition. Low dose injection- and diet-based dsRNA assays in T. castaneum and D. v. virgifera, respectively, enabled the identification of the four highly potent RNAi target genes: Rop, dre4, ncm, and RpII140. Maize was genetically engineered to express dsRNA directed against these prioritized candidate target genes. T 0 plants expressing Rop, dre4, or RpII140 RNA hairpins showed protection from D. v. virgifera larval feeding damage. dsRNA targeting Rop, dre4, ncm, and RpII140 in M. aeneus also caused high levels of mortality both by injection and feeding. In summary, high throughput systems for model organisms can be successfully used to identify potent RNA targets for difficult-to-work with agricultural insect pests.

  15. Large scale RNAi screen in Tribolium reveals novel target genes for pest control and the proteasome as prime target.

    PubMed

    Ulrich, Julia; Dao, Van Anh; Majumdar, Upalparna; Schmitt-Engel, Christian; Schwirz, Jonas; Schultheis, Dorothea; Ströhlein, Nadi; Troelenberg, Nicole; Grossmann, Daniela; Richter, Tobias; Dönitz, Jürgen; Gerischer, Lizzy; Leboulle, Gérard; Vilcinskas, Andreas; Stanke, Mario; Bucher, Gregor

    2015-09-03

    Insect pest control is challenged by insecticide resistance and negative impact on ecology and health. One promising pest specific alternative is the generation of transgenic plants, which express double stranded RNAs targeting essential genes of a pest species. Upon feeding, the dsRNA induces gene silencing in the pest resulting in its death. However, the identification of efficient RNAi target genes remains a major challenge as genomic tools and breeding capacity is limited in most pest insects impeding whole-animal-high-throughput-screening. We use the red flour beetle Tribolium castaneum as a screening platform in order to identify the most efficient RNAi target genes. From about 5,000 randomly screened genes of the iBeetle RNAi screen we identify 11 novel and highly efficient RNAi targets. Our data allowed us to determine GO term combinations that are predictive for efficient RNAi target genes with proteasomal genes being most predictive. Finally, we show that RNAi target genes do not appear to act synergistically and that protein sequence conservation does not correlate with the number of potential off target sites. Our results will aid the identification of RNAi target genes in many pest species by providing a manageable number of excellent candidate genes to be tested and the proteasome as prime target. Further, the identified GO term combinations will help to identify efficient target genes from organ specific transcriptomes. Our off target analysis is relevant for the sequence selection used in transgenic plants.

  16. Versatile control of Plasmodium falciparum gene expression with an inducible protein-RNA interaction

    PubMed Central

    Goldfless, Stephen J.; Wagner, Jeffrey C.; Niles, Jacquin C.

    2014-01-01

    The available tools for conditional gene expression in Plasmodium falciparum are limited. Here, to enable reliable control of target gene expression, we build a system to efficiently modulate translation. We overcame several problems associated with other approaches for regulating gene expression in P. falciparum. Specifically, our system functions predictably across several native and engineered promoter contexts, and affords control over reporter and native parasite proteins irrespective of their subcellular compartmentalization. Induction and repression of gene expression are rapid, homogeneous, and stable over prolonged periods. To demonstrate practical application of our system, we used it to reveal direct links between antimalarial drugs and their native parasite molecular target. This is an important out come given the rapid spread of resistance, and intensified efforts to efficiently discover and optimize new antimalarial drugs. Overall, the studies presented highlight the utility of our system for broadly controlling gene expression and performing functional genetics in P. falciparum. PMID:25370483

  17. LincRNA-p21 activates p21 in cis to promote Polycomb target gene expression and to enforce the G1/S checkpoint

    PubMed Central

    Dimitrova, Nadya; Zamudio, Jesse R.; Jong, Robyn M.; Soukup, Dylan; Resnick, Rebecca; Sarma, Kavitha; Ward, Amanda J.; Raj, Arjun; Lee, Jeannie; Sharp, Phillip A.; Jacks, Tyler

    2014-01-01

    SUMMARY The p53-regulated long non-coding RNA lincRNA-p21 has been proposed to act in trans via several mechanisms ranging from repressing genes in the p53 transcriptional network to regulating mRNA translation and protein stability. To further examine lincRNA-p21 function we generated a conditional knockout mouse model. We find that lincRNA-p21 predominantly functions in cis to activate expression of its neighboring gene, p21. Mechanistically, we show that lincRNA-p21 acts in concert with hnRNP-K as a co-activator for p53-dependent p21 transcription. Additional phenotypes of lincRNA-p21 deficiency could be attributed to diminished p21 levels, including deregulated expression and altered chromatin state of some Polycomb target genes, defective G1/S checkpoint, increased proliferation rates, and enhanced reprogramming efficiency. These findings indicate that lincRNA-p21 affects global gene expression and influences the p53 tumor suppressor pathway by acting in cis as a locus-restricted co-activator for p53-mediated p21 expression. PMID:24857549

  18. Expression-based clustering of CAZyme-encoding genes of Aspergillus niger.

    PubMed

    Gruben, Birgit S; Mäkelä, Miia R; Kowalczyk, Joanna E; Zhou, Miaomiao; Benoit-Gelber, Isabelle; De Vries, Ronald P

    2017-11-23

    The Aspergillus niger genome contains a large repertoire of genes encoding carbohydrate active enzymes (CAZymes) that are targeted to plant polysaccharide degradation enabling A. niger to grow on a wide range of plant biomass substrates. Which genes need to be activated in certain environmental conditions depends on the composition of the available substrate. Previous studies have demonstrated the involvement of a number of transcriptional regulators in plant biomass degradation and have identified sets of target genes for each regulator. In this study, a broad transcriptional analysis was performed of the A. niger genes encoding (putative) plant polysaccharide degrading enzymes. Microarray data focusing on the initial response of A. niger to the presence of plant biomass related carbon sources were analyzed of a wild-type strain N402 that was grown on a large range of carbon sources and of the regulatory mutant strains ΔxlnR, ΔaraR, ΔamyR, ΔrhaR and ΔgalX that were grown on their specific inducing compounds. The cluster analysis of the expression data revealed several groups of co-regulated genes, which goes beyond the traditionally described co-regulated gene sets. Additional putative target genes of the selected regulators were identified, based on their expression profile. Notably, in several cases the expression profile puts questions on the function assignment of uncharacterized genes that was based on homology searches, highlighting the need for more extensive biochemical studies into the substrate specificity of enzymes encoded by these non-characterized genes. The data also revealed sets of genes that were upregulated in the regulatory mutants, suggesting interaction between the regulatory systems and a therefore even more complex overall regulatory network than has been reported so far. Expression profiling on a large number of substrates provides better insight in the complex regulatory systems that drive the conversion of plant biomass by fungi. In

  19. [miR-143 inhibits cell proliferation through targeted regulating the expression of K-ras gene in HeLa cells].

    PubMed

    Qin, H X; Cui, H K; Pan, Y; Hu, R L; Zhu, L H; Wang, S J

    2016-12-23

    Objective: To explore the effect of microRNA miR-143 on the proliferation of cervical cancer HeLa cells through targeted regulating the expression of K-ras gene. Methods: The luciferase report carrier containing wild type 3'-UTR of K-ras gene (K-ras-wt) or mutated 3'-UTR of the K-ras (K-ras-mut) were co-transfected with iR-143 mimic into the HeLa cells respectively, and the targeting effect of miR-143 in the transfectants was verified by the dual luciferase report system. HeLa cells were also transfected with miR-143 mimic (miR-143 mimic group), mimic control (negative control group), and miR-143 mimic plus K-ras gene (miR-143 mimic+ K-ras group), respectively. The expression of miR-143 in the transfected HeLa cells was detected by real-time PCR (RT-PCR), and the expression of K-ras protein was detected by Western blot. The cell proliferation activity of each group was examined by MTT assay. In addition, human cervical cancer tissue samples ( n =5) and cervical intraepithelial neoplasia tissue samples ( n =5) were also examined for the expression of miR-143 and K-ras protein by RT-PCR and Western blot, respectively. Results: The luciferase report assay showed that co-transfection with miR-143 mimic decreased the luciferase activity of the K-ras-wt significantly, but did not inhibit the luciferase activity of the K-ras-mut. The expression of miR-143 in the HeLa cells transfected with miR-143 mimic was significantly higher than that in the HeLa cells transfected with the mimic control (3.31±0.45 vs 0.97±0.22, P <0.05). The MTT assay revealed that the cell proliferative activity of the miR-143 mimic group was significantly lower than that of the negative control group ( P <0.05), and the cell proliferative activity of the miR-143 mimic+ K-ras group was also significantly lower than the control group ( P <0.05) but higher than the miR-143 mimic group significantly ( P <0.05). The expression levels of K-ras protein in the miR-143 mimic group, the negative control

  20. GEsture: an online hand-drawing tool for gene expression pattern search.

    PubMed

    Wang, Chunyan; Xu, Yiqing; Wang, Xuelin; Zhang, Li; Wei, Suyun; Ye, Qiaolin; Zhu, Youxiang; Yin, Hengfu; Nainwal, Manoj; Tanon-Reyes, Luis; Cheng, Feng; Yin, Tongming; Ye, Ning

    2018-01-01

    Gene expression profiling data provide useful information for the investigation of biological function and process. However, identifying a specific expression pattern from extensive time series gene expression data is not an easy task. Clustering, a popular method, is often used to classify similar expression genes, however, genes with a 'desirable' or 'user-defined' pattern cannot be efficiently detected by clustering methods. To address these limitations, we developed an online tool called GEsture. Users can draw, or graph a curve using a mouse instead of inputting abstract parameters of clustering methods. GEsture explores genes showing similar, opposite and time-delay expression patterns with a gene expression curve as input from time series datasets. We presented three examples that illustrate the capacity of GEsture in gene hunting while following users' requirements. GEsture also provides visualization tools (such as expression pattern figure, heat map and correlation network) to display the searching results. The result outputs may provide useful information for researchers to understand the targets, function and biological processes of the involved genes.

  1. Genetics, gene expression and bioinformatics of the pituitary gland.

    PubMed

    Davis, Shannon W; Potok, Mary Anne; Brinkmeier, Michelle L; Carninci, Piero; Lyons, Robert H; MacDonald, James W; Fleming, Michelle T; Mortensen, Amanda H; Egashira, Noboru; Ghosh, Debashis; Steel, Karen P; Osamura, Robert Y; Hayashizaki, Yoshihide; Camper, Sally A

    2009-04-01

    Genetic cases of congenital pituitary hormone deficiency are common and many are caused by transcription factor defects. Mouse models with orthologous mutations are invaluable for uncovering the molecular mechanisms that lead to problems in organ development and typical patient characteristics. We are using mutant mice defective in the transcription factors PROP1 and POU1F1 for gene expression profiling to identify target genes for these critical transcription factors and candidates for cases of pituitary hormone deficiency of unknown aetiology. These studies reveal critical roles for Wnt signalling pathways, including the TCF/LEF transcription factors and interacting proteins of the groucho family, bone morphogenetic protein antagonists and targets of notch signalling. Current studies are investigating the roles of novel homeobox genes and pathways that regulate the transition from proliferation to differentiation, cell adhesion and cell migration. Pituitary adenomas are a common human health problem, yet most cases are sporadic, necessitating alternative approaches to traditional Mendelian genetic studies. Mouse models of adenoma formation offer the opportunity for gene expression profiling during progressive stages of hyperplasia, adenoma and tumorigenesis. This approach holds promise for the identification of relevant pathways and candidate genes as risk factors for adenoma formation, understanding mechanisms of progression, and identifying drug targets and clinically relevant biomarkers. Copyright 2009 S. Karger AG, Basel.

  2. Genetics, Gene Expression and Bioinformatics of the Pituitary Gland

    PubMed Central

    Davis, Shannon W; Potok, Mary Anne; Brinkmeier, Michelle L; Carninci, Piero; Lyons, Robert H; MacDonald, James W.; Fleming, Michelle T; Mortensen, Amanda H; Egashira, Noboru; Ghosh, Debashis; Steel, Karen P.; Osamura, Robert Y; Hayashizaki, Yoshihide; Camper, Sally A

    2011-01-01

    Genetic cases of congenital pituitary hormone deficiency are common and many are caused by transcription factor defects. Mouse models with orthologous mutations are invaluable for uncovering the molecular mechanisms that lead to problems in organ development and typical patient characteristics. We are using mutant mice defective in the transcription factors PROP1 and POU1F1 for gene expression profiling to identify target genes for these critical transcription factors and candidates for cases of pituitary hormone deficiency of unknown etiology. These studies reveal critical roles for Wnt signalling pathways including the TCF/LEF transcription factors and interacting proteins of the groucho family, bone morphogenetic proteins antagonists, and targets of notch signalling. Current studies are investigating roles of novel homeobox genes and pathways that regulate the transition from proliferation to differentiation, cell adhesion and cell migration. Pituitary adenomas are a common human health problem, yet most cases are sporadic, necessitating alternative approaches to traditional Mendelian genetic studies. Mouse models of adenoma formation offer the opportunity for gene expression profiling during progressive stages of hyperplasia, adenoma and tumorigenesis. This approach holds promise for identification of relevant pathways and candidate genes as risk factors for adenoma formation, understanding mechanisms of progression, and identifying drug targets and clinically relevant biomarkers. PMID:19407506

  3. New target genes of MITF-induced microRNA-211 contribute to melanoma cell invasion.

    PubMed

    Margue, Christiane; Philippidou, Demetra; Reinsbach, Susanne E; Schmitt, Martina; Behrmann, Iris; Kreis, Stephanie

    2013-01-01

    The non-coding microRNAs (miRNA) have tissue- and disease-specific expression patterns. They down-regulate target mRNAs, which likely impacts on most fundamental cellular processes. Differential expression patterns of miRNAs are currently being exploited for identification of biomarkers for early disease diagnosis, prediction of progression for melanoma and other cancers and as promising drug targets, since they can easily be inhibited or replaced in a given cellular context. Before successfully manipulating miRNAs in clinical settings, their precise expression levels, endogenous functions and thus their target genes have to be determined. MiR-211, a melanocyte lineage-specific small non-coding miRNA, is located in an intron of TRPM1, a target gene of the microphtalmia-associated transcription factor (MITF). By transcriptionally up-regulating TRPM1, MITF, which is critical for both melanocyte differentiation and survival and for melanoma progression, indirectly drives the expression of miR-211. Expression of this miRNA is often reduced in melanoma samples. Here, we investigated functional roles of miR-211 by identifying and studying new target genes. We show that MITF-correlated miR-211 expression levels are mostly but not always reduced in a panel of 11 melanoma cell lines and in primary and metastatic melanoma compared to normal melanocytes and nevi, respectively. MiR-211 itself only marginally impacted on cell invasion and migration, while perturbation of some new miR-211 target genes, such as AP1S2, SOX11, IGFBP5, and SERINC3 significantly increased invasion. These results and the variable expression levels of miR-211 raise serious doubts on the value of miR-211 as a melanoma tumor-suppressing miRNA and/or as a biomarker for melanoma.

  4. Time course of gene expression during mouse skeletal muscle hypertrophy

    PubMed Central

    Lee, Jonah D.; England, Jonathan H.; Esser, Karyn A.; McCarthy, John J.

    2013-01-01

    The purpose of this study was to perform a comprehensive transcriptome analysis during skeletal muscle hypertrophy to identify signaling pathways that are operative throughout the hypertrophic response. Global gene expression patterns were determined from microarray results on days 1, 3, 5, 7, 10, and 14 during plantaris muscle hypertrophy induced by synergist ablation in adult mice. Principal component analysis and the number of differentially expressed genes (cutoffs ≥2-fold increase or ≥50% decrease compared with control muscle) revealed three gene expression patterns during overload-induced hypertrophy: early (1 day), intermediate (3, 5, and 7 days), and late (10 and 14 days) patterns. Based on the robust changes in total RNA content and in the number of differentially expressed genes, we focused our attention on the intermediate gene expression pattern. Ingenuity Pathway Analysis revealed a downregulation of genes encoding components of the branched-chain amino acid degradation pathway during hypertrophy. Among these genes, five were predicted by Ingenuity Pathway Analysis or previously shown to be regulated by the transcription factor Kruppel-like factor-15, which was also downregulated during hypertrophy. Moreover, the integrin-linked kinase signaling pathway was activated during hypertrophy, and the downregulation of muscle-specific micro-RNA-1 correlated with the upregulation of five predicted targets associated with the integrin-linked kinase pathway. In conclusion, we identified two novel pathways that may be involved in muscle hypertrophy, as well as two upstream regulators (Kruppel-like factor-15 and micro-RNA-1) that provide targets for future studies investigating the importance of these pathways in muscle hypertrophy. PMID:23869057

  5. Time course of gene expression during mouse skeletal muscle hypertrophy.

    PubMed

    Chaillou, Thomas; Lee, Jonah D; England, Jonathan H; Esser, Karyn A; McCarthy, John J

    2013-10-01

    The purpose of this study was to perform a comprehensive transcriptome analysis during skeletal muscle hypertrophy to identify signaling pathways that are operative throughout the hypertrophic response. Global gene expression patterns were determined from microarray results on days 1, 3, 5, 7, 10, and 14 during plantaris muscle hypertrophy induced by synergist ablation in adult mice. Principal component analysis and the number of differentially expressed genes (cutoffs ≥2-fold increase or ≥50% decrease compared with control muscle) revealed three gene expression patterns during overload-induced hypertrophy: early (1 day), intermediate (3, 5, and 7 days), and late (10 and 14 days) patterns. Based on the robust changes in total RNA content and in the number of differentially expressed genes, we focused our attention on the intermediate gene expression pattern. Ingenuity Pathway Analysis revealed a downregulation of genes encoding components of the branched-chain amino acid degradation pathway during hypertrophy. Among these genes, five were predicted by Ingenuity Pathway Analysis or previously shown to be regulated by the transcription factor Kruppel-like factor-15, which was also downregulated during hypertrophy. Moreover, the integrin-linked kinase signaling pathway was activated during hypertrophy, and the downregulation of muscle-specific micro-RNA-1 correlated with the upregulation of five predicted targets associated with the integrin-linked kinase pathway. In conclusion, we identified two novel pathways that may be involved in muscle hypertrophy, as well as two upstream regulators (Kruppel-like factor-15 and micro-RNA-1) that provide targets for future studies investigating the importance of these pathways in muscle hypertrophy.

  6. Identification of potential target genes of ROR-alpha in THP1 and HUVEC cell lines.

    PubMed

    Gulec, Cagri; Coban, Neslihan; Ozsait-Selcuk, Bilge; Sirma-Ekmekci, Sema; Yildirim, Ozlem; Erginel-Unaltuna, Nihan

    2017-04-01

    ROR-alpha is a nuclear receptor, activity of which can be modulated by natural or synthetic ligands. Due to its possible involvement in, and potential therapeutic target for atherosclerosis, we aimed to identify ROR-alpha target genes in monocytic and endothelial cell lines. We performed chromatin immunoprecipitation (ChIP) followed by tiling array (ChIP-on-chip) for ROR-alpha in monocytic cell line THP1 and endothelial cell line HUVEC. Following bioinformatic analysis of the array data, we tested four candidate genes in terms of dependence of their expression level on ligand-mediated ROR-alpha activity, and two of them in terms of promoter occupancy by ROR-alpha. Bioinformatic analyses of ChIP-on-chip data suggested that ROR-alpha binds to genomic regions near the transcription start site (TSS) of more than 3000 genes in THP1 and HUVEC. Potential ROR-alpha target genes in both cell types seem to be involved mainly in membrane receptor activity, signal transduction and ion transport. While SPP1 and IKBKA were shown to be direct target genes of ROR-alpha in THP1 monocytes, inflammation related gene HMOX1 and heat shock protein gene HSPA8 were shown to be potential target genes of ROR-alpha. Our results suggest that ROR-alpha may regulate signaling receptor activity, and transmembrane transport activity through its potential target genes. ROR-alpha seems also to play role in cellular sensitivity to environmental substances like arsenite and chloroprene. Although, the expression analyses have shown that synthetic ROR-alpha ligands can modulate some of potential ROR-alpha target genes, functional significance of ligand-dependent modulation of gene expression needs to be confirmed with further analyses. Copyright © 2017 Elsevier Inc. All rights reserved.

  7. The miR172 target TOE3 represses AGAMOUS expression during Arabidopsis floral patterning.

    PubMed

    Jung, Jae-Hoon; Lee, Sangmin; Yun, Ju; Lee, Minyoung; Park, Chung-Mo

    2014-02-01

    microRNA172 (miR172) regulates phase transition and floral patterning in Arabidopsis by repressing targets that encode the APETALA2 (AP2) and AP2-like transcription factors. The miR172-mediated repression of the AP2 gene restricts AGAMOUS (AG) expression. In addition, most miR172 targets, including AP2, redundantly act as floral repressors, and the overexpression of the target genes causes delayed flowering. However, how miR172 targets other than AP2 regulate both of the developmental processes remains unclear. Here, we demonstrate that miR172-mediated repression of the TARGET OF EAT 3 (TOE3) gene is critical for floral patterning in Arabidopsis. Transgenic plants that overexpress a miR172-resistant TOE3 gene (rTOE3-ox) exhibit indeterminate flowers with numerous stamens and carpelloid organs, which is consistent with previous observations in transgenic plants that overexpress a miR172-resistant AP2 gene. TOE3 binds to the second intron of the AG gene. Accordingly, AG expression is significantly reduced in rTOE3-ox plants. TOE3 also interacts with AP2 in the nucleus. Given the major role of AP2 in floral patterning, miR172 likely regulates TOE3 in floral patterning, at least in part via AP2. In addition, a miR156 target SQUAMOSA PROMOTER BINDING PROTEIN-LIKE 3 directly activates TOE3 expression, revealing a novel signaling interaction between miR156 and miR172 in floral patterning. Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.

  8. [Progress in application of targeting viral vector regulated by microRNA in gene therapy: a review].

    PubMed

    Zhang, Guohai; Wang, Qizhao; Zhang, Jinghong; Xu, Ruian

    2010-06-01

    A safe and effective targeting viral vector is the key factor for successful clinical gene therapy. microRNA, a class of small, single-stranded endogenous RNAs, act as post-transcriptional regulators of gene expression. The discovery of these kind regulatory elements provides a new approach to regulate gene expression more accurately. In this review, we elucidated the principle of microRNA in regulation of targeting viral vector. The applications of microRNA in the fields of elimination contamination from replication competent virus, reduction of transgene-specific immunity, promotion of cancer-targeted gene therapy and development of live attenuated vaccines were also discussed.

  9. Integrated computational biology analysis to evaluate target genes for chronic myelogenous leukemia.

    PubMed

    Zheng, Yu; Wang, Yu-Ping; Cao, Hongbao; Chen, Qiusheng; Zhang, Xi

    2018-06-05

    Although hundreds of genes have been linked to chronic myelogenous leukemia (CML), many of the results lack reproducibility. In the present study, data across multiple modalities were integrated to evaluate 579 CML candidate genes, including literature‑based CML‑gene relation data, Gene Expression Omnibus RNA expression data and pathway‑based gene‑gene interaction data. The expression data included samples from 76 patients with CML and 73 healthy controls. For each target gene, four metrics were proposed and tested with case/control classification. The effectiveness of the four metrics presented was demonstrated by the high classification accuracy (94.63%; P<2x10‑4). Cross metric analysis suggested nine top candidate genes for CML: Epidermal growth factor receptor, tumor protein p53, catenin β 1, janus kinase 2, tumor necrosis factor, abelson murine leukemia viral oncogene homolog 1, vascular endothelial growth factor A, B‑cell lymphoma 2 and proto‑oncogene tyrosine‑protein kinase. In addition, 145 CML candidate pathways enriched with 485 out of 579 genes were identified (P<8.2x10‑11; q=0.005). In conclusion, weighted genetic networks generated using computational biology may be complementary to biological experiments for the evaluation of known or novel CML target genes.

  10. Gene expression studies of reference genes for quantitative real-time PCR: an overview in insects.

    PubMed

    Shakeel, Muhammad; Rodriguez, Alicia; Tahir, Urfa Bin; Jin, Fengliang

    2018-02-01

    Whenever gene expression is being examined, it is essential that a normalization process is carried out to eliminate non-biological variations. The use of reference genes, such as glyceraldehyde-3-phosphate dehydrogenase, actin, and ribosomal protein genes, is the usual method of choice for normalizing gene expression. Although reference genes are used to normalize target gene expression, a major problem is that the stability of these genes differs among tissues, developmental stages, species, and responses to abiotic factors. Therefore, the use and validation of multiple reference genes are required. This review discusses the reasons that why RT-qPCR has become the preferred method for validating results of gene expression profiles, the use of specific and non-specific dyes and the importance of use of primers and probes for qPCR as well as to discuss several statistical algorithms developed to help the validation of potential reference genes. The conflicts arising in the use of classical reference genes in gene normalization and their replacement with novel references are also discussed by citing the high stability and low stability of classical and novel reference genes under various biotic and abiotic experimental conditions by employing various methods applied for the reference genes amplification.

  11. Blue Light Modulates Murine Microglial Gene Expression in the Absence of Optogenetic Protein Expression

    PubMed Central

    Cheng, Kevin P.; Kiernan, Elizabeth A.; Eliceiri, Kevin W.; Williams, Justin C.; Watters, Jyoti J.

    2016-01-01

    Neural optogenetic applications over the past decade have steadily increased; however the effects of commonly used blue light paradigms on surrounding, non-optogenetic protein-expressing CNS cells are rarely considered, despite their simultaneous exposure. Here we report that blue light (450 nm) repetitively delivered in both long-duration boluses and rapid optogenetic bursts gene-specifically altered basal expression of inflammatory and neurotrophic genes in immortalized and primary murine wild type microglial cultures. In addition, blue light reduced pro-inflammatory gene expression in microglia activated with lipopolysaccharide. These results demonstrate previously unreported, off-target effects of blue light in cells not expressing optogenetic constructs. The unexpected gene modulatory effects of blue light on wild type CNS resident immune cells have novel and important implications for the neuro-optogenetic field. Further studies are needed to elucidate the molecular mechanisms and potential therapeutic utility of blue light modulation of the wild type CNS. PMID:26883795

  12. A whole-blood transcriptome meta-analysis identifies gene expression signatures of cigarette smoking

    PubMed Central

    Huan, Tianxiao; Joehanes, Roby; Schurmann, Claudia; Schramm, Katharina; Pilling, Luke C.; Peters, Marjolein J.; Mägi, Reedik; DeMeo, Dawn; O'Connor, George T.; Ferrucci, Luigi; Teumer, Alexander; Homuth, Georg; Biffar, Reiner; Völker, Uwe; Herder, Christian; Waldenberger, Melanie; Peters, Annette; Zeilinger, Sonja; Metspalu, Andres; Hofman, Albert; Uitterlinden, André G.; Hernandez, Dena G.; Singleton, Andrew B.; Bandinelli, Stefania; Munson, Peter J.; Lin, Honghuang; Benjamin, Emelia J.; Esko, Tõnu; Grabe, Hans J.; Prokisch, Holger; van Meurs, Joyce B.J.; Melzer, David; Levy, Daniel

    2016-01-01

    Abstract Cigarette smoking is a leading modifiable cause of death worldwide. We hypothesized that cigarette smoking induces extensive transcriptomic changes that lead to target-organ damage and smoking-related diseases. We performed a meta-analysis of transcriptome-wide gene expression using whole blood-derived RNA from 10,233 participants of European ancestry in six cohorts (including 1421 current and 3955 former smokers) to identify associations between smoking and altered gene expression levels. At a false discovery rate (FDR) <0.1, we identified 1270 differentially expressed genes in current vs. never smokers, and 39 genes in former vs. never smokers. Expression levels of 12 genes remained elevated up to 30 years after smoking cessation, suggesting that the molecular consequence of smoking may persist for decades. Gene ontology analysis revealed enrichment of smoking-related genes for activation of platelets and lymphocytes, immune response, and apoptosis. Many of the top smoking-related differentially expressed genes, including LRRN3 and GPR15, have DNA methylation loci in promoter regions that were recently reported to be hypomethylated among smokers. By linking differential gene expression with smoking-related disease phenotypes, we demonstrated that stroke and pulmonary function show enrichment for smoking-related gene expression signatures. Mediation analysis revealed the expression of several genes (e.g. ALAS2) to be putative mediators of the associations between smoking and inflammatory biomarkers (IL6 and C-reactive protein levels). Our transcriptomic study provides potential insights into the effects of cigarette smoking on gene expression in whole blood and their relations to smoking-related diseases. The results of such analyses may highlight attractive targets for treating or preventing smoking-related health effects. PMID:28158590

  13. Research Resource: Global Identification of Estrogen Receptor β Target Genes in Triple Negative Breast Cancer Cells

    PubMed Central

    Shanle, Erin K.; Zhao, Zibo; Hawse, John; Wisinski, Kari; Keles, Sunduz; Yuan, Ming

    2013-01-01

    Breast cancers that are negative for estrogen receptor α (ERα), progesterone receptor, and human epidermal growth factor receptor 2 are known as triple-negative breast cancers (TNBC). TNBCs are associated with an overall poor prognosis because they lack expression of therapeutic targets like ERα and are biologically more aggressive. A second estrogen receptor, ERβ, has been found to be expressed in 50% to 90% of ERα-negative breast cancers, and ERβ expression in TNBCs has been shown to correlate with improved disease-free survival and good prognosis. To elucidate the role of ERβ in regulating gene expression and cell proliferation in TNBC cells, the TNBC cell line MDA-MB-468 was engineered with inducible expression of full-length ERβ. In culture, ERβ expression inhibited cell growth by inducing a G1 cell cycle arrest, which was further enhanced by 17β-estradiol treatment. In xenografts, ERβ expression also inhibited tumor formation and growth, and 17β-estradiol treatment resulted in rapid tumor regression. Furthermore, genomic RNA sequencing identified both ligand-dependent and -independent ERβ target genes, some of which were also regulated by ERβ in other TNBC cell lines and correlated with ERβ expression in a cohort of TNBCs from the Cancer Genome Atlas Network. ERβ target genes were enriched in genes that regulate cell death and survival, cell movement, cell development, and growth and proliferation, as well as genes involved in the Wnt/β-catenin and the G1/S cell cycle phase checkpoint pathways. In addition to confirming the anti-proliferative effects of ERβ in TNBC cells, these data provide a comprehensive resource of ERβ target genes and suggest that ERβ may be targeted with ligands that can stimulate its growth inhibitory effects. PMID:23979844

  14. New target genes in endometrial tumors show a role for the estrogen-receptor pathway in microsatellite-unstable cancers.

    PubMed

    Ferreira, Ana M; Tuominen, Iina; Sousa, Sónia; Gerbens, Frans; van Dijk-Bos, Krista; Osinga, Jan; Kooi, Krista A; Sanjabi, Bahram; Esendam, Chris; Oliveira, Carla; Terpstra, Peter; Hardonk, Menno; van der Sluis, Tineke; Zazula, Monika; Stachura, Jerzy; van der Zee, Ate G; Hollema, Harry; Sijmons, Rolf H; Aaltonen, Lauri A; Seruca, Raquel; Hofstra, Robert M W; Westers, Helga

    2014-12-01

    Microsatellite instability (MSI) in tumors results in an accumulation of mutations in (target) genes. Previous studies suggest that the profile of target genes differs according to tumor type. This paper describes the first genome-wide search for target genes for mismatch repair-deficient endometrial cancers. Genes expressed in normal endometrium containing coding repeats were analyzed for mutations in tumors. We identified 44 possible genes of which seven are highly mutated (>15%). Some candidates were also found mutated in colorectal and gastric tumors. The most frequently mutated gene, NRIP1 encoding nuclear receptor-interacting protein 1, was silenced in an endometrial tumor cell line and expression microarray experiments were performed. Silencing of NRIP1 was associated with differences in the expression of several genes in the estrogen-receptor network. Furthermore, an enrichment of genes related to cell cycle (regulation) and replication was observed. We present a new profile of target genes, some of them tissue specific, whereas others seem to play a more general role in MSI tumors. The high-mutation frequency combined with the expression data suggest, for the first time, an involvement of NRIP1 in endometrial cancer development. © 2014 WILEY PERIODICALS, INC.

  15. Targeting MOG expression to dendritic cells delays onset of experimental autoimmune disease.

    PubMed

    Ko, Hyun-Ja; Chung, Jie-Yu; Nasa, Zeyad; Chan, James; Siatskas, Christopher; Toh, Ban-Hock; Alderuccio, Frank

    2011-05-01

    Haematopoietic stem cell (HSC) transfer coupled with gene therapy is a powerful approach to treating fatal diseases such as X-linked severe combined immunodeficiency. This ability to isolate and genetically manipulate HSCs also offers a strategy for inducing immune tolerance through ectopic expression of autoantigens. We have previously shown that retroviral transduction of bone marrow (BM) with vectors encoding the autoantigen, myelin oligodendrocyte glycoprotein (MOG), can prevent the induction of experimental autoimmune encephalomyelitis (EAE). However, ubiquitous cellular expression of autoantigen driven by retroviral promoters may not be the best approach for clinical translation and a targeted expression approach may be more acceptable. As BM-derived dendritic cells (DCs) play a major role in tolerance induction, we asked whether targeted expression of MOG, a target autoantigen in EAE, to DCs can promote tolerance induction and influence the development of EAE. Self-inactivating retroviral vectors incorporating the mouse CD11c promoter were generated and used to transduce mouse BM cells. Transplantation of gene-modified cells into irradiated recipients resulted in the generation of chimeric mice with transgene expression limited to DCs. Notably, chimeric mice transplanted with MOG-expressing BM cells manifest a significant delay in the development of EAE suggesting that targeted antigen expression to tolerogenic cell types may be a feasible approach to inducing antigen-specific tolerance.

  16. Preferential Allele Expression Analysis Identifies Shared Germline and Somatic Driver Genes in Advanced Ovarian Cancer

    PubMed Central

    Halabi, Najeeb M.; Martinez, Alejandra; Al-Farsi, Halema; Mery, Eliane; Puydenus, Laurence; Pujol, Pascal; Khalak, Hanif G.; McLurcan, Cameron; Ferron, Gwenael; Querleu, Denis; Al-Azwani, Iman; Al-Dous, Eman; Mohamoud, Yasmin A.; Malek, Joel A.; Rafii, Arash

    2016-01-01

    Identifying genes where a variant allele is preferentially expressed in tumors could lead to a better understanding of cancer biology and optimization of targeted therapy. However, tumor sample heterogeneity complicates standard approaches for detecting preferential allele expression. We therefore developed a novel approach combining genome and transcriptome sequencing data from the same sample that corrects for sample heterogeneity and identifies significant preferentially expressed alleles. We applied this analysis to epithelial ovarian cancer samples consisting of matched primary ovary and peritoneum and lymph node metastasis. We find that preferentially expressed variant alleles include germline and somatic variants, are shared at a relatively high frequency between patients, and are in gene networks known to be involved in cancer processes. Analysis at a patient level identifies patient-specific preferentially expressed alleles in genes that are targets for known drugs. Analysis at a site level identifies patterns of site specific preferential allele expression with similar pathways being impacted in the primary and metastasis sites. We conclude that genes with preferentially expressed variant alleles can act as cancer drivers and that targeting those genes could lead to new therapeutic strategies. PMID:26735499

  17. Bioinformatic identification and expression analysis of banana microRNAs and their targets.

    PubMed

    Chai, Juan; Feng, Renjun; Shi, Hourui; Ren, Mengyun; Zhang, Yindong; Wang, Jingyi

    2015-01-01

    MicroRNAs (miRNAs) represent a class of endogenous non-coding small RNAs that play important roles in multiple biological processes by degrading targeted mRNAs or repressing mRNA translation. Thousands of miRNAs have been identified in many plant species, whereas only a limited number of miRNAs have been predicted in M. acuminata (A genome) and M. balbisiana (B genome). Here, previously known plant miRNAs were BLASTed against the Expressed Sequence Tag (EST) and Genomic Survey Sequence (GSS), a database of banana genes. A total of 32 potential miRNAs belonging to 13 miRNAs families were detected using a range of filtering criteria. 244 miRNA:target pairs were subsequently predicted, most of which encode transcription factors or enzymes that participate in the regulation of development, growth, metabolism, and other physiological processes. In order to validate the predicted miRNAs and the mutual relationship between miRNAs and their target genes, qRT-PCR was applied to detect the tissue-specific expression levels of 12 putative miRNAs and 6 target genes in roots, leaves, flowers, and fruits. This study provides some important information about banana pre-miRNAs, mature miRNAs, and miRNA target genes and these findings can be applied to future research of miRNA functions.

  18. SOX11 identified by target gene evaluation of miRNAs differentially expressed in focal and non-focal brain tissue of therapy-resistant epilepsy patients.

    PubMed

    Haenisch, Sierk; Zhao, Yi; Chhibber, Aparna; Kaiboriboon, Kitti; Do, Lynn V; Vogelgesang, Silke; Barbaro, Nicholas M; Alldredge, Brian K; Lowenstein, Daniel H; Cascorbi, Ingolf; Kroetz, Deanna L

    2015-05-01

    MicroRNAs (miRNAs) are small non-coding RNAs that post-transcriptionally control the expression of their target genes via RNA interference. There is increasing evidence that expression of miRNAs is dysregulated in neuronal disorders, including epilepsy, a chronic neurological disorder characterized by spontaneous recurrent seizures. Mesial temporal lobe epilepsy (MTLE) is a common type of focal epilepsy in which disease-induced abnormalities of hippocampal neurogenesis in the subgranular zone as well as gliosis and neuronal cell loss in the cornu ammonis area are reported. We hypothesized that in MTLE altered miRNA-mediated regulation of target genes could be involved in hippocampal cell remodeling. A miRNA screen was performed in hippocampal focal and non-focal brain tissue samples obtained from the temporal neocortex (both n=8) of MTLE patients. Out of 215 detected miRNAs, two were differentially expressed (hsa-miR-34c-5p: mean increase of 5.7 fold (p=0.014), hsa-miR-212-3p: mean decrease of 76.9% (p=0.0014)). After in-silico target gene analysis and filtering, reporter gene assays confirmed RNA interference for hsa-miR-34c-5p with 3'-UTR sequences of GABRA3, GRM7 and GABBR2 and for hsa-miR-212-3p with 3'-UTR sequences of SOX11, MECP2, ADCY1 and ABCG2. Reporter gene assays with mutated 3'-UTR sequences of the transcription factor SOX11 identified two different binding sites for hsa-miR-212-3p and its primary transcript partner hsa-miR-132-3p. Additionally, there was an inverse time-dependent expression of Sox11 and miR-212-3p as well as miR-132-3p in rat neonatal cortical neurons. Transfection of neurons with anti-miRs for miR-212-3p and miR-132-3p suggest that both miRNAs work synergistically to control Sox11 expression. Taken together, these results suggest that differential miRNA expression in neurons could contribute to an altered function of the transcription factor SOX11 and other genes in the setting of epilepsy, resulting not only in impaired neural

  19. Epidrug mediated re-expression of miRNA targeting the HMGA transcripts in pituitary cells.

    PubMed

    Kitchen, Mark O; Yacqub-Usman, Kiren; Emes, Richard D; Richardson, Alan; Clayton, Richard N; Farrell, William E

    2015-10-01

    Transgenic mice overexpressing the high mobility group A (HMGA) genes, Hmga1 or Hmga2 develop pituitary tumours and their overexpression is also a frequent finding in human pituitary adenomas. In some cases, increased expression of HMGA2 but not that of HMGA1 is consequent to genetic perturbations. However, recent studies show that down-regulation of microRNA (miRNA), that contemporaneously target the HMGA1 and HMGA2 transcripts, are associated with their overexpression. In a cohort of primary pituitary adenoma we determine the impact of epigenetic modifications on the expression of HMGA-targeting miRNA. For these miRNAs, chromatin immunoprecipitations showed that transcript down-regulation is correlated with histone tail modifications associated with condensed silenced genes. The functional impact of epigenetic modification on miRNA expression was determined in the rodent pituitary cell line, GH3. In these cells, histone tail, miRNA-associated, modifications were similar to those apparent in human adenoma and likely account for their repression. Indeed, challenge of GH3 cells with the epidrugs, zebularine and TSA, led to enrichment of the histone modification, H3K9Ac, associated with active genes, and depletion of the modification, H3K27me3, associated with silent genes and re-expression of HMGA-targeting miRNA. Moreover, epidrugs challenges were also associated with a concomitant decrease in hmga1 transcript and protein levels and concurrent increase in bmp-4 expression. These findings show that the inverse relationship between HMGA expression and targeting miRNA is reversible through epidrug interventions. In addition to showing a mechanistic link between epigenetic modifications and miRNA expression these findings underscore their potential as therapeutic targets in this and other diseases.

  20. Expression of Hormonal Carcinogenesis Genes and Related Regulatory microRNAs in Uterus and Ovaries of DDT-Treated Female Rats.

    PubMed

    Kalinina, T S; Kononchuk, V V; Gulyaeva, L F

    2017-10-01

    The insecticide dichlorodiphenyltrichloroethane (DDT) is a nonmutagenic xenobiotic compound able to exert estrogen-like effects resulting in activation of estrogen receptor-α (ERα) followed by changed expression of its downstream target genes. In addition, studies performed over recent years suggest that DDT may also influence expression of microRNAs. However, an impact of DDT on expression of ER, microRNAs, and related target genes has not been fully elucidated. Here, using real-time PCR, we assessed changes in expression of key genes involved in hormonal carcinogenesis as well as potentially related regulatory oncogenic/tumor suppressor microRNAs and their target genes in the uterus and ovaries of female Wistar rats during single and chronic multiple-dose DDT exposure. We found that applying DDT results in altered expression of microRNAs-221, -222, -205, -126a, and -429, their target genes (Pten, Dicer1), as well as genes involved in hormonal carcinogenesis (Esr1, Pgr, Ccnd1, Cyp19a1). Notably, Cyp19a1 expression seems to be also regulated by microRNAs-221, -222, and -205. The data suggest that epigenetic effects induced by DDT as a potential carcinogen may be based on at least two mechanisms: (i) activation of ERα followed by altered expression of the target genes encoding receptor Pgr and Ccnd1 as well as impaired expression of Cyp19a1, affecting, thereby, cell hormone balance; and (ii) changed expression of microRNAs resulting in impaired expression of related target genes including reduced level of Cyp19a1 mRNA.

  1. MicroRNA-124-3p expression and its prospective functional pathways in hepatocellular carcinoma: A quantitative polymerase chain reaction, gene expression omnibus and bioinformatics study.

    PubMed

    He, Rong-Quan; Yang, Xia; Liang, Liang; Chen, Gang; Ma, Jie

    2018-04-01

    The present study aimed to explore the potential clinical significance of microRNA (miR)-124-3p expression in the hepatocarcinogenesis and development of hepatocellular carcinoma (HCC), as well as the potential target genes of functional HCC pathways. Reverse transcription-quantitative polymerase chain reaction was performed to evaluate the expression of miR-124-3p in 101 HCC and adjacent non-cancerous tissue samples. Additionally, the association between miR-124-3p expression and clinical parameters was also analyzed. Differentially expressed genes identified following miR-124-3p transfection, the prospective target genes predicted in silico and the key genes of HCC obtained from Natural Language Processing (NLP) were integrated to obtain potential target genes of miR-124-3p in HCC. Relevant signaling pathways were assessed with protein-protein interaction (PPI) networks, Gene Ontology (GO) enrichment analysis, Kyoto Encyclopedia of Genes and Genomes (KEGG) and Protein Annotation Through Evolutionary Relationships (PANTHER) pathway enrichment analysis. miR-124-3p expression was significantly reduced in HCC tissues compared with expression in adjacent non-cancerous liver tissues. In HCC, miR-124-3p was demonstrated to be associated with clinical stage. The mean survival time of the low miR-124-3p expression group was reduced compared with that of the high expression group. A total of 132 genes overlapped from differentially expressed genes, miR-124-3p predicted target genes and NLP identified genes. PPI network construction revealed a total of 109 nodes and 386 edges, and 20 key genes were identified. The major enriched terms of three GO categories included regulation of cell proliferation, positive regulation of cellular biosynthetic processes, cell leading edge, cytosol and cell projection, protein kinase activity, transcription activator activity and enzyme binding. KEGG analysis revealed pancreatic cancer, prostate cancer and non-small cell lung cancer as the

  2. JC virus induces altered patterns of cellular gene expression: Interferon-inducible genes as major transcriptional targets

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Verma, Saguna; Ziegler, Katja; Ananthula, Praveen

    2006-02-20

    Human polyomavirus JC (JCV) infects 80% of the population worldwide. Primary infection, typically occurring during childhood, is asymptomatic in immunocompetent individuals and results in lifelong latency and persistent infection. However, among the severely immunocompromised, JCV may cause a fatal demyelinating disease, progressive multifocal leukoencephalopathy (PML). Virus-host interactions influencing persistence and pathogenicity are not well understood, although significant regulation of JCV activity is thought to occur at the level of transcription. Regulation of the JCV early and late promoters during the lytic cycle is a complex event that requires participation of both viral and cellular factors. We have used cDNA microarraymore » technology to analyze global alterations in gene expression in JCV-permissive primary human fetal glial cells (PHFG). Expression of more than 400 cellular genes was altered, including many that influence cell proliferation, cell communication and interferon (IFN)-mediated host defense responses. Genes in the latter category included signal transducer and activator of transcription 1 (STAT1), interferon stimulating gene 56 (ISG56), myxovirus resistance 1 (MxA), 2'5'-oligoadenylate synthetase (OAS), and cig5. The expression of these genes was further confirmed in JCV-infected PHFG cells and the human glioblastoma cell line U87MG to ensure the specificity of JCV in inducing this strong antiviral response. Results obtained by real-time RT-PCR and Western blot analyses supported the microarray data and provide temporal information related to virus-induced changes in the IFN response pathway. Our data indicate that the induction of an antiviral response may be one of the cellular factors regulating/controlling JCV replication in immunocompetent hosts and therefore constraining the development of PML.« less

  3. Global gene expression profiles in brain regions reflecting abnormal neuronal and glial functions targeting myelin sheaths after 28-day exposure to cuprizone in rats

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Abe, Hajime

    Both developmental and postpubertal cuprizone (CPZ) exposure impairs hippocampal neurogenesis in rats. We previously found that developmental CPZ exposure alters the expression of genes related to neurogenesis, myelination, and synaptic transmission in specific brain regions of offspring. Here, we examined neuronal and glial toxicity profiles in response to postpubertal CPZ exposure by using expression microarray analysis in the hippocampal dentate gyrus, corpus callosum, cerebral cortex, and cerebellar vermis of 5-week-old male rats exposed to 0, 120, and 600 mg/kg CPZ for 28 days. Genes showing transcript upregulation were subjected to immunohistochemical analysis. We found transcript expression alterations at 600 mg/kgmore » for genes related to synaptic transmission, Ache and Prima1, and cell cycle regulation, Tfap4 and Cdkn1a, in the dentate gyrus, which showed aberrant neurogenesis in the subgranular zone. This dose downregulated myelination-related genes in multiple brain regions, whereas KLOTHO{sup +} oligodendrocyte density was decreased only in the corpus callosum. The corpus callosum showed an increase in transcript levels for inflammatory response-related genes and in the number of CD68{sup +} microglia, MT{sup +} astrocytes, and TUNEL{sup +} apoptotic cells. These results suggest that postpubertal CPZ exposure targets synaptic transmission and cell cycle regulation to affect neurogenesis in the dentate gyrus. CPZ suppressed myelination in multiple brain regions and KLOTHO-mediated oligodendrocyte maturation only in the corpus callosum. The increased number of CD68{sup +} microglia, MT{sup +} astrocytes, and TUNEL{sup +} apoptotic cells in the corpus callosum may be involved in the induction of KLOTHO{sup +} oligodendrocyte death and be a protective mechanism against myelin damage following CPZ exposure. - Highlights: • Target gene expression profiles were examined in rats after 28-day CPZ exposure. • Multiple brain region-specific global gene

  4. Targeted gene deletion of miRNAs in mice by TALEN system.

    PubMed

    Takada, Shuji; Sato, Tempei; Ito, Yoshiaki; Yamashita, Satoshi; Kato, Tomoko; Kawasumi, Miyuri; Kanai-Azuma, Masami; Igarashi, Arisa; Kato, Tomomi; Tamano, Moe; Asahara, Hiroshi

    2013-01-01

    Mice are among the most valuable model animal species with an enormous amount of heritage in genetic modification studies. However, targeting genes in mice is sometimes difficult, especially for small genes, such as microRNAs (miRNAs) and targeting genes in repeat sequences. Here we optimized the application of TALEN system for mice and successfully obtained gene targeting technique in mice for intergenic region and series of microRNAs. Microinjection of synthesized RNA of TALEN targeting each gene in one cell stage of embryo was carried out and injected oocytes were transferred into pseudopregnant ICR female mice, producing a high success rate of the targeted deletion of miRNA genes. In our condition, TALEN RNA without poly(A) tail worked better than that of with poly(A) tail. This mutated allele in miRNA was transmitted to the next generation, suggesting the successful germ line transmission of this targeting method. Consistent with our notion of miRNAs maturation mechanism, in homozygous mutant mice of miR-10a, the non- mutated strand of miRNAs expression was completely diminished. This method will lead us to expand and accelerate our genetic research using mice in a high throughput way.

  5. [Preliminary analysis of retinal gene expression profile of diabetic rat].

    PubMed

    Mei, Yan; Zhou, Hong-ying; Xiang, Tao; Lu, You-guang; Li, Ai-dong; Tang, En-jie; Yang, Hui-jun

    2005-10-01

    Establishing the retinal gene expression profiles of non-diabetic rat and diabetic rat and comparing the profiles in order to analyze the possible genes related with diabetic retinopathy. The whole retinal transcriptional fragments of non-diabetic rat and 8-week diabetic rat were obtained by restriction fragments differential display-PCR (RFDD-PCR). Bioinformatic analysis of retinal gene expression was performed using soft wares, including Fragment Analysis. After comparison of the expression profiles, the related gene fragments of diabetic retinopathy were initially selected as the target gene of further approach. A total of 3639 significant fragments were obtained. By means of more than 3-fold contrast of fluorescent intensity as the differential expression standard, the authors got 840 differential fragments, accounting for 23.08% of the expressed numbers and including 5 visual related genes, 13 excitatory neruotransmitter genes and 3 inhibitory neurotransmitter genes. At the 8th week, the expression of Rhodopsin kinase, beta-arrestin, Phosducinìrod photoreceptor cGMP-gated channel and Rpe65 as well as iGlu R1-4 were down-regulated. mGluRs and GABA-Rs were all up-regulated, whereas the expression of GlyR was unchanged. These results prompt again that the changes in retinal nervous layer of rat have occurred at an early stage of diabetes. The genes expression pattern of visual related genes and excitatory and inhibitory neurotransmitters in rat diabetic retina have been involved in neuro-dysfunctions of diabetic retina.

  6. Deletion and Gene Expression Analyses Define the Paxilline Biosynthetic Gene Cluster in Penicillium paxilli

    PubMed Central

    Scott, Barry; Young, Carolyn A.; Saikia, Sanjay; McMillan, Lisa K.; Monahan, Brendon J.; Koulman, Albert; Astin, Jonathan; Eaton, Carla J.; Bryant, Andrea; Wrenn, Ruth E.; Finch, Sarah C.; Tapper, Brian A.; Parker, Emily J.; Jameson, Geoffrey B.

    2013-01-01

    The indole-diterpene paxilline is an abundant secondary metabolite synthesized by Penicillium paxilli. In total, 21 genes have been identified at the PAX locus of which six have been previously confirmed to have a functional role in paxilline biosynthesis. A combination of bioinformatics, gene expression and targeted gene replacement analyses were used to define the boundaries of the PAX gene cluster. Targeted gene replacement identified seven genes, paxG, paxA, paxM, paxB, paxC, paxP and paxQ that were all required for paxilline production, with one additional gene, paxD, required for regular prenylation of the indole ring post paxilline synthesis. The two putative transcription factors, PP104 and PP105, were not co-regulated with the pax genes and based on targeted gene replacement, including the double knockout, did not have a role in paxilline production. The relationship of indole dimethylallyl transferases involved in prenylation of indole-diterpenes such as paxilline or lolitrem B, can be found as two disparate clades, not supported by prenylation type (e.g., regular or reverse). This paper provides insight into the P. paxilli indole-diterpene locus and reviews the recent advances identified in paxilline biosynthesis. PMID:23949005

  7. Turning the gene tap off; implications of regulating gene expression for cancer therapeutics

    PubMed Central

    Curtin, James F.; Candolfi, Marianela; Xiong, Weidong; Lowenstein, Pedro R.; Castro, Maria G.

    2008-01-01

    Cancer poses a tremendous therapeutic challenge worldwide, highlighting the critical need for developing novel therapeutics. A promising cancer treatment modality is gene therapy, which is a form of molecular medicine designed to introduce into target cells genetic material with therapeutic intent. Anticancer gene therapy strategies currently used in preclinical models, and in some cases in the clinic, include proapoptotic genes, oncolytic/replicative vectors, conditional cytotoxic approaches, inhibition of angiogenesis, inhibition of growth factor signaling, inactivation of oncogenes, inhibition of tumor invasion and stimulation of the immune system. The translation of these novel therapeutic modalities from the preclinical setting to the clinic has been driven by encouraging preclinical efficacy data and advances in gene delivery technologies. One area of intense research involves the ability to accurately regulate the levels of therapeutic gene expression to achieve enhanced efficacy and provide the capability to switch gene expression off completely if adverse side effects should arise. This feature could also be implemented to switch gene expression off when a successful therapeutic outcome ensues. Here, we will review recent developments related to the engineering of transcriptional switches within gene delivery systems, which could be implemented in clinical gene therapy applications directed at the treatment of cancer. PMID:18347132

  8. Genome-wide identification and characterization of microRNA genes and their targets in flax (Linum usitatissimum): Characterization of flax miRNA genes.

    PubMed

    Barvkar, Vitthal T; Pardeshi, Varsha C; Kale, Sandip M; Qiu, Shuqing; Rollins, Meaghen; Datla, Raju; Gupta, Vidya S; Kadoo, Narendra Y

    2013-04-01

    MicroRNAs (miRNAs) are small (20-24 nucleotide long) endogenous regulatory RNAs that play important roles in plant growth and development. They regulate gene expression at the post-transcriptional level by translational repression or target degradation and gene silencing. In this study, we identified 116 conserved miRNAs belonging to 23 families from the flax (Linum usitatissimum L.) genome using a computational approach. The precursor miRNAs varied in length; while most of the mature miRNAs were 21 nucleotide long, intergenic and showed conserved signatures of RNA polymerase II transcripts in their upstream regions. Promoter region analysis of the flax miRNA genes indicated prevalence of MYB transcription factor binding sites. Four miRNA gene clusters containing members of three phylogenetic groups were identified. Further, 142 target genes were predicted for these miRNAs and most of these represent transcriptional regulators. The miRNA encoding genes were expressed in diverse tissues as determined by digital expression analysis as well as real-time PCR. The expression of fourteen miRNAs and nine target genes was independently validated using the quantitative reverse transcription PCR (qRT-PCR). This study suggests that a large number of conserved plant miRNAs are also found in flax and these may play important roles in growth and development of flax.

  9. Oxygen and tissue culture affect placental gene expression.

    PubMed

    Brew, O; Sullivan, M H F

    2017-07-01

    Placental explant culture is an important model for studying placental development and functions. We investigated the differences in placental gene expression in response to tissue culture, atmospheric and physiologic oxygen concentrations. Placental explants were collected from normal term (38-39 weeks of gestation) placentae with no previous uterine contractile activity. Placental transcriptomic expressions were evaluated with GeneChip ® Human Genome U133 Plus 2.0 arrays (Affymetrix). We uncovered sub-sets of genes that regulate response to stress, induction of apoptosis programmed cell death, mis-regulation of cell growth, proliferation, cell morphogenesis, tissue viability, and protection from apoptosis in cultured placental explants. We also identified a sub-set of genes with highly unstable pattern of expression after exposure to tissue culture. Tissue culture irrespective of oxygen concentration induced dichotomous increase in significant gene expression and increased enrichment of significant pathways and transcription factor targets (TFTs) including HIF1A. The effect was exacerbated by culture at atmospheric oxygen concentration, where further up-regulation of TFTs including PPARA, CEBPD, HOXA9 and down-regulated TFTs such as JUND/FOS suggest intrinsic heightened key biological and metabolic mechanisms such as glucose use, lipid biosynthesis, protein metabolism; apoptosis, inflammatory responses; and diminished trophoblast proliferation, differentiation, invasion, regeneration, and viability. These findings demonstrate that gene expression patterns differ between pre-culture and cultured explants, and the gene expression of explants cultured at atmospheric oxygen concentration favours stressed, pro-inflammatory and increased apoptotic transcriptomic response. Copyright © 2017 Elsevier Ltd. All rights reserved.

  10. Mining, identification and function analysis of microRNAs and target genes in peanut (Arachis hypogaea L.).

    PubMed

    Zhang, Tingting; Hu, Shuhao; Yan, Caixia; Li, Chunjuan; Zhao, Xiaobo; Wan, Shubo; Shan, Shihua

    2017-02-01

    In the present investigation, a total of 60 conserved peanut (Arachis hypogaea L.) microRNA (miRNA) sequences, belonging to 16 families, were identified using bioinformatics methods. There were 392 target gene sequences, identified from 58 miRNAs with Target-align software and BLASTx analyses. Gene Ontology (GO) functional analysis suggested that these target genes were involved in mediating peanut growth and development, signal transduction and stress resistance. There were 55 miRNA sequences, verified employing a poly (A) tailing test, with a success rate of up to 91.67%. Twenty peanut target gene sequences were randomly selected, and the 5' rapid amplification of the cDNA ends (5'-RACE) method were used to validate the cleavage sites of these target genes. Of these, 14 (70%) peanut miRNA targets were verified by means of gel electrophoresis, cloning and sequencing. Furthermore, functional analysis and homologous sequence retrieval were conducted for target gene sequences, and 26 target genes were chosen as the objects for stress resistance experimental study. Real-time fluorescence quantitative PCR (qRT-PCR) technology was applied to measure the expression level of resistance-associated miRNAs and their target genes in peanut exposed to Aspergillus flavus (A. flavus) infection and drought stress, respectively. In consequence, 5 groups of miRNAs & targets were found accorded with the mode of miRNA negatively controlling the expression of target genes. This study, preliminarily determined the biological functions of some resistance-associated miRNAs and their target genes in peanut. Copyright © 2016 Elsevier Masson SAS. All rights reserved.

  11. [Inhibiting target gene expression and controlling growth of Epstein-Barr virus transformed cells by antisense RNA transcripts].

    PubMed

    Chen, Jian-jing; Raab-Traub, Nancy; Yao, Qing-yun; Zhang, Feng; Huang, Mei-ling; Kuang, Zhu-ji; Zhang, Xiao-shi; Ye, Yan-li; Gu, Li

    2002-01-01

    The latent membrane protein gene (LMP) of Epstein-Barr virus (EBV) was thought to play an important role in the carcinogenesis of nasopharyngeal carcinoma (NPC). In this study, the authors investigated the effects of antisense RNA (AsRNA) on LMP for down regulating at the target gene over expression in EBV transformed lymphoid cells, and set up an antisense system to inhibit LMP expression. Constructing the single strand antisense transcription system in vitro, the authors have got large amount of AsRNA. Designing and setting up an antisense tracing system in situ (ATSIS), the authors could observe the living particles of AsRNA/sense RNA duplex dimer. With time lapse phase-contrast microscopy, the agglutination phenotype on living cells was easily detected by MTT test, the inhibition rate on EBV transformed cells was calculated. LMP 1.9 fragment ligated into pGEM vector in reverse orientation have been constructed and produced a plentiful amount of AsLMPmRNA which could incorporated into both B95-8 and C1936 cell lines by endophagocytosis and formed the duplex dimer of As/Sense RNA. This particles have been visualized in situ when labelling 35S isotope by ATSIS. When AsLMPmRNA acted as agents for specific inhibition to LMP over expression, the transform phenotype of cell agglutination have been suppressed and MTT particle formatin was apparently reduced both two EBV tansformed cell lines. AsLMPmRNA targets at sense strand have a high effectiveness of down-regulation on EBV-LMP overexpression. This down regulating function of LMP and growth inhibition on transformed cell is demonstrated by the antisenes tracing system in situ (ATSIS). The results provide a clue to overcome the latent EBV infection in human bodies all living long time and to prevent it inducing NPC in high incidence area by antisense strategies.

  12. Selection of reliable reference genes for gene expression studies in Trichoderma afroharzianum LTR-2 under oxalic acid stress.

    PubMed

    Lyu, Yuping; Wu, Xiaoqing; Ren, He; Zhou, Fangyuan; Zhou, Hongzi; Zhang, Xinjian; Yang, Hetong

    2017-10-01

    An appropriate reference gene is required to get reliable results from gene expression analysis by quantitative real-time reverse transcription PCR (qRT-PCR). In order to identify stable and reliable reference genes in Trichoderma afroharzianum under oxalic acid (OA) stress, six commonly used housekeeping genes, i.e., elongation factor 1, ubiquitin, ubiquitin-conjugating enzyme, glyceraldehyde-3-phosphate dehydrogenase, α-tubulin, actin, from the effective biocontrol isolate T. afroharzianum strain LTR-2 were tested for their expression during growth in liquid culture amended with OA. Four in silico programs (comparative ΔCt, NormFinder, geNorm and BestKeeper) were used to evaluate the expression stabilities of six candidate reference genes. The elongation factor 1 gene EF-1 was identified as the most stably expressed reference gene, and was used as the normalizer to quantify the expression level of the oxalate decarboxylase coding gene OXDC in T. afroharzianum strain LTR-2 under OA stress. The result showed that the expression of OXDC was significantly up-regulated as expected. This study provides an effective method to quantify expression changes of target genes in T. afroharzianum under OA stress. Copyright © 2017 Elsevier B.V. All rights reserved.

  13. Analysis of differentially co-expressed genes based on microarray data of hepatocellular carcinoma.

    PubMed

    Wang, Y; Jiang, T; Li, Z; Lu, L; Zhang, R; Zhang, D; Wang, X; Tan, J

    2017-01-01

    Hepatocellular carcinoma (HCC) is the third leading cause of cancer related death worldwide. Although great progress in diagnosis and management of HCC have been made, the exact molecular mechanisms remain poorly understood. The study aims to identify potential biomarkers for HCC progression, mainly at transcription level. In this study, chip data GSE 29721 was utilized, which contains 10 HCC samples and 10 normal adjacent tissue samples. Differentially expressed genes (DEGs) between two sample types were selected by t-test method. Following, the differentially co-expressed genes (DCGs) and differentially co-expressed Links (DCLs) were identified by DCGL package in R with the threshold of q < 0.25. Afterwards, pathway enrichment analysis of the DCGs was carried out by DAVID. Then, DCLs were mapped to TRANSFAC database to reveal associations between relevant transcriptional factors (TFs) and their target genes. Quantitative real-time RT-PCR was performed for TFs or genes of interest. As a result, a total of 388 DCGs and 35,771 DCLs were obtained. The predominant pathways enriched by these genes were Cytokine-cytokine receptor interaction, ECM-receptor interaction and TGF-β signaling pathway. Three TF-target interactions, LEF1-NCAM1, EGR1-FN1 and FOS-MT2A were predicted. Compared with control, expressions of the TF genes EGR1, FOS and ETS2 were all up-regulated in the HCC cell line, HepG2; while LEF1 was down-regulated. Except NCAM1, all the target genes were up-regulated in HepG2. Our findings suggest these TFs and genes might play important roles in the pathogenesis of HCC and may be used as therapeutic targets for HCC management.

  14. Magnetic nanoparticles for targeted therapeutic gene delivery and magnetic-inducing heating on hepatoma

    NASA Astrophysics Data System (ADS)

    Yuan, Chenyan; An, Yanli; Zhang, Jia; Li, Hongbo; Zhang, Hao; Wang, Ling; Zhang, Dongsheng

    2014-08-01

    Gene therapy holds great promise for treating cancers, but their clinical applications are being hampered due to uncontrolled gene delivery and expression. To develop a targeted, safe and efficient tumor therapy system, we constructed a tissue-specific suicide gene delivery system by using magnetic nanoparticles (MNPs) as carriers for the combination of gene therapy and hyperthermia on hepatoma. The suicide gene was hepatoma-targeted and hypoxia-enhanced, and the MNPs possessed the ability to elevate temperature to the effective range for tumor hyperthermia as imposed on an alternating magnetic field (AMF). The tumoricidal effects of targeted gene therapy associated with hyperthermia were evaluated in vitro and in vivo. The experiment demonstrated that hyperthermia combined with a targeted gene therapy system proffer an effective tool for tumor therapy with high selectivity and the synergistic effect of hepatoma suppression.

  15. Gene Architectures that Minimize Cost of Gene Expression.

    PubMed

    Frumkin, Idan; Schirman, Dvir; Rotman, Aviv; Li, Fangfei; Zahavi, Liron; Mordret, Ernest; Asraf, Omer; Wu, Song; Levy, Sasha F; Pilpel, Yitzhak

    2017-01-05

    Gene expression burdens cells by consuming resources and energy. While numerous studies have investigated regulation of expression level, little is known about gene design elements that govern expression costs. Here, we ask how cells minimize production costs while maintaining a given protein expression level and whether there are gene architectures that optimize this process. We measured fitness of ∼14,000 E. coli strains, each expressing a reporter gene with a unique 5' architecture. By comparing cost-effective and ineffective architectures, we found that cost per protein molecule could be minimized by lowering transcription levels, regulating translation speeds, and utilizing amino acids that are cheap to synthesize and that are less hydrophobic. We then examined natural E. coli genes and found that highly expressed genes have evolved more forcefully to minimize costs associated with their expression. Our study thus elucidates gene design elements that improve the economy of protein expression in natural and heterologous systems. Copyright © 2017 Elsevier Inc. All rights reserved.

  16. Balancing gene expression without library construction via a reusable sRNA pool.

    PubMed

    Ghodasara, Amar; Voigt, Christopher A

    2017-07-27

    Balancing protein expression is critical when optimizing genetic systems. Typically, this requires library construction to vary the genetic parts controlling each gene, which can be expensive and time-consuming. Here, we develop sRNAs corresponding to 15nt 'target' sequences that can be inserted upstream of a gene. The targeted gene can be repressed from 1.6- to 87-fold by controlling sRNA expression using promoters of different strength. A pool is built where six sRNAs are placed under the control of 16 promoters that span a ∼103-fold range of strengths, yielding ∼107 combinations. This pool can simultaneously optimize up to six genes in a system. This requires building only a single system-specific construct by placing a target sequence upstream of each gene and transforming it with the pre-built sRNA pool. The resulting library is screened and the top clone is sequenced to determine the promoter controlling each sRNA, from which the fold-repression of the genes can be inferred. The system is then rebuilt by rationally selecting parts that implement the optimal expression of each gene. We demonstrate the versatility of this approach by using the same pool to optimize a metabolic pathway (β-carotene) and genetic circuit (XNOR logic gate). © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

  17. NFI Transcription Factors Interact with FOXA1 to Regulate Prostate-Specific Gene Expression

    PubMed Central

    Elliott, Amicia D.; DeGraff, David J.; Anderson, Philip D.; Anumanthan, Govindaraj; Yamashita, Hironobu; Sun, Qian; Friedman, David B.; Hachey, David L.; Yu, Xiuping; Sheehan, Jonathan H.; Ahn, Jung-Mo; Raj, Ganesh V.; Piston, David W.; Gronostajski, Richard M.; Matusik, Robert J.

    2014-01-01

    Androgen receptor (AR) action throughout prostate development and in maintenance of the prostatic epithelium is partly controlled by interactions between AR and forkhead box (FOX) transcription factors, particularly FOXA1. We sought to identity additional FOXA1 binding partners that may mediate prostate-specific gene expression. Here we identify the nuclear factor I (NFI) family of transcription factors as novel FOXA1 binding proteins. All four family members (NFIA, NFIB, NFIC, and NFIX) can interact with FOXA1, and knockdown studies in androgen-dependent LNCaP cells determined that modulating expression of NFI family members results in changes in AR target gene expression. This effect is probably mediated by binding of NFI family members to AR target gene promoters, because chromatin immunoprecipitation (ChIP) studies found that NFIB bound to the prostate-specific antigen enhancer. Förster resonance energy transfer studies revealed that FOXA1 is capable of bringing AR and NFIX into proximity, indicating that FOXA1 facilitates the AR and NFI interaction by bridging the complex. To determine the extent to which NFI family members regulate AR/FOXA1 target genes, motif analysis of publicly available data for ChIP followed by sequencing was undertaken. This analysis revealed that 34.4% of peaks bound by AR and FOXA1 contain NFI binding sites. Validation of 8 of these peaks by ChIP revealed that NFI family members can bind 6 of these predicted genomic elements, and 4 of the 8 associated genes undergo gene expression changes as a result of individual NFI knockdown. These observations suggest that NFI regulation of FOXA1/AR action is a frequent event, with individual family members playing distinct roles in AR target gene expression. PMID:24801505

  18. STAT3 or USF2 Contributes to HIF Target Gene Specificity

    PubMed Central

    Pawlus, Matthew R.; Wang, Liyi; Murakami, Aya; Dai, Guanhai; Hu, Cheng-Jun

    2013-01-01

    The HIF1- and HIF2-mediated transcriptional responses play critical roles in solid tumor progression. Despite significant similarities, including their binding to promoters of both HIF1 and HIF2 target genes, HIF1 and HIF2 proteins activate unique subsets of target genes under hypoxia. The mechanism for HIF target gene specificity has remained unclear. Using siRNA or inhibitor, we previously reported that STAT3 or USF2 is specifically required for activation of endogenous HIF1 or HIF2 target genes. In this study, using reporter gene assays and chromatin immuno-precipitation, we find that STAT3 or USF2 exhibits specific binding to the promoters of HIF1 or HIF2 target genes respectively even when over-expressed. Functionally, HIF1α interacts with STAT3 to activate HIF1 target gene promoters in a HIF1α HLH/PAS and N-TAD dependent manner while HIF2α interacts with USF2 to activate HIF2 target gene promoters in a HIF2α N-TAD dependent manner. Physically, HIF1α HLH and PAS domains are required for its interaction with STAT3 while both N- and C-TADs of HIF2α are involved in physical interaction with USF2. Importantly, addition of functional USF2 binding sites into a HIF1 target gene promoter increases the basal activity of the promoter as well as its response to HIF2+USF2 activation while replacing HIF binding site with HBS from a HIF2 target gene does not change the specificity of the reporter gene. Importantly, RNA Pol II on HIF1 or HIF2 target genes is primarily associated with HIF1α or HIF2α in a STAT3 or USF2 dependent manner. Thus, we demonstrate here for the first time that HIF target gene specificity is achieved by HIF transcription partners that are required for HIF target gene activation, exhibit specific binding to the promoters of HIF1 or HIF2 target genes and selectively interact with HIF1α or HIF2α protein. PMID:23991099

  19. Novel β-catenin target genes identified in thalamic neurons encode modulators of neuronal excitability

    PubMed Central

    2012-01-01

    Background LEF1/TCF transcription factors and their activator β-catenin are effectors of the canonical Wnt pathway. Although Wnt/β-catenin signaling has been implicated in neurodegenerative and psychiatric disorders, its possible role in the adult brain remains enigmatic. To address this issue, we sought to identify the genetic program activated by β-catenin in neurons. We recently showed that β-catenin accumulates specifically in thalamic neurons where it activates Cacna1g gene expression. In the present study, we combined bioinformatics and experimental approaches to find new β-catenin targets in the adult thalamus. Results We first selected the genes with at least two conserved LEF/TCF motifs within the regulatory elements. The resulting list of 428 putative LEF1/TCF targets was significantly enriched in known Wnt targets, validating our approach. Functional annotation of the presumed targets also revealed a group of 41 genes, heretofore not associated with Wnt pathway activity, that encode proteins involved in neuronal signal transmission. Using custom polymerase chain reaction arrays, we profiled the expression of these genes in the rat forebrain. We found that nine of the analyzed genes were highly expressed in the thalamus compared with the cortex and hippocampus. Removal of nuclear β-catenin from thalamic neurons in vitro by introducing its negative regulator Axin2 reduced the expression of six of the nine genes. Immunoprecipitation of chromatin from the brain tissues confirmed the interaction between β-catenin and some of the predicted LEF1/TCF motifs. The results of these experiments validated four genes as authentic and direct targets of β-catenin: Gabra3 for the receptor of GABA neurotransmitter, Calb2 for the Ca2+-binding protein calretinin, and the Cacna1g and Kcna6 genes for voltage-gated ion channels. Two other genes from the latter cluster, Cacna2d2 and Kcnh8, appeared to be regulated by β-catenin, although the binding of β-catenin to the

  20. Bioinformatic Identification and Expression Analysis of Banana MicroRNAs and Their Targets

    PubMed Central

    Shi, Hourui; Ren, Mengyun; Zhang, Yindong; Wang, Jingyi

    2015-01-01

    MicroRNAs (miRNAs) represent a class of endogenous non-coding small RNAs that play important roles in multiple biological processes by degrading targeted mRNAs or repressing mRNA translation. Thousands of miRNAs have been identified in many plant species, whereas only a limited number of miRNAs have been predicted in M. acuminata (A genome) and M. balbisiana (B genome). Here, previously known plant miRNAs were BLASTed against the Expressed Sequence Tag (EST) and Genomic Survey Sequence (GSS), a database of banana genes. A total of 32 potential miRNAs belonging to 13 miRNAs families were detected using a range of filtering criteria. 244 miRNA:target pairs were subsequently predicted, most of which encode transcription factors or enzymes that participate in the regulation of development, growth, metabolism, and other physiological processes. In order to validate the predicted miRNAs and the mutual relationship between miRNAs and their target genes, qRT-PCR was applied to detect the tissue-specific expression levels of 12 putative miRNAs and 6 target genes in roots, leaves, flowers, and fruits. This study provides some important information about banana pre-miRNAs, mature miRNAs, and miRNA target genes and these findings can be applied to future research of miRNA functions. PMID:25856313

  1. Expression and Chloroplast Targeting of Cholesterol Oxidase in Transgenic Tobacco Plants

    PubMed Central

    Corbin, David R.; Grebenok, Robert J.; Ohnmeiss, Thomas E.; Greenplate, John T.; Purcell, John P.

    2001-01-01

    Cholesterol oxidase represents a novel type of insecticidal protein with potent activity against the cotton boll weevil (Anthonomus grandis grandis Boheman). We transformed tobacco (Nicotiana tabacum) plants with the cholesterol oxidase choM gene and expressed cytosolic and chloroplast-targeted versions of the ChoM protein. Transgenic leaf tissues expressing cholesterol oxidase exerted insecticidal activity against boll weevil larvae. Our results indicate that cholesterol oxidase can metabolize phytosterols in vivo when produced cytosolically or when targeted to chloroplasts. The transgenic plants exhibiting cytosolic expression accumulated low levels of saturated sterols known as stanols, and displayed severe developmental aberrations. In contrast, the transgenic plants expressing chloroplast-targeted cholesterol oxidase maintained a greater accumulation of stanols, and appeared phenotypically and developmentally normal. These results are discussed within the context of plant sterol distribution and metabolism. PMID:11457962

  2. MicroRNAs: regulators of gene expression and cell differentiation

    PubMed Central

    Shivdasani, Ramesh A.

    2006-01-01

    The existence and roles of a class of abundant regulatory RNA molecules have recently come into sharp focus. Micro-RNAs (miRNAs) are small (approximately 22 bases), non–protein-coding RNAs that recognize target sequences of imperfect complementarity in cognate mRNAs and either destabilize them or inhibit protein translation. Although mechanisms of miRNA biogenesis have been elucidated in some detail, there is limited appreciation of their biological functions. Reported examples typically focus on miRNA regulation of a single tissue-restricted transcript, often one encoding a transcription factor, that controls a specific aspect of development, cell differentiation, or physiology. However, computational algorithms predict up to hundreds of putative targets for individual miRNAs, single transcripts may be regulated by multiple miRNAs, and miRNAs may either eliminate target gene expression or serve to finetune transcript and protein levels. Theoretical considerations and early experimental results hence suggest diverse roles for miRNAs as a class. One appealing possibility, that miRNAs eliminate low-level expression of unwanted genes and hence refine unilineage gene expression, may be especially amenable to evaluation in models of hematopoiesis. This review summarizes current understanding of miRNA mechanisms, outlines some of the important outstanding questions, and describes studies that attempt to define miRNA functions in hematopoiesis. PMID:16882713

  3. [Differential gene expression profile in ischemic myocardium of Wistar rats with acute myocardial infarction: the study on gene construction, identification and function].

    PubMed

    Guo, Chun Yu; Yin, Hui Jun; Jiang, Yue Rong; Xue, Mei; Zhang, Lu; Shi, Da Zhuo

    2008-06-18

    To construct the differential genes expressed profile in the ischemic myocardium tissue reduced from acute myocardial infarction(AMI), and determine the biological functions of target genes. AMI model was generated by ligation of the left anterior descending coronary artery in Wistar rats. Total RNA was extracted from the normal and the ischemic heart tissues under the ligation point 7 days after the operation. Differential gene expression profiles of the two samples were constructed using Long Serial Analysis of Gene Expression(LongSAGE). Real time fluorescence quantitative PCR was used to verify gene expression profile and to identify the expression of 2 functional genes. The activities of enzymes from functional genes were determined by histochemistry. A total of 15,966 tags were screened from the normal and the ischemic LongSAGE maps. The similarities of the sequences were compared using the BLAST algebra in NCBI and 7,665 novel tags were found. In the ischemic tissue 142 genes were significantly changed compared with those in the normal tissue (P<0.05). These differentially expressed genes represented the proteins which might play important roles in the pathways of oxidation and phosphorylation, ATP synthesis and glycolysis. The partial genes identified by LongSAGE were confirmed using real time fluorescence quantitative PCR. Two genes related to energy metabolism, COX5a and ATP5e, were screened and quantified. Expression of two functional genes down-regulated at their mRNA levels and the activities of correlative functional enzymes decreased compared with those in the normal tissue. AMI causes a series of changes in gene expression, in which the abnormal expression of genes related to energy metabolism could be one of the molecular mechanisms of AMI. The intervention of the expressions of COX5a and ATP5e may be a new target for AMI therapy.

  4. Gene Expression Profiling Predicts the Development of Oral Cancer

    PubMed Central

    Saintigny, Pierre; Zhang, Li; Fan, You-Hong; El-Naggar, Adel K.; Papadimitrakopoulou, Vali; Feng, Lei; Lee, J. Jack; Kim, Edward S.; Hong, Waun Ki; Mao, Li

    2011-01-01

    Patients with oral preneoplastic lesion (OPL) have high risk of developing oral cancer. Although certain risk factors such as smoking status and histology are known, our ability to predict oral cancer risk remains poor. The study objective was to determine the value of gene expression profiling in predicting oral cancer development. Gene expression profile was measured in 86 of 162 OPL patients who were enrolled in a clinical chemoprevention trial that used the incidence of oral cancer development as a prespecified endpoint. The median follow-up time was 6.08 years and 35 of the 86 patients developed oral cancer over the course. Gene expression profiles were associated with oral cancer-free survival and used to develope multivariate predictive models for oral cancer prediction. We developed a 29-transcript predictive model which showed marked improvement in terms of prediction accuracy (with 8% predicting error rate) over the models using previously known clinico-pathological risk factors. Based on the gene expression profile data, we also identified 2182 transcripts significantly associated with oral cancer risk associated genes (P-value<0.01, single variate Cox proportional hazards model). Functional pathway analysis revealed proteasome machinery, MYC, and ribosomes components as the top gene sets associated with oral cancer risk. In multiple independent datasets, the expression profiles of the genes can differentiate head and neck cancer from normal mucosa. Our results show that gene expression profiles may improve the prediction of oral cancer risk in OPL patients and the significant genes identified may serve as potential targets for oral cancer chemoprevention. PMID:21292635

  5. Circular RNA and gene expression profiles in gastric cancer based on microarray chip technology.

    PubMed

    Sui, Weiguo; Shi, Zhoufang; Xue, Wen; Ou, Minglin; Zhu, Ying; Chen, Jiejing; Lin, Hua; Liu, Fuhua; Dai, Yong

    2017-03-01

    The aim of the present study was to screen gastric cancer (GC) tissue and adjacent tissue for differences in mRNA and circular (circRNA) expression, to analyze the differences in circRNA and mRNA expression, and to investigate the circRNA expression in gastric carcinoma and its mechanism. circRNA and mRNA differential expression profiles generated using Agilent microarray technology were analyzed in the GC tissues and adjacent tissues. qRT-PCR was used to verify the differential expression of circRNAs and mRNAs according to the interactions between circRNAs and miRNAs as well as the possible existence of miRNA and mRNA interactions. We found that: i) the circRNA expression profile revealed 1,285 significant differences in circRNA expression, with circRNA expression downregulated in 594 samples and upregulated in 691 samples via interactions with miRNAs. The qRT-PCR validation experiments showed that hsa_circRNA_400071, hsa_circRNA_000543 and hsa_circRNA_001959 expression was consistent with the microarray analysis results. ii) 29,112 genes were found in the GC tissues and adjacent tissues, including 5,460 differentially expressed genes. Among them, 2,390 differentially expressed genes were upregulated and 3,070 genes were downregulated. Gene Ontology (GO) analysis of the differentially expressed genes revealed these genes involved in biological process classification, cellular component classification and molecular function classification. Pathway analysis of the differentially expressed genes identified 83 significantly enriched genes, including 28 upregulated genes and 55 downregulated genes. iii) 69 differentially expressed circRNAs were found that might adsorb specific miRNAs to regulate the expression of their target gene mRNAs. The conclusions are: i) differentially expressed circRNAs had corresponding miRNA binding sites. These circRNAs regulated the expression of target genes through interactions with miRNAs and might become new molecular biomarkers for GC

  6. Evaluation and selection of reliable reference genes for gene expression under abiotic stress in cotton (Gossypium hirsutum L.).

    PubMed

    Wang, Min; Wang, Qinglian; Zhang, Baohong

    2013-11-01

    Reference genes are critical for normalization of the gene expression level of target genes. The widely used housekeeping genes may change their expression levels at different tissue under different treatment or stress conditions. Therefore, systematical evaluation on the housekeeping genes is required for gene expression analysis. Up to date, no work was performed to evaluate the housekeeping genes in cotton under stress treatment. In this study, we chose 10 housekeeping genes to systematically assess their expression levels at two different tissues (leaves and roots) under two different abiotic stresses (salt and drought) with three different concentrations. Our results show that there is no best reference gene for all tissues at all stress conditions. The reliable reference gene should be selected based on a specific condition. For example, under salt stress, UBQ7, GAPDH and EF1A8 are better reference genes in leaves; TUA10, UBQ7, CYP1, GAPDH and EF1A8 were better in roots. Under drought stress, UBQ7, EF1A8, TUA10, and GAPDH showed less variety of expression level in leaves and roots. Thus, it is better to identify reliable reference genes first before performing any gene expression analysis. However, using a combination of housekeeping genes as reference gene may provide a new strategy for normalization of gene expression. In this study, we found that combination of four housekeeping genes worked well as reference genes under all the stress conditions. © 2013.

  7. Heterologous gene expression driven by carbonic anhydrase gene promoter in Dunaliella salina

    NASA Astrophysics Data System (ADS)

    Chai, Yurong; Lu, Yumin; Wang, Tianyun; Hou, Weihong; Xue, Lexun

    2006-12-01

    Dunaliella salina, a halotolerant unicellular green alga without a rigid cell wall, can live in salinities ranging from 0.05 to 5 mol/L NaCl. These features of D. salina make it an ideal host for the production of antibodies, oral vaccine, and commercially valuable polypeptides. To produce high level of heterologous proteins from D. salina, highly efficient promoters are required to drive expression of target genes under controlled condition. In the present study, we cloned a 5' franking region of 1.4 kb from the carbonic anhydrase ( CAH) gene of D. salina by genomic walking and PCR. The fragment was ligated to the pMD18-T vector and characterized. Sequence analysis indicated that this region contained conserved motifs, including a TATA- like box and CAAT-box. Tandem (GT)n repeats that had a potential role of transcriptional control, were also found in this region. The transcription start site (TSS) of the CAH gene was determined by 5' RACE and nested PCR method. Transformation assays showed that the 1.4 kb fragment was able to drive expression of the selectable bar (bialaphos resistance) gene when the fusion was transformed into D. salina by biolistics. Northern blotting hybridizations showed that the bar transcript was most abundant in cells grown in 2 mol/L NaCl, and less abundant in 0.5 mol/L NaCl, indicating that expression of the bar gene was induced at high salinity. These results suggest the potential use of the CAH gene promoter to induce the expression of heterologous genes in D. salina under varied salt condition.

  8. Expression of Notch pathway genes in mammalian epidermis and modulation by beta-catenin.

    PubMed

    Ambler, Carrie A; Watt, Fiona M

    2007-06-01

    The Notch pathway is required for hair follicle maintenance and is activated through beta-catenin induced transcription of the Notch ligand Jagged1. We show that hair follicles in the resting phase express low levels of Jagged1 and Hes1, and other Notch target genes are undetectable. In growing (anagen) follicles, Jagged1 and Hes1 expression increases, Hes5 and HeyL are expressed in distinct cell layers, and Hey2 is expressed in the dermal papilla. When beta-catenin is activated by means of an inducible transgene, Jagged1, Hes1, Hes5, HeyL, and Hey2 are up-regulated, the sites of expression being the same in beta-catenin induced ectopic follicles as in anagen follicles. beta-Catenin also induces Hey1 in dermal papilla cells. beta-Catenin-induced up-regulation of Jagged1 precedes induction of other Notch target genes. The different sites of expression of Hes and Hey genes suggest input from additional signaling pathways. Copyright 2007 Wiley-Liss, Inc.

  9. In Vivo Regulation of Human Skeletal Muscle Gene Expression by Thyroid Hormone

    PubMed Central

    Clément, Karine; Viguerie, Nathalie; Diehn, Maximilian; Alizadeh, Ash; Barbe, Pierre; Thalamas, Claire; Storey, John D.; Brown, Patrick O.; Barsh, Greg S.; Langin, Dominique

    2002-01-01

    Thyroid hormones are key regulators of metabolism that modulate transcription via nuclear receptors. Hyperthyroidism is associated with increased metabolic rate, protein breakdown, and weight loss. Although the molecular actions of thyroid hormones have been studied thoroughly, their pleiotropic effects are mediated by complex changes in expression of an unknown number of target genes. Here, we measured patterns of skeletal muscle gene expression in five healthy men treated for 14 days with 75 μg of triiodothyronine, using 24,000 cDNA element microarrays. To analyze the data, we used a new statistical method that identifies significant changes in expression and estimates the false discovery rate. The 381 up-regulated genes were involved in a wide range of cellular functions including transcriptional control, mRNA maturation, protein turnover, signal transduction, cellular trafficking, and energy metabolism. Only two genes were down-regulated. Most of the genes are novel targets of thyroid hormone. Cluster analysis of triiodothyronine-regulated gene expression among 19 different human tissues or cell lines revealed sets of coregulated genes that serve similar biologic functions. These results define molecular signatures that help to understand the physiology and pathophysiology of thyroid hormone action. [The list of transcripts corresponding to up-regulated and down-regulated genes is available as a web supplement at http://www.genome.org.] PMID:11827947

  10. Gene Expression in Human Accessory Lacrimal Glands of Wolfring

    PubMed Central

    Ubels, John L.; Gipson, Ilene K.; Spurr-Michaud, Sandra J.; Tisdale, Ann S.; Van Dyken, Rachel E.; Hatton, Mark P.

    2012-01-01

    Purpose. The accessory lacrimal glands are assumed to contribute to the production of tear fluid, but little is known about their function. The goal of this study was to conduct an analysis of gene expression by glands of Wolfring that would provide a more complete picture of the function of these glands. Methods. Glands of Wolfring were isolated from frozen sections of human eyelids by laser microdissection. RNA was extracted from the cells and hybridized to gene expression arrays. The expression of several of the major genes was confirmed by immunohistochemistry. Results. Of the 24 most highly expressed genes, 9 were of direct relevance to lacrimal function. These included lysozyme, lactoferrin, tear lipocalin, and lacritin. The glands of Wolfring are enriched in genes related to protein synthesis, targeting, and secretion, and a large number of genes for proteins with antimicrobial activity were detected. Ion channels and transporters, carbonic anhydrase, and aquaporins were abundantly expressed. Genes for control of lacrimal function, including cholinergic, adrenergic, vasoactive intestinal polypeptide, purinergic, androgen, and prolactin receptors were also expressed in gland of Wolfring. Conclusions. The data suggest that the function of glands of Wolfring is similar to that of main lacrimal glands and are consistent with secretion electrolytes, fluid, and protein under nervous and hormonal control. Since these glands secrete directly onto the ocular surface, their location may allow rapid response to exogenous stimuli and makes them readily accessible to topical drugs. PMID:22956620

  11. Gene expression of bovine embryos developing at the air-liquid interface on oviductal epithelial cells (ALI-BOEC).

    PubMed

    van der Weijden, Vera A; Chen, Shuai; Bauersachs, Stefan; Ulbrich, Susanne E; Schoen, Jennifer

    2017-11-25

    We recently developed an air-liquid interface long-term culture of differentiated bovine oviductal epithelial cells (ALI-BOEC). This ex vivo oviduct epithelium is capable of supporting embryo development in co-culture up to the blastocyst stage without addition of embryo culture medium. However, blastocyst rates in co-culture were markedly lower than in conventional in vitro embryo production procedures. In the present study, we assessed target gene expression of ALI-BOEC derived embryos to test their similarity to embryos from conventional in vitro embryo culture. We screened previously published data from developing bovine embryos and selected 41 genes which are either differentially expressed during embryo development, or reflect differences between various in vitro culture conditions or in vitro and in vivo embryos. Target gene expression was measured in 8-cell embryos and blastocysts using a 48.48 Dynamic Array™ on a Biomark HD instrument. For comparison with the ALI-BOEC system, we generated embryos by two different standard IVP protocols. The culture conditions lead to differential gene expression in both 8-cell embryos and blastocysts. Across the expression of all target genes the embryos developing on ALI-BOEC did not depart from conventional IVP embryos. These first results prove that gene expression in ALI-BOEC embryos is not largely aberrant. However, there was no clear indication for a more in vivo-like target gene expression of these embryos. This calls for further optimization of the ALI-BOEC system to increase its efficiency both quantitatively and qualitatively.

  12. Identification of STAT target genes in adipocytes

    PubMed Central

    Zhao, Peng; Stephens, Jacqueline M.

    2013-01-01

    Adipocytes play important roles in lipid storage, energy homeostasis and whole body insulin sensitivity. Studies in the last two decades have identified the hormones and cytokines that activate specific STATs in adipocytes in vitro and in vivo. Five of the seven STAT family members are expressed in adipocyte (STATs 1, 3, 5A, 5B and 6). Many transcription factors, including STATs, have been shown to play an important role in adipose tissue development and function. This review will summarize the importance of adipocytes, indicate the cytokines and hormones that utilize the JAK-STAT signaling pathway in fat cells and focus on the identification of STAT target genes in mature adipocytes. To date, specific target genes have been identified for STATs, 1, 5A and 5B, but not for STATs 3 and 6. PMID:24058802

  13. Expression Profiling Identifies Klf15 as a Glucocorticoid Target That Regulates Airway Hyperresponsiveness

    PubMed Central

    Masuno, Kiriko; Haldar, Saptarsi M.; Jeyaraj, Darwin; Mailloux, Christina M.; Huang, Xiaozhu; Panettieri, Rey A.; Jain, Mukesh K.

    2011-01-01

    Glucocorticoids (GCs), which activate GC receptor (GR) signaling and thus modulate gene expression, are widely used to treat asthma. GCs exert their therapeutic effects in part through modulating airway smooth muscle (ASM) structure and function. However, the effects of genes that are regulated by GCs on airway function are not fully understood. We therefore used transcription profiling to study the effects of a potent GC, dexamethasone, on human ASM (HASM) gene expression at 4 and 24 hours. After 24 hours of dexamethasone treatment, nearly 7,500 genes had statistically distinguishable changes in expression; quantitative PCR validation of a 40-gene subset of putative GR-regulated genes in 6 HASM cell lines suggested that the early transcriptional targets of GR signaling are similar in independent HASM lines. Gene ontology analysis implicated GR targets in controlling multiple aspects of ASM function. One GR-regulated gene, the transcription factor, Kruppel-like factor 15 (Klf15), was already known to modulate vascular smooth and cardiac muscle function, but had no known role in the lung. We therefore analyzed the pulmonary phenotype of Klf15−/− mice after ovalbumin sensitization and challenge. We found diminished airway responses to acetylcholine in ovalbumin-challenged Klf15−/− mice without a significant change in the induction of asthmatic inflammation. In cultured cells, overexpression of Klf15 reduced proliferation of HASM cells, whereas apoptosis in Klf15−/− murine ASM cells was increased. Together, these results further characterize the GR-regulated gene network in ASM and establish a novel role for the GR target, Klf15, in modulating airway function. PMID:21257922

  14. Evaluation of miRNA-196a2 and apoptosis-related target genes: ANXA1, DFFA and PDCD4 expression in gastrointestinal cancer patients: A pilot study

    PubMed Central

    Toraih, Eman A.; Ibrahiem, Afaf; Abdeldayem, Hala; Mohamed, Amany O.; Abdel-Daim, Mohamed M.

    2017-01-01

    Previous reports have suggested the significant association of miRNAs aberrant expression with tumor initiation, progression and metastasis in cancer, including gastrointestinal (GI) cancers. The current preliminary study aimed to evaluate the relative expression levels of miR-196a2 and three of its selected apoptosis-related targets; ANXA1, DFFA and PDCD4 in a sample of GI cancer patients. Quantitative real-time PCR for miR-196a2 and its selected mRNA targets, as well as immunohistochemical assay for annexin A1 protein expression were detected in 58 tissues with different GI cancer samples. In addition, correlation with the clinicopathological features and in silico network analysis of the selected molecular markers were analyzed. Stratified analyses by cancer site revealed elevated levels of miR-196a2 and low expression of the selected target genes. Annexin protein expression was positively correlated with its gene expression profile. In colorectal cancer, miR-196a over-expression was negatively correlated with annexin A1 protein expression (r = -0.738, p < 0.001), and both were indicators of unfavorable prognosis in terms of poor differentiation, larger tumor size, and advanced clinical stage. Taken together, aberrant expression of miR-196a2 and the selected apoptosis-related biomarkers might be involved in GI cancer development and progression and could have potential diagnostic and prognostic roles in these types of cancer; particularly colorectal cancer, provided the results experimentally validated and confirmed in larger multi-center studies. PMID:29091952

  15. Identification of reference genes and validation for gene expression studies in diverse axolotl (Ambystoma mexicanum) tissues.

    PubMed

    Guelke, Eileen; Bucan, Vesna; Liebsch, Christina; Lazaridis, Andrea; Radtke, Christine; Vogt, Peter M; Reimers, Kerstin

    2015-04-10

    For the precise quantitative RT-PCR normalization a set of valid reference genes is obligatory. Moreover have to be taken into concern the experimental conditions as they bias the regulation of reference genes. Up till now, no reference targets have been described for the axolotl (Ambystoma mexicanum). In a search in the public database SalSite for genetic information of the axolotl we identified fourteen presumptive reference genes, eleven of which were further tested for their gene expression stability. This study characterizes the expressional patterns of 11 putative endogenous control genes during axolotl limb regeneration and in an axolotl tissue panel. All 11 reference genes showed variable expression. Strikingly, ACTB was to be found most stable expressed in all comparative tissue groups, so we reason it to be suitable for all different kinds of axolotl tissue-type investigations. Moreover do we suggest GAPDH and RPLP0 as suitable for certain axolotl tissue analysis. When it comes to axolotl limb regeneration, a validated pair of reference genes is ODC and RPLP0. With these findings, new insights into axolotl gene expression profiling might be gained. Copyright © 2015 Elsevier B.V. All rights reserved.

  16. Gene expression analysis upon lncRNA DDSR1 knockdown in human fibroblasts

    PubMed Central

    Jia, Li; Sun, Zhonghe; Wu, Xiaolin; Misteli, Tom; Sharma, Vivek

    2015-01-01

    Long non-coding RNAs (lncRNAs) play important roles in regulating diverse biological processes including DNA damage and repair. We have recently reported that the DNA damage inducible lncRNA DNA damage-sensitive RNA1 (DDSR1) regulates DNA repair by homologous recombination (HR). Since lncRNAs also modulate gene expression, we identified gene expression changes upon DDSR1 knockdown in human fibroblast cells. Gene expression analysis after RNAi treatment targeted against DDSR1 revealed 119 genes that show differential expression. Here we provide a detailed description of the microarray data (NCBI GEO accession number GSE67048) and the data analysis procedure associated with the publication by Sharma et al., 2015 in EMBO Reports [1]. PMID:26697398

  17. Automated Discovery of Functional Generality of Human Gene Expression Programs

    PubMed Central

    Gerber, Georg K; Dowell, Robin D; Jaakkola, Tommi S; Gifford, David K

    2007-01-01

    An important research problem in computational biology is the identification of expression programs, sets of co-expressed genes orchestrating normal or pathological processes, and the characterization of the functional breadth of these programs. The use of human expression data compendia for discovery of such programs presents several challenges including cellular inhomogeneity within samples, genetic and environmental variation across samples, uncertainty in the numbers of programs and sample populations, and temporal behavior. We developed GeneProgram, a new unsupervised computational framework based on Hierarchical Dirichlet Processes that addresses each of the above challenges. GeneProgram uses expression data to simultaneously organize tissues into groups and genes into overlapping programs with consistent temporal behavior, to produce maps of expression programs, which are sorted by generality scores that exploit the automatically learned groupings. Using synthetic and real gene expression data, we showed that GeneProgram outperformed several popular expression analysis methods. We applied GeneProgram to a compendium of 62 short time-series gene expression datasets exploring the responses of human cells to infectious agents and immune-modulating molecules. GeneProgram produced a map of 104 expression programs, a substantial number of which were significantly enriched for genes involved in key signaling pathways and/or bound by NF-κB transcription factors in genome-wide experiments. Further, GeneProgram discovered expression programs that appear to implicate surprising signaling pathways or receptor types in the response to infection, including Wnt signaling and neurotransmitter receptors. We believe the discovered map of expression programs involved in the response to infection will be useful for guiding future biological experiments; genes from programs with low generality scores might serve as new drug targets that exhibit minimal “cross-talk,” and

  18. Selection and validation of reliable housekeeping genes to evaluate Piscirickettsia salmonis gene expression.

    PubMed

    Flores-Herrera, Patricio; Arredondo-Zelada, Oscar; Marshall, Sergio H; Gómez, Fernando A

    2018-06-01

    Piscirickettsia salmonis is a highly aggressive facultative intracellular bacterium that challenges the sustainability of Chilean salmon production. Due to the limited knowledge of its biology, there is a need to identify key molecular markers that could help define the pathogenic potential of this bacterium. We think a model system should be implemented that efficiently evaluates the expression of putative bacterial markers by using validated, stable, and highly specific housekeeping genes to properly select target genes, which could lead to identifying those responsible for infection and disease induction in naturally infected fish. Here, we selected a set of validated reference or housekeeping genes for RT-qPCR expression analyses of P. salmonis under different growth and stress conditions, including an in vitro infection kinetic. After a thorough screening, we selected sdhA as the most reliable housekeeping gene able to represent stable and highly specific host reference genes for RT-qPCR-driven P. salmonis analysis. Copyright © 2018. Published by Elsevier B.V.

  19. Inhibition of Nod2 Signaling and Target Gene Expression by Curcumin

    PubMed Central

    Huang, Shurong; Zhao, Ling; Kim, Kihoon; Lee, Dong Seok; Hwang, Daniel H.

    2008-01-01

    Nod2 is an intracellular pattern recognition receptor that detects a conserved moiety of bacterial peptidoglycan and subsequently activates proinflammatory signaling pathways. Mutations in Nod2 have been implicated to be linked to inflammatory granulomatous disorders, such as Crohn's disease and Blau syndrome. Many phytochemicals possess anti-inflammatory properties. However, it is not known whether any of these phytochemicals might modulate Nod2-mediated immune responses and thus might be of therapeutic value for the intervention of these inflammatory diseases. In this report, we demonstrate that curcumin, a polyphenol found in the plant Curcuma longa, and parthenolide, a sesquiterpene lactone, suppress both ligand-induced and lauric acid-induced Nod2 signaling, leading to the suppression of nuclear factor-κB activation and target gene interleukin-8 expression. We provide molecular and biochemical evidence that the suppression is mediated through the inhibition of Nod2 oligomerization and subsequent inhibition of downstream signaling. These results demonstrate for the first time that curcumin and parthenolide can directly inhibit Nod2-mediated signaling pathways at the receptor level and suggest that Nod2-mediated inflammatory responses can be modulated by these phytochemicals. It remains to be determined whether these phytochemicals possess protective or therapeutic efficacy against Nod2-mediated inflammatory disorders. PMID:18413660

  20. ATNT: an enhanced system for expression of polycistronic secondary metabolite gene clusters in Aspergillus niger.

    PubMed

    Geib, Elena; Brock, Matthias

    2017-01-01

    Fungi are treasure chests for yet unexplored natural products. However, exploitation of their real potential remains difficult as a significant proportion of biosynthetic gene clusters appears silent under standard laboratory conditions. Therefore, elucidation of novel products requires gene activation or heterologous expression. For heterologous gene expression, we previously developed an expression platform in Aspergillus niger that is based on the transcriptional regulator TerR and its target promoter P terA . In this study, we extended this system by regulating expression of terR  by the doxycycline inducible Tet-on system. Reporter genes cloned under the control of the target promoter P terA remained silent in the absence of doxycycline, but were strongly expressed when doxycycline was added. Reporter quantification revealed that the coupled system results in about five times higher expression rates compared to gene expression under direct control of the Tet-on system. As production of secondary metabolites generally requires the expression of several biosynthetic genes, the suitability of the self-cleaving viral peptide sequence P2A was tested in this optimised expression system. P2A allowed polycistronic expression of genes required for Asp-melanin formation in combination with the gene coding for the red fluorescent protein tdTomato. Gene expression and Asp-melanin formation was prevented in the absence of doxycycline and strongly induced by addition of doxycycline. Fluorescence studies confirmed the correct subcellular localisation of the respective enzymes. This tightly regulated but strongly inducible expression system enables high level production of secondary metabolites most likely even those with toxic potential. Furthermore, this system is compatible with polycistronic gene expression and, thus, suitable for the discovery of novel natural products.

  1. Positive correlation between ADAR expression and its targets suggests a complex regulation mediated by RNA editing in the human brain

    PubMed Central

    Liscovitch, Noa; Bazak, Lily; Levanon, Erez Y; Chechik, Gal

    2014-01-01

    A-to-I RNA editing by adenosine deaminases acting on RNA is a post-transcriptional modification that is crucial for normal life and development in vertebrates. RNA editing has been shown to be very abundant in the human transcriptome, specifically at the primate-specific Alu elements. The functional role of this wide-spread effect is still not clear; it is believed that editing of transcripts is a mechanism for their down-regulation via processes such as nuclear retention or RNA degradation. Here we combine 2 neural gene expression datasets with genome-level editing information to examine the relation between the expression of ADAR genes with the expression of their target genes. Specifically, we computed the spatial correlation across structures of post-mortem human brains between ADAR and a large set of targets that were found to be edited in their Alu repeats. Surprisingly, we found that a large fraction of the edited genes are positively correlated with ADAR, opposing the assumption that editing would reduce expression. When considering the correlations between ADAR and its targets over development, 2 gene subsets emerge, positively correlated and negatively correlated with ADAR expression. Specifically, in embryonic time points, ADAR is positively correlated with many genes related to RNA processing and regulation of gene expression. These findings imply that the suggested mechanism of regulation of expression by editing is probably not a global one; ADAR expression does not have a genome wide effect reducing the expression of editing targets. It is possible, however, that RNA editing by ADAR in non-coding regions of the gene might be a part of a more complex expression regulation mechanism. PMID:25692240

  2. Positive correlation between ADAR expression and its targets suggests a complex regulation mediated by RNA editing in the human brain.

    PubMed

    Liscovitch, Noa; Bazak, Lily; Levanon, Erez Y; Chechik, Gal

    2014-01-01

    A-to-I RNA editing by adenosine deaminases acting on RNA is a post-transcriptional modification that is crucial for normal life and development in vertebrates. RNA editing has been shown to be very abundant in the human transcriptome, specifically at the primate-specific Alu elements. The functional role of this wide-spread effect is still not clear; it is believed that editing of transcripts is a mechanism for their down-regulation via processes such as nuclear retention or RNA degradation. Here we combine 2 neural gene expression datasets with genome-level editing information to examine the relation between the expression of ADAR genes with the expression of their target genes. Specifically, we computed the spatial correlation across structures of post-mortem human brains between ADAR and a large set of targets that were found to be edited in their Alu repeats. Surprisingly, we found that a large fraction of the edited genes are positively correlated with ADAR, opposing the assumption that editing would reduce expression. When considering the correlations between ADAR and its targets over development, 2 gene subsets emerge, positively correlated and negatively correlated with ADAR expression. Specifically, in embryonic time points, ADAR is positively correlated with many genes related to RNA processing and regulation of gene expression. These findings imply that the suggested mechanism of regulation of expression by editing is probably not a global one; ADAR expression does not have a genome wide effect reducing the expression of editing targets. It is possible, however, that RNA editing by ADAR in non-coding regions of the gene might be a part of a more complex expression regulation mechanism.

  3. Genome engineering and gene expression control for bacterial strain development.

    PubMed

    Song, Chan Woo; Lee, Joungmin; Lee, Sang Yup

    2015-01-01

    In recent years, a number of techniques and tools have been developed for genome engineering and gene expression control to achieve desired phenotypes of various bacteria. Here we review and discuss the recent advances in bacterial genome manipulation and gene expression control techniques, and their actual uses with accompanying examples. Genome engineering has been commonly performed based on homologous recombination. During such genome manipulation, the counterselection systems employing SacB or nucleases have mainly been used for the efficient selection of desired engineered strains. The recombineering technology enables simple and more rapid manipulation of the bacterial genome. The group II intron-mediated genome engineering technology is another option for some bacteria that are difficult to be engineered by homologous recombination. Due to the increasing demands on high-throughput screening of bacterial strains having the desired phenotypes, several multiplex genome engineering techniques have recently been developed and validated in some bacteria. Another approach to achieve desired bacterial phenotypes is the repression of target gene expression without the modification of genome sequences. This can be performed by expressing antisense RNA, small regulatory RNA, or CRISPR RNA to repress target gene expression at the transcriptional or translational level. All of these techniques allow efficient and rapid development and screening of bacterial strains having desired phenotypes, and more advanced techniques are expected to be seen. Copyright © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  4. Successful transient expression of Cas9 and single guide RNA genes in Chlamydomonas reinhardtii.

    PubMed

    Jiang, Wenzhi; Brueggeman, Andrew J; Horken, Kempton M; Plucinak, Thomas M; Weeks, Donald P

    2014-11-01

    The clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 system has become a powerful and precise tool for targeted gene modification (e.g., gene knockout and gene replacement) in numerous eukaryotic organisms. Initial attempts to apply this technology to a model, the single-cell alga, Chlamydomonas reinhardtii, failed to yield cells containing edited genes. To determine if the Cas9 and single guide RNA (sgRNA) genes were functional in C. reinhardtii, we tested the ability of a codon-optimized Cas9 gene along with one of four different sgRNAs to cause targeted gene disruption during a 24-h period immediately following transformation. All three exogenously supplied gene targets as well as the endogenous FKB12 (rapamycin sensitivity) gene of C. reinhardtii displayed distinct Cas9/sgRNA-mediated target site modifications as determined by DNA sequencing of cloned PCR amplicons of the target site region. Success in transient expression of Cas9 and sgRNA genes contrasted with the recovery of only a single rapamycin-resistant colony bearing an appropriately modified FKB12 target site in 16 independent transformation experiments involving >10(9) cells. Failure to recover transformants with intact or expressed Cas9 genes following transformation with the Cas9 gene alone (or even with a gene encoding a Cas9 lacking nuclease activity) provided strong suggestive evidence for Cas9 toxicity when Cas9 is produced constitutively in C. reinhardtii. The present results provide compelling evidence that Cas9 and sgRNA genes function properly in C. reinhardtii to cause targeted gene modifications and point to the need for a focus on development of methods to properly stem Cas9 production and/or activity following gene editing. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

  5. Determining Physical Mechanisms of Gene Expression Regulation from Single Cell Gene Expression Data.

    PubMed

    Ezer, Daphne; Moignard, Victoria; Göttgens, Berthold; Adryan, Boris

    2016-08-01

    Many genes are expressed in bursts, which can contribute to cell-to-cell heterogeneity. It is now possible to measure this heterogeneity with high throughput single cell gene expression assays (single cell qPCR and RNA-seq). These experimental approaches generate gene expression distributions which can be used to estimate the kinetic parameters of gene expression bursting, namely the rate that genes turn on, the rate that genes turn off, and the rate of transcription. We construct a complete pipeline for the analysis of single cell qPCR data that uses the mathematics behind bursty expression to develop more accurate and robust algorithms for analyzing the origin of heterogeneity in experimental samples, specifically an algorithm for clustering cells by their bursting behavior (Simulated Annealing for Bursty Expression Clustering, SABEC) and a statistical tool for comparing the kinetic parameters of bursty expression across populations of cells (Estimation of Parameter changes in Kinetics, EPiK). We applied these methods to hematopoiesis, including a new single cell dataset in which transcription factors (TFs) involved in the earliest branchpoint of blood differentiation were individually up- and down-regulated. We could identify two unique sub-populations within a seemingly homogenous group of hematopoietic stem cells. In addition, we could predict regulatory mechanisms controlling the expression levels of eighteen key hematopoietic transcription factors throughout differentiation. Detailed information about gene regulatory mechanisms can therefore be obtained simply from high throughput single cell gene expression data, which should be widely applicable given the rapid expansion of single cell genomics.

  6. Garlic Influences Gene Expression In Vivo and In Vitro.

    PubMed

    Charron, Craig S; Dawson, Harry D; Novotny, Janet A

    2016-02-01

    There is a large body of preclinical research aimed at understanding the roles of garlic and garlic-derived preparations in the promotion of human health. Most of this research has targeted the possible functions of garlic in maintaining cardiovascular health and in preventing and treating cancer. A wide range of outcome variables has been used to investigate the bioactivity of garlic, ranging from direct measures of health status such as cholesterol concentrations, blood pressure, and changes in tumor size and number, to molecular and biochemical measures such as mRNA gene expression, protein concentration, enzyme activity, and histone acetylation status. Determination of how garlic influences mRNA gene expression has proven to be a valuable approach to elucidating the mechanisms of garlic bioactivity. Preclinical studies investigating the health benefits of garlic far outnumber human studies and have made frequent use of mRNA gene expression measurement. There is an immediate need to understand mRNA gene expression in humans as well. Although safety and ethical constraints limit the types of available human tissue, peripheral whole blood is readily accessible, and measuring mRNA gene expression in whole blood may provide a unique window to understanding how garlic intake affects human health. © 2016 American Society for Nutrition.

  7. Gene Expression Profiling Reveals Novel Candidate Markers of Ovarian Carcinoma Intraperitoneal Metastasis.

    PubMed

    Elsnerova, Katerina; Bartakova, Alena; Tihlarik, Josef; Bouda, Jiri; Rob, Lukas; Skapa, Petr; Hruda, Martin; Gut, Ivan; Mohelnikova-Duchonova, Beatrice; Soucek, Pavel; Vaclavikova, Radka

    2017-01-01

    Epithelial ovarian cancer (EOC) has the highest mortality among gynecological carcinomas. The lack of specific markers for prognostic determination of EOC progression hinders the search for novel effective therapies. The aim of the present study was (i) to explore differences in expressions of ATP-binding cassette (ABC) and solute carrier (SLC) transporter genes, genes associated with drug metabolism and cell cycle regulation between control ovarian tissues (n = 14), primary EOCs (n = 44) and intraperitoneal metastases (n = 29); (ii) to investigate associations of gene expression levels with prognosis of patients with intraperitoneal metastases. In all tissue samples, transcript levels of the above target genes were assessed using quantitative real-time PCR. Gene expression levels were compared between particular tissue types and evaluated with regard to progression-free survival (PFS) and drug-resistance status of patients with metastases. Gene expression of ABCA7 significantly increased and that of ESR2 decreased in the order control ovarian tissues - primary EOCs - metastases. High expressions of ABCA2 / 8 / 9 / 10 , ABCB1 , ABCC9 , ABCG2 , ATP7A , SLC16A14 , and SOD3 genes were significantly associated with longer progression-free survival of patients. In intraperitoneal metastases, expression of all of these genes highly correlated and indicated prognostic profile. Transporters from the ABCA family, ABCG2, and ESR2 are involved mainly in lipid metabolism, membrane transport, and cell proliferation. These processes are thus probably the most important for EOC progression. Based on these results, we have proposed novel markers of ovarian carcinoma progression and metastatic spread which might be potentially useful as therapeutic targets. Their significance should be further explored on a larger independent set of patients.

  8. Inhibition of histone deacetylation and DNA methylation improves gene expression mediated by the adeno-associated virus/phage in cancer cells.

    PubMed

    Kia, Azadeh; Yata, Teerapong; Hajji, Nabil; Hajitou, Amin

    2013-10-22

    Bacteriophage (phage), viruses that infect bacteria only, have become promising vectors for targeted systemic delivery of genes to cancer, although, with poor efficiency. We previously designed an improved phage vector by incorporating cis genetic elements of adeno-associated virus (AAV). This novel AAV/phage hybrid (AAVP) specifically targeted systemic delivery of therapeutic genes into tumors. To advance the AAVP vector, we recently introduced the stress-inducible Grp78 tumor specific promoter and found that this dual tumor-targeted AAVP provides persistent gene expression, over time, in cancer cells compared to silenced gene expression from the CMV promoter in the parental AAVP. Herein, we investigated the effect of histone deacetylation and DNA methylation on AAVP-mediated gene expression in cancer cells and explored the effect of cell confluence state on AAVP gene expression efficacy. Using a combination of AAVP expressing the GFP reporter gene, flow cytometry, inhibitors of histone deacetylation, and DNA methylation, we have demonstrated that histone deacetylation and DNA methylation are associated with silencing of gene expression from the CMV promoter in the parental AAVP. Importantly, inhibitors of histone deacetylases boost gene expression in cancer cells from the Grp78 promoter in the dual tumor-targeted AAVP. However, cell confluence had no effect on AAVP-guided gene expression. Our findings prove that combination of histone deacetylase inhibitor drugs with the Grp78 promoter is an effective approach to improve AAVP-mediated gene expression in cancer cells and should be considered for AAVP-based clinical cancer gene therapy.

  9. Annotation of gene function in citrus using gene expression information and co-expression networks

    PubMed Central

    2014-01-01

    Background The genus Citrus encompasses major cultivated plants such as sweet orange, mandarin, lemon and grapefruit, among the world’s most economically important fruit crops. With increasing volumes of transcriptomics data available for these species, Gene Co-expression Network (GCN) analysis is a viable option for predicting gene function at a genome-wide scale. GCN analysis is based on a “guilt-by-association” principle whereby genes encoding proteins involved in similar and/or related biological processes may exhibit similar expression patterns across diverse sets of experimental conditions. While bioinformatics resources such as GCN analysis are widely available for efficient gene function prediction in model plant species including Arabidopsis, soybean and rice, in citrus these tools are not yet developed. Results We have constructed a comprehensive GCN for citrus inferred from 297 publicly available Affymetrix Genechip Citrus Genome microarray datasets, providing gene co-expression relationships at a genome-wide scale (33,000 transcripts). The comprehensive citrus GCN consists of a global GCN (condition-independent) and four condition-dependent GCNs that survey the sweet orange species only, all citrus fruit tissues, all citrus leaf tissues, or stress-exposed plants. All of these GCNs are clustered using genome-wide, gene-centric (guide) and graph clustering algorithms for flexibility of gene function prediction. For each putative cluster, gene ontology (GO) enrichment and gene expression specificity analyses were performed to enhance gene function, expression and regulation pattern prediction. The guide-gene approach was used to infer novel roles of genes involved in disease susceptibility and vitamin C metabolism, and graph-clustering approaches were used to investigate isoprenoid/phenylpropanoid metabolism in citrus peel, and citric acid catabolism via the GABA shunt in citrus fruit. Conclusions Integration of citrus gene co-expression networks

  10. miR-638 regulates gene expression networks associated with emphysematous lung destruction

    PubMed Central

    2013-01-01

    Background Chronic obstructive pulmonary disease (COPD) is a heterogeneous disease characterized by varying degrees of emphysematous lung destruction and small airway disease, each with distinct effects on clinical outcomes. There is little known about how microRNAs contribute specifically to the emphysema phenotype. We examined how genome-wide microRNA expression is altered with regional emphysema severity and how these microRNAs regulate disease-associated gene expression networks. Methods We profiled microRNAs in different regions of the lung with varying degrees of emphysema from 6 smokers with COPD and 2 controls (8 regions × 8 lungs = 64 samples). Regional emphysema severity was quantified by mean linear intercept. Whole genome microRNA and gene expression data were integrated in the same samples to build co-expression networks. Candidate microRNAs were perturbed in human lung fibroblasts in order to validate these networks. Results The expression levels of 63 microRNAs (P < 0.05) were altered with regional emphysema. A subset, including miR-638, miR-30c, and miR-181d, had expression levels that were associated with those of their predicted mRNA targets. Genes correlated with these microRNAs were enriched in pathways associated with emphysema pathophysiology (for example, oxidative stress and accelerated aging). Inhibition of miR-638 expression in lung fibroblasts led to modulation of these same emphysema-related pathways. Gene targets of miR-638 in these pathways were amongst those negatively correlated with miR-638 expression in emphysema. Conclusions Our findings demonstrate that microRNAs are altered with regional emphysema severity and modulate disease-associated gene expression networks. Furthermore, miR-638 may regulate gene expression pathways related to the oxidative stress response and aging in emphysematous lung tissue and lung fibroblasts. PMID:24380442

  11. Prostate cancer-associated gene expression alterations determined from needle biopsies.

    PubMed

    Qian, David Z; Huang, Chung-Ying; O'Brien, Catherine A; Coleman, Ilsa M; Garzotto, Mark; True, Lawrence D; Higano, Celestia S; Vessella, Robert; Lange, Paul H; Nelson, Peter S; Beer, Tomasz M

    2009-05-01

    To accurately identify gene expression alterations that differentiate neoplastic from normal prostate epithelium using an approach that avoids contamination by unwanted cellular components and is not compromised by acute gene expression changes associated with tumor devascularization and resulting ischemia. Approximately 3,000 neoplastic and benign prostate epithelial cells were isolated using laser capture microdissection from snap-frozen prostate biopsy specimens provided by 31 patients who subsequently participated in a clinical trial of preoperative chemotherapy. cDNA synthesized from amplified total RNA was hybridized to custom-made microarrays composed of 6,200 clones derived from the Prostate Expression Database. Expression differences for selected genes were verified using quantitative reverse transcription-PCR. Comparative analyses identified 954 transcript alterations associated with cancer (q < 0.01%), including 149 differentially expressed genes with no known functional roles. Gene expression changes associated with ischemia and surgical removal of the prostate gland were absent. Genes up-regulated in prostate cancer were statistically enriched in categories related to cellular metabolism, energy use, signal transduction, and molecular transport. Genes down-regulated in prostate cancers were enriched in categories related to immune response, cellular responses to pathogens, and apoptosis. A heterogeneous pattern of androgen receptor expression changes was noted. In exploratory analyses, androgen receptor down-regulation was associated with a lower probability of cancer relapse after neoadjuvant chemotherapy followed by radical prostatectomy. Assessments of tumor phenotypes based on gene expression for treatment stratification and drug targeting of oncogenic alterations may best be ascertained using biopsy-based analyses where the effects of ischemia do not complicate interpretation.

  12. Prostate Cancer-Associated Gene Expression Alterations Determined from Needle Biopsies

    PubMed Central

    Qian, David Z.; Huang, Chung-Ying; O'Brien, Catherine A.; Coleman, Ilsa M.; Garzotto, Mark; True, Lawrence D.; Higano, Celestia S.; Vessella, Robert; Lange, Paul H.; Nelson, Peter S.; Beer, Tomasz M.

    2010-01-01

    Purpose To accurately identify gene expression alterations that differentiate neoplastic from normal prostate epithelium using an approach that avoids contamination by unwanted cellular components and is not compromised by acute gene expression changes associated with tumor devascularization and resulting ischemia. Experimental Design Approximately 3,000 neoplastic and benign prostate epithelial cells were isolated using laser capture microdissection from snap-frozen prostate biopsy specimens provided by 31 patients who subsequently participated in a clinical trial of preoperative chemotherapy. cDNA synthesized from amplified total RNA was hybridized to custom-made microarrays comprised of 6200 clones derived from the Prostate Expression Database. Expression differences for selected genes were verified using quantitative RT-PCR. Results Comparative analyses identified 954 transcript alterations associated with cancer (q value <0.01%) including 149 differentially expressed genes with no known functional roles. Gene expression changes associated with ischemia and surgical removal of the prostate gland were absent. Genes up-regulated in prostate cancer were statistically enriched in categories related to cellular metabolism, energy utilization, signal transduction, and molecular transport. Genes down-regulated in prostate cancers were enriched in categories related to immune response, cellular responses to pathogens, and apoptosis. A heterogeneous pattern of AR expression changes was noted. In exploratory analyses, AR down regulation was associated with a lower probability of cancer relapse after neoadjuvant chemotherapy followed by radical prostatectomy. Conclusions Assessments of tumor phenotypes based on gene expression for treatment stratification and drug targeting of oncogenic alterations may best be ascertained using biopsy-based analyses where the effects of ischemia do not complicate interpretation. PMID:19366833

  13. Bioinformatics approaches to predict target genes from transcription factor binding data.

    PubMed

    Essebier, Alexandra; Lamprecht, Marnie; Piper, Michael; Bodén, Mikael

    2017-12-01

    Transcription factors regulate gene expression and play an essential role in development by maintaining proliferative states, driving cellular differentiation and determining cell fate. Transcription factors are capable of regulating multiple genes over potentially long distances making target gene identification challenging. Currently available experimental approaches to detect distal interactions have multiple weaknesses that have motivated the development of computational approaches. Although an improvement over experimental approaches, existing computational approaches are still limited in their application, with different weaknesses depending on the approach. Here, we review computational approaches with a focus on data dependency, cell type specificity and usability. With the aim of identifying transcription factor target genes, we apply available approaches to typical transcription factor experimental datasets. We show that approaches are not always capable of annotating all transcription factor binding sites; binding sites should be treated disparately; and a combination of approaches can increase the biological relevance of the set of genes identified as targets. Copyright © 2017 Elsevier Inc. All rights reserved.

  14. Gene Expression in Bone

    NASA Astrophysics Data System (ADS)

    D'Ambrogio, A.

    Skeletal system has two main functions, to provide mechanical integrity for both locomotion and protection and to play an important role in mineral homeostasis. There is extensive evidence showing loss of bone mass during long-term Space-Flights. The loss is due to a break in the equilibrium between the activity of osteoblasts (the cells that forms bone) and the activity of osteoclasts (the cells that resorbs bone). Surprisingly, there is scanty information about the possible altered gene expression occurring in cells that form bone in microgravity.(Just 69 articles result from a "gene expression in microgravity" MedLine query.) Gene-chip or microarray technology allows to screen thousands of genes at the same time: the use of this technology on samples coming from cells exposed to microgravity could provide us with many important informations. For example, the identification of the molecules or structures which are the first sensors of the mechanical stress derived from lack of gravity, could help in understanding which is the first event leading to bone loss due to long-term exposure to microgravity. Consequently, this structure could become a target for a custom-designed drug. It is evident that bone mass loss, observed during long-time stay in Space, represents an accelerated model of what happens in aging osteoporosis. Therefore, the discovery and design of drugs able to interfere with the bone-loss process, could help also in preventing negative physiological processes normally observed on Earth. Considering the aims stated above, my research is designed to:

  15. Peptide-conjugated micelles as a targeting nanocarrier for gene delivery

    NASA Astrophysics Data System (ADS)

    Lin, Wen Jen; Chien, Wei Hsuan

    2015-09-01

    The aim of this study was to develop peptide-conjugated micelles possessing epidermal growth factor receptor (EGFR) targeting ability for gene delivery. A sequence-modified dodecylpeptide, GE11(2R), with enhancing EGF receptor binding affinity, was applied in this study as a targeting ligand. The active targeting micelles were composed of poly( d,l-lactide- co-glycolide)-poly(ethylene glycol) (PLGA-PEG) copolymer conjugated with GE11(2R)-peptide. The particle sizes of peptide-free and peptide-conjugated micelles were 277.0 ± 5.1 and 308.7 ± 14.5 nm, respectively. The peptide-conjugated micelles demonstrated the cellular uptake significantly higher than peptide-free micelles in EGFR high-expressed MDA-MB-231 and MDA-MB-468 cells due to GE11(2R)-peptide specificity. Furthermore, the peptide-conjugated micelles were able to encapsulate plasmid DNA and expressed cellular transfection higher than peptide-free micelles in EGFR high-expressed cells. The EGFR-targeting delivery micelles enhanced DNA internalized into cells and achieved higher cellular transfection in EGFR high-expressed cells.

  16. Heterologous expression of proteins from Plasmodium falciparum: results from 1000 genes.

    PubMed

    Mehlin, Christopher; Boni, Erica; Buckner, Frederick S; Engel, Linnea; Feist, Tiffany; Gelb, Michael H; Haji, Lutfiyah; Kim, David; Liu, Colleen; Mueller, Natascha; Myler, Peter J; Reddy, J T; Sampson, Joshua N; Subramanian, E; Van Voorhis, Wesley C; Worthey, Elizabeth; Zucker, Frank; Hol, Wim G J

    2006-08-01

    As part of a structural genomics initiative, 1000 open reading frames from Plasmodium falciparum, the causative agent of the most deadly form of malaria, were tested in an E. coli protein expression system. Three hundred and thirty-seven of these targets were observed to express, although typically the protein was insoluble. Sixty-three of the targets provided soluble protein in yields ranging from 0.9 to 406.6 mg from one liter of rich media. Higher molecular weight, greater protein disorder (segmental analysis, SEG), more basic isoelectric point (pI), and a lack of homology to E. coli proteins were all highly and independently correlated with difficulties in expression. Surprisingly, codon usage and the percentage of adenosines and thymidines (%AT) did not appear to play a significant role. Of those proteins which expressed, high pI and a hypothetical annotation were both strongly and independently correlated with insolubility. The overwhelmingly important role of pI in both expression and solubility appears to be a surprising and fundamental issue in the heterologous expression of P. falciparum proteins in E. coli. Twelve targets which did not express in E. coli from the native gene sequence were codon-optimized through whole gene synthesis, resulting in the (insoluble) expression of three of these proteins. Seventeen targets which were expressed insolubly in E. coli were moved into a baculovirus/Sf-21 system, resulting in the soluble expression of one protein at a high level and six others at a low level. A variety of factors conspire to make the heterologous expression of P. falciparum proteins challenging, and these observations lay the groundwork for a rational approach to prioritizing and, ultimately, eliminating these impediments.

  17. Gene expression signatures differentiate ovarian/peritoneal serous carcinoma from breast carcinoma in effusions

    PubMed Central

    Davidson, Ben; Stavnes, Helene Tuft; Holth, Arild; Chen, Xu; Yang, Yanqin; Shih, Ie-Ming; Wang, Tian-Li

    2011-01-01

    Abstract Ovarian/primary peritoneal carcinoma and breast carcinoma are the gynaecological cancers that most frequently involve the serosal cavities. With the objective of improving on the limited diagnostic panel currently available for the differential diagnosis of these two malignancies, as well as to define tumour-specific biological targets, we compared their global gene expression patterns. Gene expression profiles of 10 serous ovarian/peritoneal and eight ductal breast carcinoma effusions were analysed using the HumanRef-8 BeadChip from Illumina. Differentially expressed candidate genes were validated using quantitative real-time PCR and immunohistochemistry. Unsupervised hierarchical clustering using all 54,675 genes in the array separated ovarian from breast carcinoma samples. We identified 288 unique probes that were significantly differentially expressed in the two cancers by greater than 3.5-fold, of which 81 and 207 were overexpressed in breast and ovarian/peritoneal carcinoma, respectively. SAM analysis identified 1078 differentially expressed probes with false discovery rate less than 0.05. Genes overexpressed in breast carcinoma included TFF1, TFF3, FOXA1, CA12, GATA3, SDC1, PITX1, TH, EHFD1, EFEMP1, TOB1 and KLF2. Genes overexpressed in ovarian/peritoneal carcinoma included SPON1, RBP1, MFGE8, TM4SF12, MMP7, KLK5/6/7, FOLR1/3, PAX8, APOL2 and NRCAM. The differential expression of 14 genes was validated by quantitative real-time PCR, and differences in 5 gene products were confirmed by immunohistochemistry. Expression profiling distinguishes ovarian/peritoneal carcinoma from breast carcinoma and identifies genes that are differentially expressed in these two tumour types. The molecular signatures unique to these cancers may facilitate their differential diagnosis and may provide a molecular basis for therapeutic target discovery. PMID:20132413

  18. Targeted Adenoviral Vector Demonstrates Enhanced Efficacy for In Vivo Gene Therapy of Uterine Leiomyoma.

    PubMed

    Abdelaziz, Mohamed; Sherif, Lotfy; ElKhiary, Mostafa; Nair, Sanjeeta; Shalaby, Shahinaz; Mohamed, Sara; Eziba, Noura; El-Lakany, Mohamed; Curiel, David; Ismail, Nahed; Diamond, Michael P; Al-Hendy, Ayman

    2016-04-01

    Gene therapy is a potentially effective non-surgical approach for the treatment of uterine leiomyoma. We demonstrated that targeted adenovirus vector, Ad-SSTR-RGD-TK/GCV, was highly effective in selectively inducing apoptosis and inhibiting proliferation of human leiomyoma cells in vitro while sparing normal myometrial cells. An in-vivo study, to compare efficacy and safety of modified adenovirus vector Ad-SSTR-RGD-TK/GCV versus untargeted vector for treatment of leiomyoma. Female nude mice were implanted with rat leiomyoma cells subcutaneously. Then mice were randomized into three groups. Group 1 received Ad-LacZ (marker gene), Group 2 received untargeted Ad-TK, and Group 3 received the targeted Ad-SSTR-RGD-TK. Tumors were measured weekly for 4 weeks. Then mice were sacrificed and tissue samples were collected. Evaluation of markers of apoptosis, proliferation, extracellular matrix, and angiogenesis was performed using Western Blot & Immunohistochemistry. Statistical analysis was done using ANOVA. Dissemination of adenovirus was assessed by PCR. In comparison with the untargeted vector, the targeted adenoviral vector significantly shrank leiomyoma size (P < 0.05), reduced expression of proliferation marker (PCNA) (P < 0.05), induced expression of apoptotic protein, c-PARP-1, (P < 0.05) and inhibited expression of extracellular matrix-related genes (TGF beta 3) and angiogenesis-related genes (VEGF & IGF-1) (P < 0.01). There were no detectable adenovirus in tested tissues other than leiomyoma lesions with both targeted and untargeted adenovirus. Targeted adenovirus, effectively reduces tumor size in leiomyoma without dissemination to other organs. Further evaluation of this localized targeted strategy for gene therapy is needed in appropriate preclinical humanoid animal models in preparation for a future pilot human trial. © The Author(s) 2016.

  19. In silico analysis and expression profiling of miRNAs targeting genes of steviol glycosides biosynthetic pathway and their relationship with steviol glycosides content in different tissues of Stevia rebaudiana.

    PubMed

    Saifi, Monica; Nasrullah, Nazima; Ahmad, Malik Mobeen; Ali, Athar; Khan, Jawaid A; Abdin, M Z

    2015-09-01

    miRNAs are emerging as potential regulators of the gene expression. Their proven promising role in regulating biosynthetic pathways related gene networks may hold the key to understand the genetic regulation of these pathways which may assist in selection and manipulation to get high performing plant genotypes with better secondary metabolites yields and increased biomass. miRNAs associated with genes of steviol glycosides biosynthetic pathway, however, have not been identified so far. In this study miRNAs targeting genes of steviol glycosides biosynthetic pathway were identified for the first time whose precursors were potentially generated from ESTs and nucleotide sequences of Stevia rebaudiana. Thereafter, stem-loop coupled real time PCR based expressions of these miRNAs in different tissues of Stevia rebaudiana were investigated and their relationship pattern was analysed with the expression levels of their target mRNAs as well as steviol glycoside contents. All the miRNAs investigated showed differential expressions in all the three tissues studied, viz. leaves, flowers and stems. Out of the eleven miRNAs validated, the expression levels of nine miRNAs (miR319a, miR319b, miR319c, miR319d, miR319e, miR319f, miR319h, miRstv_7, miRstv_9) were found to be inversely related, while expression levels of the two, i.e. miR319g and miRstv_11 on the contrary, showed direct relation with the expression levels of their target mRNAs and steviol glycoside contents in the leaves, flowers and stems. This study provides a platform for better understanding of the steviol glycosides biosynthetic pathway and these miRNAs can further be employed to manipulate the biosynthesis of these metabolites to enhance their contents and yield in S. rebaudiana. Copyright © 2015 Elsevier Masson SAS. All rights reserved.

  20. Identification of Candidate B-Lymphoma Genes by Cross-Species Gene Expression Profiling

    PubMed Central

    Tompkins, Van S.; Han, Seong-Su; Olivier, Alicia; Syrbu, Sergei; Bair, Thomas; Button, Anna; Jacobus, Laura; Wang, Zebin; Lifton, Samuel; Raychaudhuri, Pradip; Morse, Herbert C.; Weiner, George; Link, Brian; Smith, Brian J.; Janz, Siegfried

    2013-01-01

    Comparative genome-wide expression profiling of malignant tumor counterparts across the human-mouse species barrier has a successful track record as a gene discovery tool in liver, breast, lung, prostate and other cancers, but has been largely neglected in studies on neoplasms of mature B-lymphocytes such as diffuse large B cell lymphoma (DLBCL) and Burkitt lymphoma (BL). We used global gene expression profiles of DLBCL-like tumors that arose spontaneously in Myc-transgenic C57BL/6 mice as a phylogenetically conserved filter for analyzing the human DLBCL transcriptome. The human and mouse lymphomas were found to have 60 concordantly deregulated genes in common, including 8 genes that Cox hazard regression analysis associated with overall survival in a published landmark dataset of DLBCL. Genetic network analysis of the 60 genes followed by biological validation studies indicate FOXM1 as a candidate DLBCL and BL gene, supporting a number of studies contending that FOXM1 is a therapeutic target in mature B cell tumors. Our findings demonstrate the value of the “mouse filter” for genomic studies of human B-lineage neoplasms for which a vast knowledge base already exists. PMID:24130802

  1. Weighted gene co-expression network analysis of expression data of monozygotic twins identifies specific modules and hub genes related to BMI.

    PubMed

    Wang, Weijing; Jiang, Wenjie; Hou, Lin; Duan, Haiping; Wu, Yili; Xu, Chunsheng; Tan, Qihua; Li, Shuxia; Zhang, Dongfeng

    2017-11-13

    The therapeutic management of obesity is challenging, hence further elucidating the underlying mechanisms of obesity development and identifying new diagnostic biomarkers and therapeutic targets are urgent and necessary. Here, we performed differential gene expression analysis and weighted gene co-expression network analysis (WGCNA) to identify significant genes and specific modules related to BMI based on gene expression profile data of 7 discordant monozygotic twins. In the differential gene expression analysis, it appeared that 32 differentially expressed genes (DEGs) were with a trend of up-regulation in twins with higher BMI when compared to their siblings. Categories of positive regulation of nitric-oxide synthase biosynthetic process, positive regulation of NF-kappa B import into nucleus, and peroxidase activity were significantly enriched within GO database and NF-kappa B signaling pathway within KEGG database. DEGs of NAMPT, TLR9, PTGS2, HBD, and PCSK1N might be associated with obesity. In the WGCNA, among the total 20 distinct co-expression modules identified, coral1 module (68 genes) had the strongest positive correlation with BMI (r = 0.56, P = 0.04) and disease status (r = 0.56, P = 0.04). Categories of positive regulation of phospholipase activity, high-density lipoprotein particle clearance, chylomicron remnant clearance, reverse cholesterol transport, intermediate-density lipoprotein particle, chylomicron, low-density lipoprotein particle, very-low-density lipoprotein particle, voltage-gated potassium channel complex, cholesterol transporter activity, and neuropeptide hormone activity were significantly enriched within GO database for this module. And alcoholism and cell adhesion molecules pathways were significantly enriched within KEGG database. Several hub genes, such as GAL, ASB9, NPPB, TBX2, IL17C, APOE, ABCG4, and APOC2 were also identified. The module eigengene of saddlebrown module (212 genes) was also significantly

  2. Automated Protocol for Large-Scale Modeling of Gene Expression Data.

    PubMed

    Hall, Michelle Lynn; Calkins, David; Sherman, Woody

    2016-11-28

    With the continued rise of phenotypic- and genotypic-based screening projects, computational methods to analyze, process, and ultimately make predictions in this field take on growing importance. Here we show how automated machine learning workflows can produce models that are predictive of differential gene expression as a function of a compound structure using data from A673 cells as a proof of principle. In particular, we present predictive models with an average accuracy of greater than 70% across a highly diverse ∼1000 gene expression profile. In contrast to the usual in silico design paradigm, where one interrogates a particular target-based response, this work opens the opportunity for virtual screening and lead optimization for desired multitarget gene expression profiles.

  3. STUDIES OF NORMAL GENE EXPRESSION IN THE RAT NASAL EPITHELIUM USNG CDNA ARRAY TECHNOLOGY

    EPA Science Inventory


    Studies of Normal Gene Expression in the Rat Nasal Epithelium Using cDNA Array

    The nasal epithelium is an important target site for chemically-induced toxicity and carcinogenicity .Gene expression data are being used increasingly for studies of such conditions. In or...

  4. The Transcription Factors Islet and Lim3 Combinatorially Regulate Ion Channel Gene Expression

    PubMed Central

    Wolfram, Verena; Southall, Tony D.; Günay, Cengiz; Prinz, Astrid A.; Brand, Andrea H.

    2014-01-01

    Expression of appropriate ion channels is essential to allow developing neurons to form functional networks. Our previous studies have identified LIM-homeodomain (HD) transcription factors (TFs), expressed by developing neurons, that are specifically able to regulate ion channel gene expression. In this study, we use the technique of DNA adenine methyltransferase identification (DamID) to identify putative gene targets of four such TFs that are differentially expressed in Drosophila motoneurons. Analysis of targets for Islet (Isl), Lim3, Hb9, and Even-skipped (Eve) identifies both ion channel genes and genes predicted to regulate aspects of dendritic and axonal morphology. Significantly, some ion channel genes are bound by more than one TF, consistent with the possibility of combinatorial regulation. One such gene is Shaker (Sh), which encodes a voltage-dependent fast K+ channel (Kv1.1). DamID reveals that Sh is bound by both Isl and Lim3. We used body wall muscle as a test tissue because in conditions of low Ca2+, the fast K+ current is carried solely by Sh channels (unlike neurons in which a second fast K+ current, Shal, also contributes). Ectopic expression of isl, but not Lim3, is sufficient to reduce both Sh transcript and Sh current level. By contrast, coexpression of both TFs is additive, resulting in a significantly greater reduction in both Sh transcript and current compared with isl expression alone. These observations provide evidence for combinatorial activity of Isl and Lim3 in regulating ion channel gene expression. PMID:24523544

  5. FUNDAMENTALS OF VITAMIN D HORMONE-REGULATED GENE EXPRESSION

    PubMed Central

    Pike, J. Wesley; Meyer, Mark B.

    2014-01-01

    Initial research focused upon several known genetic targets provided early insight into the mechanism of action of the vitamin D hormone (1,25-dihydroxyvitamin D3 (1,25(OH)2D3)). Recently, however, a series of technical advances involving the coupling of chromatin immunoprecipitation (ChIP) to unbiased methodologies that initially involved tiled DNA microarrays (ChIP-chip analysis) and now Next Generation DNA Sequencing techniques (ChIP-Seq analysis) has opened new avenues of research into the mechanisms through which 1,25(OH)2D3 regulates gene expression. In this review, we summarize briefly the results of this early work and then focus on more recent studies in which ChIP-chip and ChIP-seq analyses have been used to explore the mechanisms of 1,25(OH)2D3 action on a genome-wide scale providing specific target genes as examples. The results of this work have advanced our understanding of the mechanisms involved at both genetic and epigenetic levels and have revealed a series of new principles through which the vitamin D hormone functions to control the expression of genes. PMID:24239506

  6. Targeted Resequencing of 29 Candidate Genes and Mouse Expression Studies Implicate ZIC3 and FOXF1 in Human VATER/VACTERL Association.

    PubMed

    Hilger, Alina C; Halbritter, Jan; Pennimpede, Tracie; van der Ven, Amelie; Sarma, Georgia; Braun, Daniela A; Porath, Jonathan D; Kohl, Stefan; Hwang, Daw-Yang; Dworschak, Gabriel C; Hermann, Bernhard G; Pavlova, Anna; El-Maarri, Osman; Nöthen, Markus M; Ludwig, Michael; Reutter, Heiko; Hildebrandt, Friedhelm

    2015-12-01

    The VATER/VACTERL association describes the combination of congenital anomalies including vertebral defects, anorectal malformations, cardiac defects, tracheoesophageal fistula with or without esophageal atresia, renal malformations, and limb defects. As mutations in ciliary genes were observed in diseases related to VATER/VACTERL, we performed targeted resequencing of 25 ciliary candidate genes as well as disease-associated genes (FOXF1, HOXD13, PTEN, ZIC3) in 123 patients with VATER/VACTERL or VATER/VACTERL-like phenotype. We detected no biallelic mutation in any of the 25 ciliary candidate genes; however, identified an identical, probably disease-causing ZIC3 missense mutation (p.Gly17Cys) in four patients and a FOXF1 de novo mutation (p.Gly220Cys) in a further patient. In situ hybridization analyses in mouse embryos between E9.5 and E14.5 revealed Zic3 expression in limb and prevertebral structures, and Foxf1 expression in esophageal, tracheal, vertebral, anal, and genital tubercle tissues, hence VATER/VACTERL organ systems. These data provide strong evidence that mutations in ZIC3 or FOXF1 contribute to VATER/VACTERL. © 2015 WILEY PERIODICALS, INC.

  7. Stress amplifies sex differences in primate prefrontal profiles of gene expression.

    PubMed

    Lee, Alex G; Hagenauer, Megan; Absher, Devin; Morrison, Kathleen E; Bale, Tracy L; Myers, Richard M; Watson, Stanley J; Akil, Huda; Schatzberg, Alan F; Lyons, David M

    2017-11-02

    Stress is a recognized risk factor for mood and anxiety disorders that occur more often in women than men. Prefrontal brain regions mediate stress coping, cognitive control, and emotion. Here, we investigate sex differences and stress effects on prefrontal cortical profiles of gene expression in squirrel monkey adults. Dorsolateral, ventrolateral, and ventromedial prefrontal cortical regions from 18 females and 12 males were collected after stress or no-stress treatment conditions. Gene expression profiles were acquired using HumanHT-12v4.0 Expression BeadChip arrays adapted for squirrel monkeys. Extensive variation between prefrontal cortical regions was discerned in the expression of numerous autosomal and sex chromosome genes. Robust sex differences were also identified across prefrontal cortical regions in the expression of mostly autosomal genes. Genes with increased expression in females compared to males were overrepresented in mitogen-activated protein kinase and neurotrophin signaling pathways. Many fewer genes with increased expression in males compared to females were discerned, and no molecular pathways were identified. Effect sizes for sex differences were greater in stress compared to no-stress conditions for ventromedial and ventrolateral prefrontal cortical regions but not dorsolateral prefrontal cortex. Stress amplifies sex differences in gene expression profiles for prefrontal cortical regions involved in stress coping and emotion regulation. Results suggest molecular targets for new treatments of stress disorders in human mental health.

  8. Identification of unique expression signatures and therapeutic targets in esophageal squamous cell carcinoma

    PubMed Central

    2012-01-01

    Background Esophageal squamous cell carcinoma (ESCC), the predominant histological subtype of esophageal cancer, is characterized by high mortality. Previous work identified important mRNA expression differences between normal and tumor cells; however, to date there are limited ex vivo studies examining expression changes occurring during normal esophageal squamous cell differentiation versus those associated with tumorigenesis. In this study, we used a unique tissue microdissection strategy and microarrays to measure gene expression profiles associated with cell differentiation versus tumorigenesis in twelve cases of patient-matched normal basal squamous epithelial cells (NB), normal differentiated squamous epithelium (ND), and squamous cell cancer. Class comparison and pathway analysis were used to compare NB versus tumor in a search for unique therapeutic targets. Results As a first step towards this goal, gene expression profiles and pathways were evaluated. Overall, ND expression patterns were markedly different from NB and tumor; whereas, tumor and NB were more closely related. Tumor showed a general decrease in differentially expressed genes relative to NB as opposed to ND that exhibited the opposite trend. FSH and IgG networks were most highly dysregulated in normal differentiation and tumorigenesis, respectively. DNA repair pathways were generally elevated in NB and tumor relative to ND indicating involvement in both normal and pathological growth. PDGF signaling pathway and 12 individual genes unique to the tumor/NB comparison were identified as therapeutic targets, and 10 associated ESCC gene-drug pairs were identified. We further examined the protein expression level and the distribution patterns of four genes: ODC1, POSTN, ASPA and IGF2BP3. Ultimately, three genes (ODC1, POSTN, ASPA) were verified to be dysregulated in the same pattern at both the mRNA and protein levels. Conclusions These data reveal insight into genes and molecular pathways mediating

  9. Intracellular high cholesterol content disorders the clock genes, apoptosis-related genes and fibrinolytic-related genes rhythmic expressions in human plaque-derived vascular smooth muscle cells.

    PubMed

    Lin, Changpo; Tang, Xiao; Xu, Lirong; Qian, Ruizhe; Shi, Zhenyu; Wang, Lixin; Cai, Tingting; Yan, Dong; Fu, Weiguo; Guo, Daqiao

    2017-07-10

    The clock genes are involved in regulating cardiovascular functions, and their expression disorders would lead to circadian rhythm disruptions of clock-controlled genes (CCGs), resulting in atherosclerotic plaque formation and rupture. Our previous study revealed the rhythmic expression of clock genes were attenuated in human plaque-derived vascular smooth muscle cells (PVSMCs), but failed to detect the downstream CCGs expressions and the underlying molecular mechanism. In this study, we examined the difference of CCGs rhythmic expression between human normal carotid VSMCs (NVSMCs) and PVSMCs. Furthermore, we compared the cholesterol and triglycerides levels between two groups and the link to clock genes and CCGs expressions. Seven health donors' normal carotids and 19 carotid plaques yielded viable cultured NVSMCs and PVSMCs. The expression levels of target genes were measured by quantitative real-time PCR and Western-blot. The intracellular cholesterol and triglycerides levels were measured by kits. The circadian expressions of apoptosis-related genes and fibrinolytic-related genes were disordered. Besides, the cholesterol levels were significant higher in PVSMCs. After treated with cholesterol or oxidized low density lipoprotein (ox-LDL), the expressions of clock genes were inhibited; and the rhythmic expressions of clock genes, apoptosis-related genes and fibrinolytic-related genes were disturbed in NVSMCs, which were similar to PVSMCs. The results suggested that intracellular high cholesterol content of PVSMCs would lead to the disorders of clock genes and CCGs rhythmic expressions. And further studies should be conducted to demonstrate the specific molecular mechanisms involved.

  10. A Tupaia paramyxovirus vector system for targeting and transgene expression.

    PubMed

    Engeland, Christine E; Bossow, Sascha; Hudacek, Andrew W; Hoyler, Birgit; Förster, Judith; Veinalde, Rūta; Jäger, Dirk; Cattaneo, Roberto; Ungerechts, Guy; Springfeld, Christoph

    2017-09-01

    Viruses from the diverse family of Paramyxoviridae include important pathogens and are applied in gene therapy and for cancer treatment. The Tupaia paramyxovirus (TPMV), isolated from the kidney of a tree shrew, does not infect human cells and neutralizing antibodies against other Paramyxoviridae do not cross-react with TPMV. Here, we present a vector system for de novo generation of infectious TPMV that allows for insertion of additional genes as well as targeting using antibody single-chain variable fragments. We show that the recombinant TPMV specifically infect cells expressing the targeted receptor and replicate in human cells. This vector system provides a valuable tool for both basic research and therapeutic applications.

  11. Smooth Muscle Cell Genome Browser: Enabling the Identification of Novel Serum Response Factor Target Genes

    PubMed Central

    Lee, Moon Young; Park, Chanjae; Berent, Robyn M.; Park, Paul J.; Fuchs, Robert; Syn, Hannah; Chin, Albert; Townsend, Jared; Benson, Craig C.; Redelman, Doug; Shen, Tsai-wei; Park, Jong Kun; Miano, Joseph M.; Sanders, Kenton M.; Ro, Seungil

    2015-01-01

    Genome-scale expression data on the absolute numbers of gene isoforms offers essential clues in cellular functions and biological processes. Smooth muscle cells (SMCs) perform a unique contractile function through expression of specific genes controlled by serum response factor (SRF), a transcription factor that binds to DNA sites known as the CArG boxes. To identify SRF-regulated genes specifically expressed in SMCs, we isolated SMC populations from mouse small intestine and colon, obtained their transcriptomes, and constructed an interactive SMC genome and CArGome browser. To our knowledge, this is the first online resource that provides a comprehensive library of all genetic transcripts expressed in primary SMCs. The browser also serves as the first genome-wide map of SRF binding sites. The browser analysis revealed novel SMC-specific transcriptional variants and SRF target genes, which provided new and unique insights into the cellular and biological functions of the cells in gastrointestinal (GI) physiology. The SRF target genes in SMCs, which were discovered in silico, were confirmed by proteomic analysis of SMC-specific Srf knockout mice. Our genome browser offers a new perspective into the alternative expression of genes in the context of SRF binding sites in SMCs and provides a valuable reference for future functional studies. PMID:26241044

  12. Differential gene expression patterns in developing sexually dimorphic rat brain regions exposed to antiandrogenic, estrogenic, or complex endocrine disruptor mixtures: glutamatergic synapses as target.

    PubMed

    Lichtensteiger, Walter; Bassetti-Gaille, Catherine; Faass, Oliver; Axelstad, Marta; Boberg, Julie; Christiansen, Sofie; Rehrauer, Hubert; Georgijevic, Jelena Kühn; Hass, Ulla; Kortenkamp, Andreas; Schlumpf, Margret

    2015-04-01

    The study addressed the question whether gene expression patterns induced by different mixtures of endocrine disrupting chemicals (EDCs) administered in a higher dose range, corresponding to 450×, 200×, and 100× high-end human exposure levels, could be characterized in developing brain with respect to endocrine activity of mixture components, and which developmental processes were preferentially targeted. Three EDC mixtures, A-Mix (anti-androgenic mixture) with 8 antiandrogenic chemicals (di-n-butylphthalate, diethylhexylphthalate, vinclozolin, prochloraz, procymidone, linuron, epoxiconazole, and DDE), E-Mix (estrogenic mixture) with 4 estrogenic chemicals (bisphenol A, 4-methylbenzylidene camphor, 2-ethylhexyl 4-methoxycinnamate, and butylparaben), a complex mixture, AEP-Mix, containing the components of A-Mix and E-Mix plus paracetamol, and paracetamol alone, were administered by oral gavage to rat dams from gestation day 7 until weaning. General developmental endpoints were not affected by EDC mixtures or paracetamol. Gene expression was analyzed on postnatal day 6, during sexual brain differentiation, by exon microarray in medial preoptic area in the high-dose group, and by real-time RT-PCR in medial preoptic area and ventromedial hypothalamus in all dose groups. Expression patterns were mixture, sex, and region specific. Effects of the analgesic drug paracetamol, which exhibits antiandrogenic activity in peripheral systems, differed from those of A-Mix. All mixtures had a strong, mixture-specific impact on genes encoding for components of excitatory glutamatergic synapses and genes controlling migration and pathfinding of glutamatergic and GABAergic neurons, as well as genes linked with increased risk of autism spectrum disorders. Because development of glutamatergic synapses is regulated by sex steroids also in hippocampus, this may represent a general target of ECD mixtures.

  13. Desensitization and Incomplete Recovery of Hepatic Target Genes After Chronic Thyroid Hormone Treatment and Withdrawal in Male Adult Mice

    PubMed Central

    Ohba, Kenji; Singh, Brijesh Kumar; Sinha, Rohit Anthony; Lesmana, Ronny; Liao, Xiao-Hui; Ghosh, Sujoy; Refetoff, Samuel

    2016-01-01

    Clinical symptoms may vary and not necessarily reflect serum thyroid hormone (TH) levels during acute and chronic hyperthyroidism as well as recovery from hyperthyroidism. We thus examined changes in hepatic gene expression and serum TH/TSH levels in adult male mice treated either with a single T3 (20 μg per 100 g body weight) injection (acute T3) or daily injections for 14 days (chronic T3) followed by 10 days of withdrawal. Gene expression arrays from livers harvested at these time points showed that among positively-regulated target genes, 320 were stimulated acutely and 429 chronically by T3. Surprisingly, only 69 of 680 genes (10.1%) were induced during both periods, suggesting desensitization of the majority of acutely stimulated target genes. About 90% of positively regulated target genes returned to baseline expression levels after 10 days of withdrawal; however, 67 of 680 (9.9%) did not return to baseline despite normalization of serum TH/TSH levels. Similar findings also were observed for negatively regulated target genes. Chromatin immunoprecipitation analysis of representative positively regulated target genes suggested that acetylation of H3K9/K14 was associated with acute stimulation, whereas trimethylation of H3K4 was associated with chronic stimulation. In an in vivo model of chronic intrahepatic hyperthyroidism since birth, adult male monocarboxylate transporter-8 knockout mice also demonstrated desensitization of most acutely stimulated target genes that were examined. In summary, we have identified transcriptional desensitization and incomplete recovery of gene expression during chronic hyperthyroidism and recovery. Our findings may be a potential reason for discordance between clinical symptoms and serum TH levels observed in these conditions. PMID:26866609

  14. Hypoxia regulates alternative splicing of HIF and non-HIF target genes.

    PubMed

    Sena, Johnny A; Wang, Liyi; Heasley, Lynn E; Hu, Cheng-Jun

    2014-09-01

    Hypoxia is a common characteristic of many solid tumors. The hypoxic microenvironment stabilizes hypoxia-inducible transcription factor 1α (HIF1α) and 2α (HIF2α/EPAS1) to activate gene transcription, which promotes tumor cell survival. The majority of human genes are alternatively spliced, producing RNA isoforms that code for functionally distinct proteins. Thus, an effective hypoxia response requires increased HIF target gene expression as well as proper RNA splicing of these HIF-dependent transcripts. However, it is unclear if and how hypoxia regulates RNA splicing of HIF targets. This study determined the effects of hypoxia on alternative splicing (AS) of HIF and non-HIF target genes in hepatocellular carcinoma cells and characterized the role of HIF in regulating AS of HIF-induced genes. The results indicate that hypoxia generally promotes exon inclusion for hypoxia-induced, but reduces exon inclusion for hypoxia-reduced genes. Mechanistically, HIF activity, but not hypoxia per se is found to be necessary and sufficient to increase exon inclusion of several HIF targets, including pyruvate dehydrogenase kinase 1 (PDK1). PDK1 splicing reporters confirm that transcriptional activation by HIF is sufficient to increase exon inclusion of PDK1 splicing reporter. In contrast, transcriptional activation of a PDK1 minigene by other transcription factors in the absence of endogenous HIF target gene activation fails to alter PDK1 RNA splicing. This study demonstrates a novel function of HIF in regulating RNA splicing of HIF target genes. ©2014 American Association for Cancer Research.

  15. tCRISPRi: tunable and reversible, one-step control of gene expression

    NASA Astrophysics Data System (ADS)

    Li, Xin-Tian; Jun, Yonggun; Erickstad, Michael J.; Brown, Steven D.; Parks, Adam; Court, Donald L.; Jun, Suckjoon

    2016-12-01

    The ability to control the level of gene expression is a major quest in biology. A widely used approach employs deletion of a nonessential gene of interest (knockout), or multi-step recombineering to move a gene of interest under a repressible promoter (knockdown). However, these genetic methods are laborious, and limited for quantitative study. Here, we report a tunable CRISPR-cas system, “tCRISPRi”, for precise and continuous titration of gene expression by more than 30-fold. Our tCRISPRi system employs various previous advancements into a single strain: (1) We constructed a new strain containing a tunable arabinose operon promoter PBAD to quantitatively control the expression of CRISPR-(d)Cas protein over two orders of magnitude in a plasmid-free system. (2) tCRISPRi is reversible, and gene expression is repressed under knockdown conditions. (3) tCRISPRi shows significantly less than 10% leaky expression. (4) Most important from a practical perspective, construction of tCRISPRi to target a new gene requires only one-step of oligo recombineering. Our results show that tCRISPRi, in combination with recombineering, provides a simple and easy-to-implement tool for gene expression control, and is ideally suited for construction of both individual strains and high-throughput tunable knockdown libraries.

  16. Candidate EDA targets revealed by expression profiling of primary keratinocytes from Tabby mutant mice

    PubMed Central

    Esibizione, Diana; Cui, Chang-Yi; Schlessinger, David

    2009-01-01

    EDA, the gene mutated in anhidrotic ectodermal dysplasia, encodes ectodysplasin, a TNF superfamily member that activates NF-kB mediated transcription. To identify EDA target genes, we have earlier used expression profiling to infer genes differentially expressed at various developmental time points in Tabby (Eda-deficient) compared to wild-type mouse skin. To increase the resolution to find genes whose expression may be restricted to epidermal cells, we have now extended studies to primary keratinocyte cultures established from E19 wild-type and Tabby skin. Using microarrays bearing 44,000 gene probes, we found 385 preliminary candidate genes whose expression was significantly affected by Eda loss. By comparing expression profiles to those from Eda-A1 transgenic skin, we restricted the list to 38 “candidate EDA targets”, 14 of which were already known to be expressed in hair follicles or epidermis. We confirmed expression changes for 3 selected genes, Tbx1, Bmp7, and Jag1, both in keratinocytes and in whole skin, by Q-PCR and Western blotting analyses. Thus, by the analysis of keratinocytes, novel candidate pathways downstream of EDA were detected. PMID:18848976

  17. Rhythmic expression of miR-27b-3p targets the clock gene Bmal1 at the posttranscriptional level in the mouse liver.

    PubMed

    Zhang, Wenxiang; Wang, Peng; Chen, Siyu; Zhang, Zhao; Liang, Tingming; Liu, Chang

    2016-06-01

    Circadian clocks orchestrate daily oscillations in mammalian behaviors, physiology, and gene expression. MicroRNAs (miRNAs) play a crucial role in fine-tuning of the circadian system. However, little is known about the direct regulation of the clock genes by specific miRNAs. In this study, we found that miR-27b-3p exhibits rhythmic expression in the metabolic tissues of the mice subjected to constant darkness. MiR-27b-3p's expression is induced in livers of unfed and ob/ob mice. In addition, the oscillation phases of miR-27b-3p can be reversed by restricted feeding, suggesting a role of peripheral clock in regulating its rhythmicity. Bioinformatics analysis indicated that aryl hydrocarbon receptor nuclear translocator-like (also known as Bmal1) may be a direct target of miR-27b-3p. Luciferase reporter assay showed that miR-27b-3p suppressed Bmal1 3' UTR activity in a dose-dependent manner, and mutagenesis of their binding site abolished this suppression. Furthermore, overexpression of miR-27b-3p dose-dependently reduced the protein expression levels of BMAL1 and impaired the endogenous BMAL1 and gluconeogenic protein rhythmicity. Collectively, our results suggest that miR-27b-3p plays an important role in the posttranscriptional regulation of BMAL1 protein in the liver. MiR-27b-3p may serve as a novel node to integrate the circadian clock and energy metabolism.-Zhang, W., Wang, P., Chen, S., Zhang, Z., Liang, T., Liu, C. Rhythmic expression of miR-27b-3p targets the clock gene Bmal1 at the posttranscriptional level in the mouse liver. © FASEB.

  18. Creating and validating cis-regulatory maps of tissue-specific gene expression regulation

    PubMed Central

    O'Connor, Timothy R.; Bailey, Timothy L.

    2014-01-01

    Predicting which genomic regions control the transcription of a given gene is a challenge. We present a novel computational approach for creating and validating maps that associate genomic regions (cis-regulatory modules–CRMs) with genes. The method infers regulatory relationships that explain gene expression observed in a test tissue using widely available genomic data for ‘other’ tissues. To predict the regulatory targets of a CRM, we use cross-tissue correlation between histone modifications present at the CRM and expression at genes within 1 Mbp of it. To validate cis-regulatory maps, we show that they yield more accurate models of gene expression than carefully constructed control maps. These gene expression models predict observed gene expression from transcription factor binding in the CRMs linked to that gene. We show that our maps are able to identify long-range regulatory interactions and improve substantially over maps linking genes and CRMs based on either the control maps or a ‘nearest neighbor’ heuristic. Our results also show that it is essential to include CRMs predicted in multiple tissues during map-building, that H3K27ac is the most informative histone modification, and that CAGE is the most informative measure of gene expression for creating cis-regulatory maps. PMID:25200088

  19. [Effect of gene optimization on the expression and purification of HDV small antigen produced by genetic engineering].

    PubMed

    Ding, Jun-Ying; Meng, Qing-Ling; Guo, Min-Zhuo; Yi, Yao; Su, Qiu-Dong; Lu, Xue-Xin; Qiu, Feng; Bi, Sheng-Li

    2012-10-01

    To study the effect of gene optimization on the expression and purification of HDV small antigen produced by genetic engineering. Based on the colon preference of E. coli, the HDV small antigen original gene from GenBank was optimized. Both the original gene and the optimized gene expressed in prokaryotic cells, SDS-PAGE was made to analyze the protein expression yield and to decide which protein expression style was more proportion than the other. Furthermore, two antigens were purified by chromatography in order to compare the purity by SDS-PAGE and Image Lab software. SDS-PAGE indicated that the molecular weight of target proteins from two groups were the same as we expected. Gene optimization resulted in the higher yield and it could make the product more soluble. After chromatography, the purity of target protein from optimized gene was up to 96.3%, obviously purer than that from original gene. Gene optimization could increase the protein expression yield and solubility of genetic engineering HDV small antigen. In addition, the product from the optimized gene group was easier to be purified for diagnosis usage.

  20. Salmonella induces prominent gene expression in the rat colon

    PubMed Central

    Rodenburg, Wendy; Keijer, Jaap; Kramer, Evelien; Roosing, Susanne; Vink, Carolien; Katan, Martijn B; van der Meer, Roelof; Bovee-Oudenhoven, Ingeborg MJ

    2007-01-01

    Background Salmonella enteritidis is suggested to translocate in the small intestine. In vivo it induces gene expression changes in the ileal mucosa and Peyer's patches. Stimulation of Salmonella translocation by dietary prebiotics fermented in colon suggests involvement of the colon as well. However, effects of Salmonella on colonic gene expression in vivo are largely unknown. We aimed to characterize time dependent Salmonella-induced changes of colonic mucosal gene expression in rats using whole genome microarrays. For this, rats were orally infected with Salmonella enteritidis to mimic a foodborne infection and colonic gene expression was determined at days 1, 3 and 6 post-infection (n = 8 rats per time-point). As fructo-oligosaccharides (FOS) affect colonic physiology, we analyzed colonic mucosal gene expression of FOS-fed versus cellulose-fed rats infected with Salmonella in a separate experiment. Colonic mucosal samples were isolated at day 2 post-infection. Results Salmonella affected transport (e.g. Chloride channel calcium activated 6, H+/K+ transporting Atp-ase), antimicrobial defense (e.g. Lipopolysaccharide binding protein, Defensin 5 and phospholipase A2), inflammation (e.g. calprotectin), oxidative stress related genes (e.g. Dual oxidase 2 and Glutathione peroxidase 2) and Proteolysis (e.g. Ubiquitin D and Proteosome subunit beta type 9). Furthermore, Salmonella translocation increased serum IFNγ and many interferon-related genes in colonic mucosa. The gene most strongly induced by Salmonella infection was Pancreatitis Associated Protein (Pap), showing >100-fold induction at day 6 after oral infection. Results were confirmed by Q-PCR in individual rats. Stimulation of Salmonella translocation by dietary FOS was accompanied by enhancement of the Salmonella-induced mucosal processes, not by induction of other processes. Conclusion We conclude that the colon is a target tissue for Salmonella, considering the abundant changes in mucosal gene expression

  1. Salmonella induces prominent gene expression in the rat colon.

    PubMed

    Rodenburg, Wendy; Keijer, Jaap; Kramer, Evelien; Roosing, Susanne; Vink, Carolien; Katan, Martijn B; van der Meer, Roelof; Bovee-Oudenhoven, Ingeborg M J

    2007-09-12

    Salmonella enteritidis is suggested to translocate in the small intestine. In vivo it induces gene expression changes in the ileal mucosa and Peyer's patches. Stimulation of Salmonella translocation by dietary prebiotics fermented in colon suggests involvement of the colon as well. However, effects of Salmonella on colonic gene expression in vivo are largely unknown. We aimed to characterize time dependent Salmonella-induced changes of colonic mucosal gene expression in rats using whole genome microarrays. For this, rats were orally infected with Salmonella enteritidis to mimic a foodborne infection and colonic gene expression was determined at days 1, 3 and 6 post-infection (n = 8 rats per time-point). As fructo-oligosaccharides (FOS) affect colonic physiology, we analyzed colonic mucosal gene expression of FOS-fed versus cellulose-fed rats infected with Salmonella in a separate experiment. Colonic mucosal samples were isolated at day 2 post-infection. Salmonella affected transport (e.g. Chloride channel calcium activated 6, H+/K+ transporting Atp-ase), antimicrobial defense (e.g. Lipopolysaccharide binding protein, Defensin 5 and phospholipase A2), inflammation (e.g. calprotectin), oxidative stress related genes (e.g. Dual oxidase 2 and Glutathione peroxidase 2) and Proteolysis (e.g. Ubiquitin D and Proteosome subunit beta type 9). Furthermore, Salmonella translocation increased serum IFN gamma and many interferon-related genes in colonic mucosa. The gene most strongly induced by Salmonella infection was Pancreatitis Associated Protein (Pap), showing >100-fold induction at day 6 after oral infection. Results were confirmed by Q-PCR in individual rats. Stimulation of Salmonella translocation by dietary FOS was accompanied by enhancement of the Salmonella-induced mucosal processes, not by induction of other processes. We conclude that the colon is a target tissue for Salmonella, considering the abundant changes in mucosal gene expression.

  2. The prognostic significance of specific HOX gene expression patterns in ovarian cancer.

    PubMed

    Kelly, Zoe; Moller-Levet, Carla; McGrath, Sophie; Butler-Manuel, Simon; Kavitha Madhuri, Thumuluru; Kierzek, Andrzej M; Pandha, Hardev; Morgan, Richard; Michael, Agnieszka

    2016-10-01

    HOX genes are vital for all aspects of mammalian growth and differentiation, and their dysregulated expression is related to ovarian carcinogenesis. The aim of the current study was to establish the prognostic value of HOX dysregulation as well as its role in platinum resistance. The potential to target HOX proteins through the HOX/PBX interaction was also explored in the context of platinum resistance. HOX gene expression was determined in ovarian cancer cell lines and primary EOCs by QPCR, and compared to expression in normal ovarian epithelium and fallopian tube tissue samples. Statistical analysis included one-way ANOVA and t-tests, using statistical software R and GraphPad. The analysis identified 36 of the 39 HOX genes as being overexpressed in high grade serous EOC compared to normal tissue. We detected a molecular HOX gene-signature that predicted poor outcome. Overexpression of HOXB4 and HOXB9 was identified in high grade serous cell lines after platinum resistance developed. Targeting the HOX/PBX dimer with the HXR9 peptide enhanced the cytotoxicity of cisplatin in platinum-resistant ovarian cancer. In conclusion, this study has shown the HOX genes are highly dysregulated in ovarian cancer with high expression of HOXA13, B6, C13, D1 and D13 being predictive of poor clinical outcome. Targeting the HOX/PBX dimer in platinum-resistant cancer represents a potentially new therapeutic option that should be further developed and tested in clinical trials. © 2016 The Authors International Journal of Cancer published by John Wiley & Sons Ltd on behalf of UICC.

  3. Digital gene expression analysis of gene expression differences within Brassica diploids and allopolyploids.

    PubMed

    Jiang, Jinjin; Wang, Yue; Zhu, Bao; Fang, Tingting; Fang, Yujie; Wang, Youping

    2015-01-27

    Brassica includes many successfully cultivated crop species of polyploid origin, either by ancestral genome triplication or by hybridization between two diploid progenitors, displaying complex repetitive sequences and transposons. The U's triangle, which consists of three diploids and three amphidiploids, is optimal for the analysis of complicated genomes after polyploidization. Next-generation sequencing enables the transcriptome profiling of polyploids on a global scale. We examined the gene expression patterns of three diploids (Brassica rapa, B. nigra, and B. oleracea) and three amphidiploids (B. napus, B. juncea, and B. carinata) via digital gene expression analysis. In total, the libraries generated between 5.7 and 6.1 million raw reads, and the clean tags of each library were mapped to 18547-21995 genes of B. rapa genome. The unambiguous tag-mapped genes in the libraries were compared. Moreover, the majority of differentially expressed genes (DEGs) were explored among diploids as well as between diploids and amphidiploids. Gene ontological analysis was performed to functionally categorize these DEGs into different classes. The Kyoto Encyclopedia of Genes and Genomes analysis was performed to assign these DEGs into approximately 120 pathways, among which the metabolic pathway, biosynthesis of secondary metabolites, and peroxisomal pathway were enriched. The non-additive genes in Brassica amphidiploids were analyzed, and the results indicated that orthologous genes in polyploids are frequently expressed in a non-additive pattern. Methyltransferase genes showed differential expression pattern in Brassica species. Our results provided an understanding of the transcriptome complexity of natural Brassica species. The gene expression changes in diploids and allopolyploids may help elucidate the morphological and physiological differences among Brassica species.

  4. Casein Kinase II Regulation of the Hot1 Transcription Factor Promotes Stochastic Gene Expression*

    PubMed Central

    Burns, Laura T.; Wente, Susan R.

    2014-01-01

    In Saccharomyces cerevisiae, Hog1 MAPK is activated and induces a transcriptional program in response to hyperosmotic stress. Several Hog1-responsive genes exhibit stochastic transcription, resulting in cell-to-cell variability in mRNA and protein levels. However, the mechanisms governing stochastic gene activity are not fully defined. Here we uncover a novel role for casein kinase II (CK2) in the cellular response to hyperosmotic stress. CK2 interacts with and phosphorylates the Hot1 transcription factor; however, Hot1 phosphorylation is not sufficient for controlling the stochastic response. The CK2 protein itself is required to negatively regulate mRNA expression of Hot1-responsive genes and Hot1 enrichment at target promoters. Single-cell gene expression analysis reveals altered activation of Hot1-targeted STL1 in ck2 mutants, resulting in a bimodal to unimodal shift in expression. Together, this work reveals a novel CK2 function during the hyperosmotic stress response that promotes cell-to-cell variability in gene expression. PMID:24817120

  5. CRISPR/Cas9-mediated gene knockout screens and target identification via whole-genome sequencing uncover host genes required for picornavirus infection.

    PubMed

    Kim, Heon Seok; Lee, Kyungjin; Bae, Sangsu; Park, Jeongbin; Lee, Chong-Kyo; Kim, Meehyein; Kim, Eunji; Kim, Minju; Kim, Seokjoong; Kim, Chonsaeng; Kim, Jin-Soo

    2017-06-23

    Several groups have used genome-wide libraries of lentiviruses encoding small guide RNAs (sgRNAs) for genetic screens. In most cases, sgRNA expression cassettes are integrated into cells by using lentiviruses, and target genes are statistically estimated by the readout of sgRNA sequences after targeted sequencing. We present a new virus-free method for human gene knockout screens using a genome-wide library of CRISPR/Cas9 sgRNAs based on plasmids and target gene identification via whole-genome sequencing (WGS) confirmation of authentic mutations rather than statistical estimation through targeted amplicon sequencing. We used 30,840 pairs of individually synthesized oligonucleotides to construct the genome-scale sgRNA library, collectively targeting 10,280 human genes ( i.e. three sgRNAs per gene). These plasmid libraries were co-transfected with a Cas9-expression plasmid into human cells, which were then treated with cytotoxic drugs or viruses. Only cells lacking key factors essential for cytotoxic drug metabolism or viral infection were able to survive. Genomic DNA isolated from cells that survived these challenges was subjected to WGS to directly identify CRISPR/Cas9-mediated causal mutations essential for cell survival. With this approach, we were able to identify known and novel genes essential for viral infection in human cells. We propose that genome-wide sgRNA screens based on plasmids coupled with WGS are powerful tools for forward genetics studies and drug target discovery. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  6. Aberrant Gene Expression in Humans

    PubMed Central

    Yang, Ence; Ji, Guoli; Brinkmeyer-Langford, Candice L.; Cai, James J.

    2015-01-01

    Gene expression as an intermediate molecular phenotype has been a focus of research interest. In particular, studies of expression quantitative trait loci (eQTL) have offered promise for understanding gene regulation through the discovery of genetic variants that explain variation in gene expression levels. Existing eQTL methods are designed for assessing the effects of common variants, but not rare variants. Here, we address the problem by establishing a novel analytical framework for evaluating the effects of rare or private variants on gene expression. Our method starts from the identification of outlier individuals that show markedly different gene expression from the majority of a population, and then reveals the contributions of private SNPs to the aberrant gene expression in these outliers. Using population-scale mRNA sequencing data, we identify outlier individuals using a multivariate approach. We find that outlier individuals are more readily detected with respect to gene sets that include genes involved in cellular regulation and signal transduction, and less likely to be detected with respect to the gene sets with genes involved in metabolic pathways and other fundamental molecular functions. Analysis of polymorphic data suggests that private SNPs of outlier individuals are enriched in the enhancer and promoter regions of corresponding aberrantly-expressed genes, suggesting a specific regulatory role of private SNPs, while the commonly-occurring regulatory genetic variants (i.e., eQTL SNPs) show little evidence of involvement. Additional data suggest that non-genetic factors may also underlie aberrant gene expression. Taken together, our findings advance a novel viewpoint relevant to situations wherein common eQTLs fail to predict gene expression when heritable, rare inter-individual variation exists. The analytical framework we describe, taking into consideration the reality of differential phenotypic robustness, may be valuable for investigating

  7. Reliable reference genes for normalization of gene expression data in tea plants (Camellia sinensis) exposed to metal stresses.

    PubMed

    Wang, Ming-Le; Li, Qing-Hui; Xin, Hua-Hong; Chen, Xuan; Zhu, Xu-Jun; Li, Xing-Hui

    2017-01-01

    Tea plants [Camellia sinensis (L.) O. Kuntze] are an important leaf-type crop that are widely used for the production of non-alcoholic beverages in the world. Exposure to excessive amounts of heavy metals adversely affects the quality and yield of tea leaves. To analyze the molecular responses of tea plants to heavy metals, a reliable quantification of gene expression is important and of major importance herein is the normalization of the measured expression levels for the target genes. Ideally, stably expressed reference genes should be evaluated in all experimental systems. In this study, 12 candidate reference genes (i.e., 18S rRNA, Actin, CYP, EF-1α, eIF-4α, GAPDH, MON1, PP2AA3, TBP, TIP41, TUA, and UBC) were cloned from tea plants, and the stability of their expression was examined systematically in 60 samples exposed to diverse heavy metals (i.e., manganese, aluminum, copper, iron, and zinc). Three Excel-based algorithms (geNorm, NormFinder, and BestKeeper) were used to evaluate the expression stability of these genes. PP2AA3 and 18S rRNA were the most stably expressed genes, even though their expression profiles exhibited some variability. Moreover, commonly used reference genes (i.e., GAPDH and TBP) were the least appropriate reference genes for most samples. To further validate the suitability of the analyzed reference genes, the expression level of a phytochelatin synthase gene (i.e., CsPCS1) was determined using the putative reference genes for data normalizations. Our results may be beneficial for future studies involving the quantification of relative gene expression levels in tea plants.

  8. Reliable reference genes for normalization of gene expression data in tea plants (Camellia sinensis) exposed to metal stresses

    PubMed Central

    Wang, Ming-Le; Li, Qing-Hui; Xin, Hua-Hong; Chen, Xuan; Zhu, Xu-Jun

    2017-01-01

    Tea plants [Camellia sinensis (L.) O. Kuntze] are an important leaf-type crop that are widely used for the production of non-alcoholic beverages in the world. Exposure to excessive amounts of heavy metals adversely affects the quality and yield of tea leaves. To analyze the molecular responses of tea plants to heavy metals, a reliable quantification of gene expression is important and of major importance herein is the normalization of the measured expression levels for the target genes. Ideally, stably expressed reference genes should be evaluated in all experimental systems. In this study, 12 candidate reference genes (i.e., 18S rRNA, Actin, CYP, EF-1α, eIF-4α, GAPDH, MON1, PP2AA3, TBP, TIP41, TUA, and UBC) were cloned from tea plants, and the stability of their expression was examined systematically in 60 samples exposed to diverse heavy metals (i.e., manganese, aluminum, copper, iron, and zinc). Three Excel-based algorithms (geNorm, NormFinder, and BestKeeper) were used to evaluate the expression stability of these genes. PP2AA3 and 18S rRNA were the most stably expressed genes, even though their expression profiles exhibited some variability. Moreover, commonly used reference genes (i.e., GAPDH and TBP) were the least appropriate reference genes for most samples. To further validate the suitability of the analyzed reference genes, the expression level of a phytochelatin synthase gene (i.e., CsPCS1) was determined using the putative reference genes for data normalizations. Our results may be beneficial for future studies involving the quantification of relative gene expression levels in tea plants. PMID:28453515

  9. Genome-wide STAT3 binding analysis after histone deacetylase inhibition reveals novel target genes in dendritic cells

    PubMed Central

    Sun, Yaping; Iyer, Matthew; McEachin, Richard; Zhao, Meng; Wu, Yi-Mi; Cao, Xuhong; Oravecz-Wilson, Katherine; Zajac, Cynthia; Mathewson, Nathan; Wu, Shin-Rong Julia; Rossi, Corinne; Toubai, Tomomi; Qin, Zhaohui S.; Chinnaiya, Arul M.; Reddy, Pavan

    2016-01-01

    STAT3 is a master transcriptional regulator that plays an important role in the induction of both immune activation and immune tolerance in dendritic cells (DCs). The transcriptional targets of STAT3 in promoting DC activation are becoming increasingly understood; however, the mechanisms underpinning its role in causing DC suppression remain largely unknown. To determine the functional gene targets of STAT3, we compared the genome-wide binding of STAT3 using ChIP-seq coupled with gene expression microarrays to determine STAT3-dependent gene regulation in DCs after histone deacetylase (HDAC) inhibition. HDAC inhibition boosted the ability of STAT3 to bind to distinct DNA targets and regulate gene expression. Among the top 500 STAT3 binding sites, the frequency of canonical motifs was significantly higher than that of non-canonical motifs. Functional analysis revealed that after treatment with an HDAC inhibitor, the upregulated STAT3 target genes were those that were primarily the negative regulators of pro-inflammatory cytokines and those in the IL-10 signaling pathway. The downregulated STAT3-dependent targets were those involved in immune effector processes and antigen processing/presentation. The expression and functional relevance of these genes were validated. Specifically, functional studies confirmed that the upregulation of IL-10Ra by STAT3 contributed to the suppressive function of DCs following HDAC inhibition. PMID:27866206

  10. Gene expression profile of isolated rat adipocytes treated with anthocyanins.

    PubMed

    Tsuda, Takanori; Ueno, Yuki; Kojo, Hitoshi; Yoshikawa, Toshikazu; Osawa, Toshihiko

    2005-04-15

    Adipocyte dysfunction is strongly associated with the development of obesity and insulin resistance. It is accepted that the regulation of adipocytokine secretion or the adipocyte specific gene expression is one of the most important targets for the prevention of obesity and amelioration of insulin sensitivity. Recently, we demonstrated that anthocyanins, which are pigments widespread in the plant kingdom, have the potency of anti-obesity in mice and the enhancement adipocytokine secretion and adipocyte gene expression in adipocytes. In this study, we have shown for the first time the gene expression profile in isolated rat adipocytes treated with anthocyanins (cyanidin 3-glucoside; C3G or cyanidin; Cy). The rat adipocytes were treated with 100 muM C3G, Cy or vehicle for 24 h. The total RNA from the adipocytes was isolated and carried out GeneChip microarray analysis. A total of 633 or 427 genes was up-regulated (>1.5-fold) by the treatment of adipocytes with C3G or Cy, respectively. The up-regulated genes include lipid metabolism and signal transduction-related genes, however, the altered genes were partly different between the C3G- and Cy-treated groups. Based on the gene expression profile, we demonstrated the up-regulation of hormone sensitive lipase and enhancement of the lipolytic activity by the treatment of adipocytes with C3G or Cy. These data have provided an overview of the gene expression profiles in adipocytes treated with anthocyanins and identified new responsive genes with potentially important functions in adipocytes related with obesity and diabetes that merit further investigation.

  11. Fusion genes in solid tumors: an emerging target for cancer diagnosis and treatment.

    PubMed

    Parker, Brittany C; Zhang, Wei

    2013-11-01

    Studies over the past decades have uncovered fusion genes, a class of oncogenes that provide immense diagnostic and therapeutic advantages because of their tumor-specific expression. Originally associated with hemotologic cancers, fusion genes have recently been discovered in a wide array of solid tumors, including sarcomas, carcinomas, and tumors of the central nervous system. Fusion genes are attractive as both therapeutic targets and diagnostic tools due to their inherent expression in tumor tissue alone. Therefore, the discovery and elucidation of fusion genes in various cancer types may provide more effective therapies in the future for cancer patients.

  12. Suitable Reference Genes for Accurate Gene Expression Analysis in Parsley (Petroselinum crispum) for Abiotic Stresses and Hormone Stimuli

    PubMed Central

    Li, Meng-Yao; Song, Xiong; Wang, Feng; Xiong, Ai-Sheng

    2016-01-01

    Parsley, one of the most important vegetables in the Apiaceae family, is widely used in the food, medicinal, and cosmetic industries. Recent studies on parsley mainly focus on its chemical composition, and further research involving the analysis of the plant's gene functions and expressions is required. qPCR is a powerful method for detecting very low quantities of target transcript levels and is widely used to study gene expression. To ensure the accuracy of results, a suitable reference gene is necessary for expression normalization. In this study, four software, namely geNorm, NormFinder, BestKeeper, and RefFinder were used to evaluate the expression stabilities of eight candidate reference genes of parsley (GAPDH, ACTIN, eIF-4α, SAND, UBC, TIP41, EF-1α, and TUB) under various conditions, including abiotic stresses (heat, cold, salt, and drought) and hormone stimuli treatments (GA, SA, MeJA, and ABA). Results showed that EF-1α and TUB were the most stable genes for abiotic stresses, whereas EF-1α, GAPDH, and TUB were the top three choices for hormone stimuli treatments. Moreover, EF-1α and TUB were the most stable reference genes among all tested samples, and UBC was the least stable one. Expression analysis of PcDREB1 and PcDREB2 further verified that the selected stable reference genes were suitable for gene expression normalization. This study can guide the selection of suitable reference genes in gene expression in parsley. PMID:27746803

  13. Suitable Reference Genes for Accurate Gene Expression Analysis in Parsley (Petroselinum crispum) for Abiotic Stresses and Hormone Stimuli.

    PubMed

    Li, Meng-Yao; Song, Xiong; Wang, Feng; Xiong, Ai-Sheng

    2016-01-01

    Parsley, one of the most important vegetables in the Apiaceae family, is widely used in the food, medicinal, and cosmetic industries. Recent studies on parsley mainly focus on its chemical composition, and further research involving the analysis of the plant's gene functions and expressions is required. qPCR is a powerful method for detecting very low quantities of target transcript levels and is widely used to study gene expression. To ensure the accuracy of results, a suitable reference gene is necessary for expression normalization. In this study, four software, namely geNorm, NormFinder, BestKeeper, and RefFinder were used to evaluate the expression stabilities of eight candidate reference genes of parsley ( GAPDH, ACTIN, eIF-4 α, SAND, UBC, TIP41, EF-1 α, and TUB ) under various conditions, including abiotic stresses (heat, cold, salt, and drought) and hormone stimuli treatments (GA, SA, MeJA, and ABA). Results showed that EF-1 α and TUB were the most stable genes for abiotic stresses, whereas EF-1 α, GAPDH , and TUB were the top three choices for hormone stimuli treatments. Moreover, EF-1 α and TUB were the most stable reference genes among all tested samples, and UBC was the least stable one. Expression analysis of PcDREB1 and PcDREB2 further verified that the selected stable reference genes were suitable for gene expression normalization. This study can guide the selection of suitable reference genes in gene expression in parsley.

  14. Gene expression in lung adenocarcinomas of smokers and nonsmokers.

    PubMed

    Powell, Charles A; Spira, Avrum; Derti, Adnan; DeLisi, Charles; Liu, Gang; Borczuk, Alain; Busch, Steve; Sahasrabudhe, Sudhir; Chen, Yangde; Sugarbaker, David; Bueno, Raphael; Richards, William G; Brody, Jerome S

    2003-08-01

    Adenocarcinoma (AC) has become the most frequent type of lung cancer in men and women, and is the major form of lung cancer in nonsmokers. Our goal in this paper was to determine if AC in smokers and nonsmokers represents the same genetic disease. We compared gene expression profiles in resected samples of nonmalignant lung tissue and tumor tissue in six never-smokers with AC and in six smokers with AC, who were matched for clinical staging and histologic criteria of cell differentiation. Results were analyzed using a variety of bioinformatic tools. Four times as many genes changed expression in the transition from noninvolved lung to tumor in nonsmokers as in smokers, suggesting that AC in nonsmokers evolves locally, whereas AC in smokers evolves in a field of genetically altered tissue. There were some similarities in gene expression in smokers and nonsmokers, but many differences, suggesting different pathways of cell transformation and tumor formation. Gene expression in the noninvolved lungs of smokers differed from that of nonsmokers, and multidimensional scaling showed that noninvolved lungs of smokers groups with tumors rather than noninvolved lungs of nonsmokers. In addition, expression of a number of genes correlated with smoking intensity. Our findings, although limited by small sample size, suggest that additional studies comparing noninvolved to tumor tissue may identify pathogenetic mechanisms and therapeutic targets that differ in AC of smokers and nonsmokers.

  15. PiiL: visualization of DNA methylation and gene expression data in gene pathways.

    PubMed

    Moghadam, Behrooz Torabi; Zamani, Neda; Komorowski, Jan; Grabherr, Manfred

    2017-08-02

    DNA methylation is a major mechanism involved in the epigenetic state of a cell. It has been observed that the methylation status of certain CpG sites close to or within a gene can directly affect its expression, either by silencing or, in some cases, up-regulating transcription. However, a vertebrate genome contains millions of CpG sites, all of which are potential targets for methylation, and the specific effects of most sites have not been characterized to date. To study the complex interplay between methylation status, cellular programs, and the resulting phenotypes, we present PiiL, an interactive gene expression pathway browser, facilitating analyses through an integrated view of methylation and expression on multiple levels. PiiL allows for specific hypothesis testing by quickly assessing pathways or gene networks, where the data is projected onto pathways that can be downloaded directly from the online KEGG database. PiiL provides a comprehensive set of analysis features that allow for quick and specific pattern searches. Individual CpG sites and their impact on host gene expression, as well as the impact on other genes present in the regulatory network, can be examined. To exemplify the power of this approach, we analyzed two types of brain tumors, Glioblastoma multiform and lower grade gliomas. At a glance, we could confirm earlier findings that the predominant methylation and expression patterns separate perfectly by mutations in the IDH genes, rather than by histology. We could also infer the IDH mutation status for samples for which the genotype was not known. By applying different filtering methods, we show that a subset of CpG sites exhibits consistent methylation patterns, and that the status of sites affect the expression of key regulator genes, as well as other genes located downstream in the same pathways. PiiL is implemented in Java with focus on a user-friendly graphical interface. The source code is available under the GPL license from https://github.com/behroozt/PiiL.git .

  16. Targeted Gene Editing of Glia Maturation Factor in Microglia: a Novel Alzheimer's Disease Therapeutic Target.

    PubMed

    Raikwar, Sudhanshu P; Thangavel, Ramasamy; Dubova, Iuliia; Selvakumar, Govindhasamy Pushpavathi; Ahmed, Mohammad Ejaz; Kempuraj, Duraisamy; Zaheer, Smita A; Iyer, Shankar S; Zaheer, Asgar

    2018-04-27

    Alzheimer's disease (AD) is a devastating, progressive neurodegenerative disorder that leads to severe cognitive impairment in elderly patients. Chronic neuroinflammation plays an important role in the AD pathogenesis. Glia maturation factor (GMF), a proinflammatory molecule discovered in our laboratory, is significantly upregulated in various regions of AD brains. We have previously reported that GMF is predominantly expressed in the reactive glial cells surrounding the amyloid plaques (APs) in the mouse and human AD brain. Microglia are the major source of proinflammatory cytokines and chemokines including GMF. Recently clustered regularly interspaced short palindromic repeats (CRISPR) based genome editing has been recognized to study the functions of genes that are implicated in various diseases. Here, we investigated if CRISPR-Cas9-mediated GMF gene editing leads to inhibition of GMF expression and suppression of microglial activation. Confocal microscopy of murine BV2 microglial cell line transduced with an adeno-associated virus (AAV) coexpressing Staphylococcus aureus (Sa) Cas9 and a GMF-specific guide RNA (GMF-sgRNA) revealed few cells expressing SaCas9 while lacking GMF expression, thereby confirming successful GMF gene editing. To further improve GMF gene editing efficiency, we developed lentiviral vectors (LVs) expressing either Streptococcus pyogenes (Sp) Cas9 or GMF-sgRNAs. BV2 cells cotransduced with LVs expressing SpCas9 and GMF-sgRNAs revealed reduced GMF expression and the presence of indels in the exons 2 and 3 of the GMF coding sequence. Lipopolysaccharide (LPS) treatment of GMF-edited cells led to reduced microglial activation as shown by reduced p38 MAPK phosphorylation. We believe that targeted in vivo GMF gene editing has a significant potential for developing a unique and novel AD therapy.

  17. Stability evaluation of reference genes for gene expression analysis by RT-qPCR in soybean under different conditions.

    PubMed

    Wan, Qiao; Chen, Shuilian; Shan, Zhihui; Yang, Zhonglu; Chen, Limiao; Zhang, Chanjuan; Yuan, Songli; Hao, Qinnan; Zhang, Xiaojuan; Qiu, Dezhen; Chen, Haifeng; Zhou, Xinan

    2017-01-01

    Real-time quantitative reverse transcription PCR is a sensitive and widely used technique to quantify gene expression. To achieve a reliable result, appropriate reference genes are highly required for normalization of transcripts in different samples. In this study, 9 previously published reference genes (60S, Fbox, ELF1A, ELF1B, ACT11, TUA5, UBC4, G6PD, CYP2) of soybean [Glycine max (L.) Merr.] were selected. The expression stability of the 9 genes was evaluated under conditions of biotic stress caused by infection with soybean mosaic virus, nitrogen stress, across different cultivars and developmental stages. ΔCt and geNorm algorithms were used to evaluate and rank the expression stability of the 9 reference genes. Results obtained from two algorithms showed high consistency. Moreover, results of pairwise variation showed that two reference genes were sufficient to normalize the expression levels of target genes under each experimental setting. For virus infection, ELF1A and ELF1B were the most stable reference genes for accurate normalization. For different developmental stages, Fbox and G6PD had the highest expression stability between two soybean cultivars (Tanlong No. 1 and Tanlong No. 2). ELF1B and ACT11 were identified as the most stably expressed reference genes both under nitrogen stress and among different cultivars. The results showed that none of the candidate reference genes were uniformly expressed at different conditions, and selecting appropriate reference genes was pivotal for gene expression studies with particular condition and tissue. The most stable combination of genes identified in this study will help to achieve more accurate and reliable results in a wide variety of samples in soybean.

  18. DGEM--a microarray gene expression database for primary human disease tissues.

    PubMed

    Xia, Yuni; Campen, Andrew; Rigsby, Dan; Guo, Ying; Feng, Xingdong; Su, Eric W; Palakal, Mathew; Li, Shuyu

    2007-01-01

    Gene expression patterns can reflect gene regulations in human tissues under normal or pathologic conditions. Gene expression profiling data from studies of primary human disease samples are particularly valuable since these studies often span many years in order to collect patient clinical information and achieve a large sample size. Disease-to-Gene Expression Mapper (DGEM) provides a beneficial community resource to access and analyze these data; it currently includes Affymetrix oligonucleotide array datasets for more than 40 human diseases and 1400 samples. The data are normalized to the same scale and stored in a relational database. A statistical-analysis pipeline was implemented to identify genes abnormally expressed in disease tissues or genes whose expressions are associated with clinical parameters such as cancer patient survival. Data-mining results can be queried through a web-based interface at http://dgem.dhcp.iupui.edu/. The query tool enables dynamic generation of graphs and tables that are further linked to major gene and pathway resources that connect the data to relevant biology, including Entrez Gene and Kyoto Encyclopedia of Genes and Genomes (KEGG). In summary, DGEM provides scientists and physicians a valuable tool to study disease mechanisms, to discover potential disease biomarkers for diagnosis and prognosis, and to identify novel gene targets for drug discovery. The source code is freely available for non-profit use, on request to the authors.

  19. In silico identification and characterization of conserved miRNAs and their target genes in sweet potato (Ipomoea batatas L.) Expressed Sequence Tags (ESTs)

    PubMed Central

    Dehury, Budheswar; Panda, Debashis; Sahu, Jagajjit; Sahu, Mousumi; Sarma, Kishore; Barooah, Madhumita; Sen, Priyabrata; Modi, Mahendra Kumar

    2013-01-01

    The endogenous small non-coding micro RNAs (miRNAs), which are typically ~21–24 nt nucleotides, play a crucial role in regulating the intrinsic normal growth of cells and development of the plants as well as in maintaining the integrity of genomes. These small non-coding RNAs function as the universal specificity factors in post-transcriptional gene silencing. Discovering miRNAs, identifying their targets, and further inferring miRNA functions is a routine process to understand normal biological processes of miRNAs and their roles in the development of plants. Comparative genomics based approach using expressed sequence tags (EST) and genome survey sequences (GSS) offer a cost-effective platform for identification and characterization of miRNAs and their target genes in plants. Despite the fact that sweet potato (Ipomoea batatas L.) is an important staple food source for poor small farmers throughout the world, the role of miRNA in various developmental processes remains largely unknown. In this paper, we report the computational identification of miRNAs and their target genes in sweet potato from their ESTs. Using comparative genomics-based approach, 8 potential miRNA candidates belonging to miR168, miR2911, and miR156 families were identified from 23 406 ESTs in sweet potato. A total of 42 target genes were predicted and their probable functions were illustrated. Most of the newly identified miRNAs target transcription factors as well as genes involved in plant growth and development, signal transduction, metabolism, defense, and stress response. The identification of miRNAs and their targets is expected to accelerate the pace of miRNA discovery, leading to an improved understanding of the role of miRNA in development and physiology of sweet potato, as well as stress response. PMID:24067297

  20. Three gene expression vector sets for concurrently expressing multiple genes in Saccharomyces cerevisiae.

    PubMed

    Ishii, Jun; Kondo, Takashi; Makino, Harumi; Ogura, Akira; Matsuda, Fumio; Kondo, Akihiko

    2014-05-01

    Yeast has the potential to be used in bulk-scale fermentative production of fuels and chemicals due to its tolerance for low pH and robustness for autolysis. However, expression of multiple external genes in one host yeast strain is considerably labor-intensive due to the lack of polycistronic transcription. To promote the metabolic engineering of yeast, we generated systematic and convenient genetic engineering tools to express multiple genes in Saccharomyces cerevisiae. We constructed a series of multi-copy and integration vector sets for concurrently expressing two or three genes in S. cerevisiae by embedding three classical promoters. The comparative expression capabilities of the constructed vectors were monitored with green fluorescent protein, and the concurrent expression of genes was monitored with three different fluorescent proteins. Our multiple gene expression tool will be helpful to the advanced construction of genetically engineered yeast strains in a variety of research fields other than metabolic engineering. © 2014 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved.

  1. Targeted Adenoviral Vector Demonstrates Enhanced Efficacy for In Vivo Gene Therapy of Uterine Leiomyoma

    PubMed Central

    Abdelaziz, Mohamed; Sherif, Lotfy; ElKhiary, Mostafa; Nair, Sanjeeta; Shalaby, Shahinaz; Mohamed, Sara; Eziba, Noura; El-Lakany, Mohamed; Curiel, David; Ismail, Nahed; Diamond, Michael P.; Al-Hendy, Ayman

    2016-01-01

    Background: Gene therapy is a potentially effective non-surgical approach for the treatment of uterine leiomyoma. We demonstrated that targeted adenovirus vector, Ad-SSTR-RGD-TK/GCV, was highly effective in selectively inducing apoptosis and inhibiting proliferation of human leiomyoma cells in vitro while sparing normal myometrial cells. Study design: An in-vivo study, to compare efficacy and safety of modified adenovirus vector Ad-SSTR-RGD-TK/GCV versus untargeted vector for treatment of leiomyoma. Materials and methods: Female nude mice were implanted with rat leiomyoma cells subcutaneously. Then mice were randomized into three groups. Group 1 received Ad-LacZ (marker gene), Group 2 received untargeted Ad-TK, and Group 3 received the targeted Ad-SSTR-RGD-TK. Tumors were measured weekly for 4 weeks. Then mice were sacrificed and tissue samples were collected. Evaluation of markers of apoptosis, proliferation, extracellular matrix, and angiogenesis was performed using Western Blot & Immunohistochemistry. Statistical analysis was done using ANOVA. Dissemination of adenovirus was assessed by PCR. Results: In comparison with the untargeted vector, the targeted adenoviral vector significantly shrank leiomyoma size (P < 0.05), reduced expression of proliferation marker (PCNA) (P < 0.05), induced expression of apoptotic protein, c-PARP-1, (P < 0.05) and inhibited expression of extracellular matrix-related genes (TGF beta 3) and angiogenesis-related genes (VEGF & IGF-1) (P < 0.01). There were no detectable adenovirus in tested tissues other than leiomyoma lesions with both targeted and untargeted adenovirus. Conclusion: Targeted adenovirus, effectively reduces tumor size in leiomyoma without dissemination to other organs. Further evaluation of this localized targeted strategy for gene therapy is needed in appropriate preclinical humanoid animal models in preparation for a future pilot human trial. PMID:26884457

  2. Inhibition of Gene Expression in Escherichia coli by Antisense Phosphorodiamidate Morpholino Oligomers

    PubMed Central

    Geller, B. L.; Deere, J. D.; Stein, D. A.; Kroeker, A. D.; Moulton, H. M.; Iversen, P. L.

    2003-01-01

    Antisense phosphorodiamidate morpholino oligomers (PMOs) were tested for the ability to inhibit gene expression in Escherichia coli. PMOs targeted to either a myc-luciferase reporter gene product or 16S rRNA did not inhibit luciferase expression or growth. However, in a strain with defective lipopolysaccharide (lpxA mutant), which has a leaky outer membrane, PMOs targeted to the myc-luciferase or acyl carrier protein (acpP) mRNA significantly inhibited their targets in a dose-dependent response. A significant improvement was made by covalently joining the peptide (KFF)3KC to the end of PMOs. In strains with an intact outer membrane, (KFF)3KC-myc PMO inhibited luciferase expression by 63%. A second (KFF)3KC-PMO conjugate targeted to lacI mRNA induced β-galactosidase in a dose-dependent response. The end of the PMO to which (KFF)3KC is attached affected the efficiency of target inhibition but in various ways depending on the PMO. Another peptide-lacI PMO conjugate was synthesized with the cationic peptide CRRRQRRKKR and was found not to induce β-galactosidase. We conclude that the outer membrane of E. coli inhibits entry of PMOs and that (KFF)3KC-PMO conjugates are transported across both membranes and specifically inhibit expression of their genetic targets. PMID:14506035

  3. Multilevel Regulation of Bacterial Gene Expression with the Combined STAR and Antisense RNA System.

    PubMed

    Lee, Young Je; Kim, Soo-Jung; Moon, Tae Seok

    2018-03-16

    Synthetic small RNA regulators have emerged as a versatile tool to predictably control bacterial gene expression. Owing to their simple design principles, small size, and highly orthogonal behavior, these engineered genetic parts have been incorporated into genetic circuits. However, efforts to achieve more sophisticated cellular functions using RNA regulators have been hindered by our limited ability to integrate different RNA regulators into complex circuits. Here, we present a combined RNA regulatory system in Escherichia coli that uses small transcription activating RNA (STAR) and antisense RNA (asRNA) to activate or deactivate target gene expression in a programmable manner. Specifically, we demonstrated that the activated target output by the STAR system can be deactivated by expressing two different types of asRNAs: one binds to and sequesters the STAR regulator, affecting the transcription process, while the other binds to the target mRNA, affecting the translation process. We improved deactivation efficiencies (up to 96%) by optimizing each type of asRNA and then integrating the two optimized asRNAs into a single circuit. Furthermore, we demonstrated that the combined STAR and asRNA system can control gene expression in a reversible way and can regulate expression of a gene in the genome. Lastly, we constructed and simultaneously tested two A AND NOT B logic gates in the same cell to show sophisticated multigene regulation by the combined system. Our approach establishes a methodology for integrating multiple RNA regulators to rationally control multiple genes.

  4. Murine Hyperglycemic Vasculopathy and Cardiomyopathy: Whole-Genome Gene Expression Analysis Predicts Cellular Targets and Regulatory Networks Influenced by Mannose Binding Lectin

    PubMed Central

    Zou, Chenhui; La Bonte, Laura R.; Pavlov, Vasile I.; Stahl, Gregory L.

    2012-01-01

    Hyperglycemia, in the absence of type 1 or 2 diabetes, is an independent risk factor for cardiovascular disease. We have previously demonstrated a central role for mannose binding lectin (MBL)-mediated cardiac dysfunction in acute hyperglycemic mice. In this study, we applied whole-genome microarray data analysis to investigate MBL’s role in systematic gene expression changes. The data predict possible intracellular events taking place in multiple cellular compartments such as enhanced insulin signaling pathway sensitivity, promoted mitochondrial respiratory function, improved cellular energy expenditure and protein quality control, improved cytoskeleton structure, and facilitated intracellular trafficking, all of which may contribute to the organismal health of MBL null mice against acute hyperglycemia. Our data show a tight association between gene expression profile and tissue function which might be a very useful tool in predicting cellular targets and regulatory networks connected with in vivo observations, providing clues for further mechanistic studies. PMID:22375142

  5. The Schizophrenia Risk Gene MIR137 Acts as a Hippocampal Gene Network Node Orchestrating the Expression of Genes Relevant to Nervous System Development and Function

    PubMed Central

    Loohuis, Nikkie FM Olde; Kasri, Nael Nadif; Glennon, Jeffrey C; van Bokhoven, Hans; Hébert, Sébastien S; Kaplan, Barry B.; Martens, Gerard JM; Aschrafi, Armaz

    2016-01-01

    MicroRNAs (miRs) are small regulatory molecules, which orchestrate neuronal development and plasticity through modulation of complex gene networks. microRNA-137 (miR-137) is a brain-enriched RNA with a critical role in regulating brain development and in mediating synaptic plasticity. Importantly, mutations in this miR are associated with the pathoetiology of schizophrenia (SZ), and there is a widespread assumption that disruptions in miR-137 expression lead to aberrant expression of gene regulatory networks associated with SZ. To systematically identify the mRNA targets for this miR, we performed miR-137 gain- and loss-of-function experiments in primary rat hippocampal neurons and profiled differentially expressed mRNAs through next-generation sequencing. We identified 500 genes that were bidirectionally activated or repressed in their expression by the modulation of miR-137 levels. Gene ontology analysis using two independent software resources suggested functions for these miR-137-regulated genes in neurodevelopmental processes, neuronal maturation processes and cell maintenance, all of which known to be critical for proper brain circuitry formation. Since many of the putative miR-137 targets identified here also have been previously shown to be associated with SZ, we propose that this miR acts as a critical gene network hub contributing to the pathophysiology of this neurodevelopmental disorder. PMID:26925706

  6. A Meta-Analysis: Identification of Common Mir-145 Target Genes that have Similar Behavior in Different GEO Datasets.

    PubMed

    Pashaei, Elnaz; Guzel, Esra; Ozgurses, Mete Emir; Demirel, Goksun; Aydin, Nizamettin; Ozen, Mustafa

    MicroRNAs, which are small regulatory RNAs, post-transcriptionally regulate gene expression by binding 3'-UTR of their mRNA targets. Their deregulation has been shown to cause increased proliferation, migration, invasion, and apoptosis. miR-145, an important tumor supressor microRNA, has shown to be downregulated in many cancer types and has crucial roles in tumor initiation, progression, metastasis, invasion, recurrence, and chemo-radioresistance. Our aim is to investigate potential common target genes of miR-145, and to help understanding the underlying molecular pathways of tumor pathogenesis in association with those common target genes. Eight published microarray datasets, where targets of mir-145 were investigated in cell lines upon mir-145 over expression, were included into this study for meta-analysis. Inter group variabilities were assessed by box-plot analysis. Microarray datasets were analyzed using GEOquery package in Bioconducter 3.2 with R version 3.2.2 and two-way Hierarchical Clustering was used for gene expression data analysis. Meta-analysis of different GEO datasets showed that UNG, FUCA2, DERA, GMFB, TF, and SNX2 were commonly downregulated genes, whereas MYL9 and TAGLN were found to be commonly upregulated upon mir-145 over expression in prostate, breast, esophageal, bladder cancer, and head and neck squamous cell carcinoma. Biological process, molecular function, and pathway analysis of these potential targets of mir-145 through functional enrichments in PPI network demonstrated that those genes are significantly involved in telomere maintenance, DNA binding and repair mechanisms. As a conclusion, our results indicated that mir-145, through targeting its common potential targets, may significantly contribute to tumor pathogenesis in distinct cancer types and might serve as an important target for cancer therapy.

  7. Assessment of α-fetoprotein targeted HSV1-tk expression in hepatocellular carcinoma with in vivo imaging.

    PubMed

    Park, Ju Hui; Kim, Kwang Il; Lee, Kyo Chul; Lee, Yong Jin; Lee, Tae Sup; Chung, Wee Sup; Lim, Sang Moo; Kang, Joo Hyun

    2015-02-01

    Tumor-specific enhancer/promoter is applicable for targeting gene expression in tumors and helpful for tumor-targeting imaging and therapy. We aimed to acquire α-fetoprotein (AFP)-producing hepatocellular carcinoma (HCC) specific images using adenovirus containing HSV1-tk gene controlled by AFP enhancer/promoter and evaluate in vivo ganciclovir (GCV)-medicated therapeutic effects on AFP-targeted HSV1-tk expression with (18)F-FDG positron emission tomography (PET). Recombinant adenovirus expressing HSV1-tk under AFP enhancer/promoter was produced (AdAFP-TK) and the expression levels were evaluated by RT-PCR and (125)I-IVDU uptake. GCV-mediated HSV1-tk cytotoxicity was determined by MTT assay. After the mixture of AdAFP-fLuc and AdAFP-TK was administrated, bioluminescent images (BLIs) and (18)F-FHBG PET images were obtained in tumor-bearing mice. In vivo therapeutic effects of AdAFP-TK and GCV in the HuH-7 xenograft model were monitored by (18)F-FDG PET. When infected with AdAFP-TK, cell viability in HuH-7 was reduced, but those in HT-29 and SK-Hep-1 were not significantly decreased at any GCV concentration less than 100 μM. AFP-targeted fLuc and HSV1-tk expression were clearly visualized by BLI and (18)F-FHBG PET images in AFP-producing HCC, respectively. In vivo GCV-mediated tumor growth inhibition by AFP-targeted HSV1-tk expression was monitored by (18)F-FDG PET. Recombinant AdAFP-TK could be applied for AFP-targeted HCC gene therapy and imaging in AFP-producing HCC.

  8. Enhancer connectome in primary human cells identifies target genes of disease-associated DNA elements

    PubMed Central

    Mumbach, Maxwell R; Satpathy, Ansuman T; Boyle, Evan A; Dai, Chao; Gowen, Benjamin G; Cho, Seung Woo; Nguyen, Michelle L; Rubin, Adam J; Granja, Jeffrey M; Kazane, Katelynn R; Wei, Yuning; Nguyen, Trieu; Greenside, Peyton G; Corces, M Ryan; Tycko, Josh; Simeonov, Dimitre R; Suliman, Nabeela; Li, Rui; Xu, Jin; Flynn, Ryan A; Kundaje, Anshul; Khavari, Paul A; Marson, Alexander; Corn, Jacob E; Quertermous, Thomas; Greenleaf, William J; Chang, Howard Y

    2018-01-01

    The challenge of linking intergenic mutations to target genes has limited molecular understanding of human diseases. Here we show that H3K27ac HiChIP generates high-resolution contact maps of active enhancers and target genes in rare primary human T cell subtypes and coronary artery smooth muscle cells. Differentiation of naive T cells into T helper 17 cells or regulatory T cells creates subtype-specific enhancer–promoter interactions, specifically at regions of shared DNA accessibility. These data provide a principled means of assigning molecular functions to autoimmune and cardiovascular disease risk variants, linking hundreds of noncoding variants to putative gene targets. Target genes identified with HiChIP are further supported by CRISPR interference and activation at linked enhancers, by the presence of expression quantitative trait loci, and by allele-specific enhancer loops in patient-derived primary cells. The majority of disease-associated enhancers contact genes beyond the nearest gene in the linear genome, leading to a fourfold increase in the number of potential target genes for autoimmune and cardiovascular diseases. PMID:28945252

  9. Cytokinin oxidase/dehydrogenase genes in barley and wheat: cloning and heterologous expression.

    PubMed

    Galuszka, Petr; Frébortová, Jitka; Werner, Tomás; Yamada, Mamoru; Strnad, Miroslav; Schmülling, Thomas; Frébort, Ivo

    2004-10-01

    The cloning of two novel genes that encode cytokinin oxidase/dehydrogenase (CKX) in barley is described in this work. Transformation of both genes into Arabidopsis and tobacco showed that at least one of the genes codes for a functional enzyme, as its expression caused a cytokinin-deficient phenotype in the heterologous host plants. Additional cloning of two gene fragments, and an in silico search in the public expressed sequence tag clone databases, revealed the presence of at least 13 more members of the CKX gene family in barley and wheat. The expression of three selected barley genes was analyzed by RT-PCR and found to be organ-specific with peak expression in mature kernels. One barley CKX (HvCKX2) was characterized in detail after heterologous expression in tobacco. Interestingly, this enzyme shows a pH optimum at 4.5 and a preference for cytokinin ribosides as substrates, which may indicate its vacuolar targeting. Different substrate specificities, and the pH profiles of cytokinin-degrading enzymes extracted from different barley tissues, are also presented.

  10. Effect of melatonin and tetrapeptide on gene expression in mouse brain.

    PubMed

    Anisimov, S V; Khavinson, V Kh; Anisimov, V N

    2004-11-01

    A microchip technique was used to study expression of 16,897 clones from a cDNA library in the brain of mice receiving melatonin or tetrapeptide Epithalon (Ala-Glu-Asp-Gly). Expression of 53 transcripts in mouse brain underwent significant changes after treatment with the preparations. Melatonin and Epithalon modified expression of 38 and 22 transcripts, respectively. These preparations produced similar changes in the expression of 6 transcripts. Expression of 1 transcript (Rp119) was inhibited by melatonin, but induced by Epithalon. The target genes are physiologically related to the cell cycle, apoptosis, biosynthesis, processing, and transport of nucleic acids. Comparative study of gene expression in the brain and heart of CBA mice receiving melatonin and Epithalon suggest that these preparations have a tissue-specific biological effect.

  11. Validation of reference genes for quantitative RT-PCR studies of gene expression in perennial ryegrass (Lolium perenne L.)

    PubMed Central

    2010-01-01

    Background Perennial ryegrass (Lolium perenne L.) is an important pasture and turf crop. Biotechniques such as gene expression studies are being employed to improve traits in this temperate grass. Quantitative reverse transcription-polymerase chain reaction (qRT-PCR) is among the best methods available for determining changes in gene expression. Before analysis of target gene expression, it is essential to select an appropriate normalisation strategy to control for non-specific variation between samples. Reference genes that have stable expression at different biological and physiological states can be effectively used for normalisation; however, their expression stability must be validated before use. Results Existing Serial Analysis of Gene Expression data were queried to identify six moderately expressed genes that had relatively stable gene expression throughout the year. These six candidate reference genes (eukaryotic elongation factor 1 alpha, eEF1A; TAT-binding protein homolog 1, TBP-1; eukaryotic translation initiation factor 4 alpha, eIF4A; YT521-B-like protein family protein, YT521-B; histone 3, H3; ubiquitin-conjugating enzyme, E2) were validated for qRT-PCR normalisation in 442 diverse perennial ryegrass (Lolium perenne L.) samples sourced from field- and laboratory-grown plants under a wide range of experimental conditions. Eukaryotic EF1A is encoded by members of a multigene family exhibiting differential expression and necessitated the expression analysis of different eEF1A encoding genes; a highly expressed eEF1A (h), a moderately, but stably expressed eEF1A (s), and combined expression of multigene eEF1A (m). NormFinder identified eEF1A (s) and YT521-B as the best combination of two genes for normalisation of gene expression data in perennial ryegrass following different defoliation management in the field. Conclusions This study is unique in the magnitude of samples tested with the inclusion of numerous field-grown samples, helping pave the way to

  12. Intraperitoneal AAV9-shRNA inhibits target expression in neonatal skeletal and cardiac muscles.

    PubMed

    Mayra, Azat; Tomimitsu, Hiroyuki; Kubodera, Takayuki; Kobayashi, Masaki; Piao, Wenying; Sunaga, Fumiko; Hirai, Yukihiko; Shimada, Takashi; Mizusawa, Hidehiro; Yokota, Takanori

    2011-02-11

    Systemic injections of AAV vectors generally transduce to the liver more effectively than to cardiac and skeletal muscles. The short hairpin RNA (shRNA)-expressing AAV9 (shRNA-AAV9) can also reduce target gene expression in the liver, but not enough in cardiac or skeletal muscles. Higher doses of shRNA-AAV9 required for inhibiting target genes in cardiac and skeletal muscles often results in shRNA-related toxicity including microRNA oversaturation that can induce fetal liver failure. In this study, we injected high-dose shRNA-AAV9 to neonates and efficiently silenced genes in cardiac and skeletal muscles without inducing liver toxicity. This is because AAV is most likely diluted or degraded in the liver than in cardiac or skeletal muscle during cell division after birth. We report that this systemically injected shRNA-AAV method does not induce any major side effects, such as liver dysfunction, and the dose of shRNA-AAV is sufficient for gene silencing in skeletal and cardiac muscle tissues. This novel method may be useful for generating gene knockdown in skeletal and cardiac mouse tissues, thus providing mouse models useful for analyzing diseases caused by loss-of-function of target genes. Copyright © 2011 Elsevier Inc. All rights reserved.

  13. Cell-Free Optogenetic Gene Expression System.

    PubMed

    Jayaraman, Premkumar; Yeoh, Jing Wui; Jayaraman, Sudhaghar; Teh, Ai Ying; Zhang, Jingyun; Poh, Chueh Loo

    2018-04-20

    Optogenetic tools provide a new and efficient way to dynamically program gene expression with unmatched spatiotemporal precision. To date, their vast potential remains untapped in the field of cell-free synthetic biology, largely due to the lack of simple and efficient light-switchable systems. Here, to bridge the gap between cell-free systems and optogenetics, we studied our previously engineered one component-based blue light-inducible Escherichia coli promoter in a cell-free environment through experimental characterization and mathematical modeling. We achieved >10-fold dynamic expression and demonstrated rapid and reversible activation of the target gene to generate oscillatory response. The deterministic model developed was able to recapitulate the system behavior and helped to provide quantitative insights to optimize dynamic response. This in vitro optogenetic approach could be a powerful new high-throughput screening technology for rapid prototyping of complex biological networks in both space and time without the need for chemical induction.

  14. Systemic analysis of genome-wide expression profiles identified potential therapeutic targets of demethylation drugs for glioblastoma.

    PubMed

    Ning, Tongbo; Cui, Hao; Sun, Feng; Zou, Jidian

    2017-09-05

    Glioblastoma represents one of the most aggressive malignant brain tumors with high morbidity and motility. Demethylation drugs have been developed for its treatment with little efficacy has been observed. The purpose of this study was to screen therapeutic targets of demethylation drugs or bioactive molecules for glioblastoma through systemic bioinformatics analysis. We firstly downloaded genome-wide expression profiles from the Gene Expression Omnibus (GEO) and conducted the primary analysis through R software, mainly including preprocessing of raw microarray data, transformation between probe ID and gene symbol and identification of differential expression genes (DEGs). Secondly, functional enrichment analysis was conducted via the Database for Annotation, Visualization and Integrated Discovery (DAVID) to explore biological processes involved in the development of glioblastoma. Thirdly, we constructed protein-protein interaction (PPI) network of interested genes and conducted cross analysis for multi datasets to obtain potential therapeutic targets for glioblastoma. Finally, we further confirmed the therapeutic targets through real-time RT-PCR. As a result, biological processes that related to cancer development, amino metabolism, immune response and etc. were found to be significantly enriched in genes that differential expression in glioblastoma and regulated by 5'aza-dC. Besides, network and cross analysis identified ACAT2, UFC1 and CYB5R1 as novel therapeutic targets of demethylation drugs which also confirmed by real time RT-PCR. In conclusions, our study identified several biological processes and genes that involved in the development of glioblastoma and regulated by 5'aza-dC, which would be helpful for the treatment of glioblastoma. Copyright © 2017 Elsevier B.V. All rights reserved.

  15. The SET1 Complex Selects Actively Transcribed Target Genes via Multivalent Interaction with CpG Island Chromatin.

    PubMed

    Brown, David A; Di Cerbo, Vincenzo; Feldmann, Angelika; Ahn, Jaewoo; Ito, Shinsuke; Blackledge, Neil P; Nakayama, Manabu; McClellan, Michael; Dimitrova, Emilia; Turberfield, Anne H; Long, Hannah K; King, Hamish W; Kriaucionis, Skirmantas; Schermelleh, Lothar; Kutateladze, Tatiana G; Koseki, Haruhiko; Klose, Robert J

    2017-09-05

    Chromatin modifications and the promoter-associated epigenome are important for the regulation of gene expression. However, the mechanisms by which chromatin-modifying complexes are targeted to the appropriate gene promoters in vertebrates and how they influence gene expression have remained poorly defined. Here, using a combination of live-cell imaging and functional genomics, we discover that the vertebrate SET1 complex is targeted to actively transcribed gene promoters through CFP1, which engages in a form of multivalent chromatin reading that involves recognition of non-methylated DNA and histone H3 lysine 4 trimethylation (H3K4me3). CFP1 defines SET1 complex occupancy on chromatin, and its multivalent interactions are required for the SET1 complex to place H3K4me3. In the absence of CFP1, gene expression is perturbed, suggesting that normal targeting and function of the SET1 complex are central to creating an appropriately functioning vertebrate promoter-associated epigenome. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

  16. Genetic analysis of Ikaros target genes and tumor suppressor function in BCR-ABL1+ pre–B ALL

    PubMed Central

    Aghajanirefah, Ali; McLaughlin, Jami; Cheng, Donghui; Geng, Huimin; Eggesbø, Linn M.; Smale, Stephen T.; Müschen, Markus

    2017-01-01

    Inactivation of the tumor suppressor gene encoding the transcriptional regulator Ikaros (IKZF1) is a hallmark of BCR-ABL1+ precursor B cell acute lymphoblastic leukemia (pre–B ALL). However, the mechanisms by which Ikaros functions as a tumor suppressor in pre–B ALL remain poorly understood. Here, we analyzed a mouse model of BCR-ABL1+ pre–B ALL together with a new model of inducible expression of wild-type Ikaros in IKZF1 mutant human BCR-ABL1+ pre–B ALL. We performed integrated genome-wide chromatin and expression analyses and identified Ikaros target genes in mouse and human BCR-ABL1+ pre–B ALL, revealing novel conserved gene pathways associated with Ikaros tumor suppressor function. Notably, genetic depletion of different Ikaros targets, including CTNND1 and the early hematopoietic cell surface marker CD34, resulted in reduced leukemic growth. Our results suggest that Ikaros mediates tumor suppressor function by enforcing proper developmental stage–specific expression of multiple genes through chromatin compaction at its target genes. PMID:28190001

  17. Validation of reference genes for gene expression analysis in olive (Olea europaea) mesocarp tissue by quantitative real-time RT-PCR

    PubMed Central

    2014-01-01

    Background Gene expression analysis using quantitative reverse transcription PCR (qRT-PCR) is a robust method wherein the expression levels of target genes are normalised using internal control genes, known as reference genes, to derive changes in gene expression levels. Although reference genes have recently been suggested for olive tissues, combined/independent analysis on different cultivars has not yet been tested. Therefore, an assessment of reference genes was required to validate the recent findings and select stably expressed genes across different olive cultivars. Results A total of eight candidate reference genes [glyceraldehyde 3-phosphate dehydrogenase (GAPDH), serine/threonine-protein phosphatase catalytic subunit (PP2A), elongation factor 1 alpha (EF1-alpha), polyubiquitin (OUB2), aquaporin tonoplast intrinsic protein (TIP2), tubulin alpha (TUBA), 60S ribosomal protein L18-3 (60S RBP L18-3) and polypyrimidine tract-binding protein homolog 3 (PTB)] were chosen based on their stability in olive tissues as well as in other plants. Expression stability was examined by qRT-PCR across 12 biological samples, representing mesocarp tissues at various developmental stages in three different olive cultivars, Barnea, Frantoio and Picual, independently and together during the 2009 season with two software programs, GeNorm and BestKeeper. Both software packages identified GAPDH, EF1-alpha and PP2A as the three most stable reference genes across the three cultivars and in the cultivar, Barnea. GAPDH, EF1-alpha and 60S RBP L18-3 were found to be most stable reference genes in the cultivar Frantoio while 60S RBP L18-3, OUB2 and PP2A were found to be most stable reference genes in the cultivar Picual. Conclusions The analyses of expression stability of reference genes using qRT-PCR revealed that GAPDH, EF1-alpha, PP2A, 60S RBP L18-3 and OUB2 are suitable reference genes for expression analysis in developing Olea europaea mesocarp tissues, displaying the highest level

  18. Gene expression profiling combined with bioinformatics analysis identify biomarkers for Parkinson disease.

    PubMed

    Diao, Hongyu; Li, Xinxing; Hu, Sheng; Liu, Yunhui

    2012-01-01

    Parkinson disease (PD) progresses relentlessly and affects approximately 4% of the population aged over 80 years old. It is difficult to diagnose in its early stages. The purpose of our study is to identify molecular biomarkers for PD initiation using a computational bioinformatics analysis of gene expression. We downloaded the gene expression profile of PD from Gene Expression Omnibus and identified differentially coexpressed genes (DCGs) and dysfunctional pathways in PD patients compared to controls. Besides, we built a regulatory network by mapping the DCGs to known regulatory data between transcription factors (TFs) and target genes and calculated the regulatory impact factor of each transcription factor. As the results, a total of 1004 genes associated with PD initiation were identified. Pathway enrichment of these genes suggests that biological processes of protein turnover were impaired in PD. In the regulatory network, HLF, E2F1 and STAT4 were found have altered expression levels in PD patients. The expression levels of other transcription factors, NKX3-1, TAL1, RFX1 and EGR3, were not found altered. However, they regulated differentially expressed genes. In conclusion, we suggest that HLF, E2F1 and STAT4 may be used as molecular biomarkers for PD; however, more work is needed to validate our result.

  19. Gene Expression Profiling Combined with Bioinformatics Analysis Identify Biomarkers for Parkinson Disease

    PubMed Central

    Diao, Hongyu; Li, Xinxing; Hu, Sheng; Liu, Yunhui

    2012-01-01

    Parkinson disease (PD) progresses relentlessly and affects approximately 4% of the population aged over 80 years old. It is difficult to diagnose in its early stages. The purpose of our study is to identify molecular biomarkers for PD initiation using a computational bioinformatics analysis of gene expression. We downloaded the gene expression profile of PD from Gene Expression Omnibus and identified differentially coexpressed genes (DCGs) and dysfunctional pathways in PD patients compared to controls. Besides, we built a regulatory network by mapping the DCGs to known regulatory data between transcription factors (TFs) and target genes and calculated the regulatory impact factor of each transcription factor. As the results, a total of 1004 genes associated with PD initiation were identified. Pathway enrichment of these genes suggests that biological processes of protein turnover were impaired in PD. In the regulatory network, HLF, E2F1 and STAT4 were found have altered expression levels in PD patients. The expression levels of other transcription factors, NKX3-1, TAL1, RFX1 and EGR3, were not found altered. However, they regulated differentially expressed genes. In conclusion, we suggest that HLF, E2F1 and STAT4 may be used as molecular biomarkers for PD; however, more work is needed to validate our result. PMID:23284986

  20. Microgravity and immunity: Changes in lymphocyte gene expression.

    NASA Astrophysics Data System (ADS)

    Risin, D.; Ward, N. E.; Risin, S. A.; Pellis, N. R.

    Earlier studies had shown that modeled and true microgravity MG cause multiple direct effects on human lymphocytes MG inhibits lymphocyte locomotion suppresses polyclonal and antigen-specific activation affects signal transduction mechanisms as well as activation-induced apoptosis In this study we assessed changes in gene expression associated with lymphocyte exposure to microgravity in an attempt to identify microgravity-sensitive genes MGSG in general and specifically those genes that might be responsible for the functional and structural changes observed earlier Two sets of experiments targeting different goals were conducted In the first set T-lymphocytes from normal donors were activated with anti-CD3 and IL2 and then cultured in 1g static and modeled MG MMG conditions Rotating Wall Vessel bioreactor for 24 hours This setting allowed searching for MGSG by comparison of gene expression patterns in zero and 1 g gravity In the second set - activated T-cells after culturing for 24 hours in 1g and MMG were exposed three hours before harvesting to a secondary activation stimulus PHA thus triggering the apoptotic pathway Total RNA was extracted using the RNeasy isolation kit Qiagen Valencia CA Affymetrix Gene Chips U133A allowing testing for 18 400 human genes were used for microarray analysis The experiments were performed in triplicates with T-cells obtained from different blood donors to minimize the possible input of biological variation in gene expression and discriminate changes that are associated with the

  1. Bioinformatics approach for choosing the correct reference genes when studying gene expression in human keratinocytes.

    PubMed

    Beer, Lucian; Mlitz, Veronika; Gschwandtner, Maria; Berger, Tanja; Narzt, Marie-Sophie; Gruber, Florian; Brunner, Patrick M; Tschachler, Erwin; Mildner, Michael

    2015-10-01

    Reverse transcription polymerase chain reaction (qRT-PCR) has become a mainstay in many areas of skin research. To enable quantitative analysis, it is necessary to analyse expression of reference genes (RGs) for normalization of target gene expression. The selection of reliable RGs therefore has an important impact on the experimental outcome. In this study, we aimed to identify and validate the best suited RGs for qRT-PCR in human primary keratinocytes (KCs) over a broad range of experimental conditions using the novel bioinformatics tool 'RefGenes', which is based on a manually curated database of published microarray data. Expression of 6 RGs identified by RefGenes software and 12 commonly used RGs were validated by qRT-PCR. We assessed whether these 18 markers fulfilled the requirements for a valid RG by the comprehensive ranking of four bioinformatics tools and the coefficient of variation (CV). In an overall ranking, we found GUSB to be the most stably expressed RG, whereas the expression values of the commonly used RGs, GAPDH and B2M were significantly affected by varying experimental conditions. Our results identify RefGenes as a powerful tool for the identification of valid RGs and suggest GUSB as the most reliable RG for KCs. © 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  2. Mechanisms of epigenetic and cell-type specific regulation of Hey target genes in ES cells and cardiomyocytes.

    PubMed

    Weber, David; Heisig, Julia; Kneitz, Susanne; Wolf, Elmar; Eilers, Martin; Gessler, Manfred

    2015-02-01

    Hey bHLH transcription factors are critical effectors of Notch signaling. During mammalian heart development they are expressed in atrial and ventricular cardiomyocytes and in the developing endocardium. Hey knockout mice suffer from lethal cardiac defects, such as ventricular septum defects, valve defects and cardiomyopathy. Despite this functional relevance, little is known about the regulation of downstream targets in relevant cell types. The objective of this study was to elucidate the regulatory mechanisms by which Hey proteins affect gene expression in a cell type specific manner. We used an in vitro cardiomyocyte differentiation system with inducible Hey1 or Hey2 expression to study target gene regulation in cardiomyocytes (CM) generated from murine embryonic stem cells (ESC). The effects of Hey1 and Hey2 are largely redundant, but cell type specific. The number of regulated genes is comparable between ESC and CM, but the total number of binding sites is much higher, especially in ESC, targeting mainly genes involved in transcriptional regulation and developmental processes. Repression by Hey proteins generally correlates with the extent of Hey-binding to target promoters, Hdac recruitment and lower histone acetylation. Functionally, treatment with the Hdac inhibitor TSA abolished Hey target gene regulation. However, in CM the repressive effect of Hey-binding is lost for a subset of genes. These also lack Hey-dependent histone deacetylation in CM and are enriched for binding sites of cardiac specific activators like Srf, Nkx2-5, and Gata4. Ectopic Nkx2-5 overexpression in ESC blocks Hey-mediated repression of these genes. Thus, Hey proteins mechanistically repress target genes via Hdac recruitment and histone deacetylation. In CM Hey-repression is counteracted by cardiac activators, which recruit histone acetylases and prevent Hey mediated deacetylation and subsequent repression for a subset of genes. Copyright © 2014 Elsevier Ltd. All rights reserved.

  3. Rat lung metallothionein and heme oxygenase gene expression following ozone and zinc oxide exposure.

    PubMed

    Cosma, G; Fulton, H; DeFeo, T; Gordon, T

    1992-11-01

    We have conducted exposures in rats to determine pulmonary responses following inhalation of two common components of welding fumes, zinc oxide and ozone. To examine their effects on target-inducible gene expression, we measured mRNA levels of two metal-responsive genes, metallothionein (MT) and heme oxygenase (HO), in lung tissue by RNA slot-blot analysis. A 3-hr exposure to ZnO fume via a combustion furnace caused a substantial elevation in lung MT mRNA at all concentrations tested. Exposures to 5 and 2.5 mg/m3 ZnO resulted in peak 8-fold increases in MT mRNA levels (compared to air-exposed control animal values) immediately after exposure, while 1 mg/m3 ZnO exposure caused a 3.5-fold elevation in MT mRNA. These levels returned to approximate control gene expression values 24 hr after exposure. In addition, ZnO exposure caused an immediate elevation in lung HO gene expression levels, with 8-, 11-, and 5-fold increases observed after the same ZnO exposure levels (p < 0.05). Like MT gene induction, HO mRNA values returned to approximate control levels 24 hr after exposure. In striking contrast to the induction of MT and HO gene expression after ZnO exposures, there was no elevation in gene expression following a 6-hr exposure to 0.5 and 1 ppm ozone, even when lungs were examined as late as 72 hr after exposure. Our results demonstrate the induction of target gene expression following the inhalation of ZnO at concentrations equal to, and below, the current recommended threshold limit value of 5 mg/m3 ZnO. Furthermore, the lack of effect of ozone exposure on MT and HO gene expression suggests no involvement of these genes in the acute respiratory response to this oxidant compound.

  4. Gene expression characterizes different nutritional strategies among three mixotrophic protists.

    PubMed

    Liu, Zhenfeng; Campbell, Victoria; Heidelberg, Karla B; Caron, David A

    2016-07-01

    Mixotrophic protists, i.e. protists that can carry out both phototrophy and heterotrophy, are a group of organisms with a wide range of nutritional strategies. The ecological and biogeochemical importance of these species has recently been recognized. In this study, we investigated and compared the gene expression of three mixotrophic protists, Prymnesium parvum, Dinobyron sp. and Ochromonas sp. under light and dark conditions in the presence of prey using RNA-Seq. Gene expression of the obligately phototrophic P. parvum and Dinobryon sp. changed significantly between light and dark treatments, while that of primarily heterotrophic Ochromonas sp. was largely unchanged. Gene expression of P. parvum and Dinobryon sp. shared many similarities, especially in the expression patterns of genes related to reproduction. However, key genes involved in central carbon metabolism and phagotrophy had different expression patterns between these two species, suggesting differences in prey consumption and heterotrophic nutrition in the dark. Transcriptomic data also offered clues to other physiological traits of these organisms such as preference of nitrogen sources and photo-oxidative stress. These results provide potential target genes for further exploration of the mechanisms of mixotrophic physiology and demonstrate the potential usefulness of molecular approaches in characterizing the nutritional modes of mixotrophic protists. © FEMS 2016. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  5. Production of Prnp-/- goats by gene targeting in adult fibroblasts.

    PubMed

    Zhu, Caihong; Li, Bei; Yu, Guohua; Chen, Jianquan; Yu, Huiqing; Chen, Juan; Xu, Xujun; Wu, Youbing; Zhang, Aimin; Cheng, Guoxiang

    2009-04-01

    Homozygous mice devoid of functional Prnp are resistant to scrapie and prion propagation, but heterozygous mice for Prnp disruption still suffer from prion disease and prion deposition. We have previously generated heterozygous cloned goats with one allele of Prnp functional disruption. To obtain goats with both alleles of Prnp be disrupted which would be resistant to scrapie completely, a second-round gene targeting was applied to disrupt the wild type allele of Prnp in the heterozygous goats. By second-round gene targeting, we successfully disrupted the wild type allele of Prnp in primary Prnp (+/-) goat skin fibroblasts and obtained a Prnp (-/-) cell line without Prnp expression. This is the first report on successful targeting modification in primary adult somatic cells of animals. These cells were used as nuclear donors for somatic cell cloning to produce Prnp (-/-) goats. A total of 57 morulae or blastocytes developed from the reconstructed embryos were transferred to 31 recipients, which produced 7 pregnancies at day 35. At 73 days of gestation, we obtained one cloned fetus with Prnp (-/-) genotype. Our research not only indicated that multiple genetic modifications could be accomplished by multi-round gene targeting in primary somatic cells, but also provided strong evidence that gene targeting in adult cells other than fetal cells could be applied to introduce precise genetic modifications in animals without destroying the embryos.

  6. Genome-wide analysis reveals inositol, not choline, as the major effector of Ino2p-Ino4p and unfolded protein response target gene expression in yeast.

    PubMed

    Jesch, Stephen A; Zhao, Xin; Wells, Martin T; Henry, Susan A

    2005-03-11

    In the yeast Saccharomyces cerevisiae, the transcription of many genes encoding enzymes of phospholipid biosynthesis are repressed in cells grown in the presence of the phospholipid precursors inositol and choline. A genome-wide approach using cDNA microarray technology was used to profile the changes in the expression of all genes in yeast that respond to the exogenous presence of inositol and choline. We report that the global response to inositol is completely distinct from the effect of choline. Whereas the effect of inositol on gene expression was primarily repressing, the effect of choline on gene expression was activating. Moreover, the combination of inositol and choline increased the number of repressed genes compared with inositol alone and enhanced the repression levels of a subset of genes that responded to inositol. In all, 110 genes were repressed in the presence of inositol and choline. Two distinct sets of genes exhibited differential expression in response to inositol or the combination of inositol and choline in wild-type cells. One set of genes contained the UASINO sequence and were bound by Ino2p and Ino4p. Many of these genes were also negatively regulated by OPI1, suggesting a common regulatory mechanism for Ino2p, Ino4p, and Opi1p. Another nonoverlapping set of genes was coregulated by the unfolded protein response pathway, an ER-localized stress response pathway, but was not dependent on OPI1 and did not show further repression when choline was present together with inositol. These results suggest that inositol is the major effector of target gene expression, whereas choline plays a minor role.

  7. Genome Wide Analysis Reveals Inositol, not Choline, as the Major Effector of Ino2p-Ino4p and Unfolded Protein Response Target Gene Expression in Yeast

    PubMed Central

    Jesch, Stephen A.; Zhao, Xin; Wells, Martin T.; Henry, Susan A.

    2005-01-01

    SUMMARY In the yeast Saccharomyces cerevisiae the transcription of many genes encoding enzymes of phospholipid biosynthesis are repressed in cells grown in the presence of the phospholipid precursors inositol and choline. A genome-wide approach using cDNA microarray technology was utilized to profile the changes in the expression of all genes in yeast that respond to the exogenous presence of inositol and choline. We report that the global response to inositol is completely distinct from the effect of choline. Whereas the effect of inositol on gene expression was primarily repressing, the effect of choline on gene expression was activating. Moreover, the combination inositol and choline increased the number of repressed genes compared to inositol alone and enhanced the repression levels of a subset of genes that responded to inositol. In all, 110 genes were repressed in the presence of inositol and choline. Two distinct sets of genes exhibited differential expression in response to inositol or the combination of inositol and choline in wild type cells. One set of genes contained the UASINO sequence and were bound by Ino2p and Ino4p. Many of these genes were also negatively regulated by OPI1, suggesting a common regulatory mechanism for Ino2p, Ino4p, and Opi1p. Another non-overlapping set of genes were coregulated by the unfolded protein response pathway, an ER-localized stress response pathway, but were not dependent on OPI1 and did not show further repression when choline was present together with inositol. These results suggest that inositol is the major effector of target gene expression, while choline plays a minor role. PMID:15611057

  8. Identification of an expressed gene in Dipylidium caninum.

    PubMed

    Miranda, Rodrigo R C; Costa-Júnior, Livio M; Campos, Artur K; Santos, Hudson A; Rabelo, Elida M L

    2004-10-01

    Recombinant DNA studies have been focused on developing vaccines to different cestodes. But few studies involving Dipylidium caninum molecular biology and genes have been done. Only partial sequences of mitochondrial DNA and ribosomal RNA gene are available in databases. Any molecular work with this parasite, including epidemiology, study of drug-resistant strains, and vaccine development, is hampered by the lack of knowledge of its genome. Thus, the knowledge of specific genes of different developmental stages of D. caninum is crucial to locate potential targets to be used as candidates to develop a vaccine and/or new drugs against this parasite. Here we report, for the first time, the sequencing of a fragment of a D. caninum expressed gene.

  9. Antisense imaging of gene expression in the brain in vivo

    NASA Astrophysics Data System (ADS)

    Shi, Ningya; Boado, Ruben J.; Pardridge, William M.

    2000-12-01

    Antisense radiopharmaceuticals could be used to image gene expression in the brain in vivo, should these polar molecules be made transportable through the blood-brain barrier. The present studies describe an antisense imaging agent comprised of an iodinated peptide nucleic acid (PNA) conjugated to a monoclonal antibody to the rat transferrin receptor by using avidin-biotin technology. The PNA was a 16-mer antisense to the sequence around the methionine initiation codon of the luciferase mRNA. C6 rat glioma cells were permanently transfected with a luciferase expression plasmid, and C6 experimental brain tumors were developed in adult rats. The expression of the luciferase transgene in the tumors in vivo was confirmed by measurement of luciferase enzyme activity in the tumor extract. The [125I]PNA conjugate was injected intravenously in anesthetized animals with brain tumors and killed 2 h later for frozen sectioning of brain and film autoradiography. No image of the luciferase gene expression was obtained after the administration of either the unconjugated antiluciferase PNA or a PNA conjugate that was antisense to the mRNA of a viral transcript. In contrast, tumors were imaged in all rats administered the [125I]PNA that was antisense to the luciferase sequence and was conjugated to the targeting antibody. In conclusion, these studies demonstrate gene expression in the brain in vivo can be imaged with antisense radiopharmaceuticals that are conjugated to a brain drug-targeting system.

  10. Ossification of the posterior longitudinal ligament related genes identification using microarray gene expression profiling and bioinformatics analysis.

    PubMed

    He, Hailong; Mao, Lingzhou; Xu, Peng; Xi, Yanhai; Xu, Ning; Xue, Mingtao; Yu, Jiangming; Ye, Xiaojian

    2014-01-10

    Ossification of the posterior longitudinal ligament (OPLL) is a kind of disease with physical barriers and neurological disorders. The objective of this study was to explore the differentially expressed genes (DEGs) in OPLL patient ligament cells and identify the target sites for the prevention and treatment of OPLL in clinic. Gene expression data GSE5464 was downloaded from Gene Expression Omnibus; then DEGs were screened by limma package in R language, and changed functions and pathways of OPLL cells compared to normal cells were identified by DAVID (The Database for Annotation, Visualization and Integrated Discovery); finally, an interaction network of DEGs was constructed by string. A total of 1536 DEGs were screened, with 31 down-regulated and 1505 up-regulated genes. Response to wounding function and Toll-like receptor signaling pathway may involve in the development of OPLL. Genes, such as PDGFB, PRDX2 may involve in OPLL through response to wounding function. Toll-like receptor signaling pathway enriched genes such as TLR1, TLR5, and TLR7 may involve in spine cord injury in OPLL. PIK3R1 was the hub gene in the network of DEGs with the highest degree; INSR was one of the most closely related genes of it. OPLL related genes screened by microarray gene expression profiling and bioinformatics analysis may be helpful for elucidating the mechanism of OPLL. © 2013.

  11. Targeted gene delivery in the cricket brain, using in vivo electroporation.

    PubMed

    Matsumoto, Chihiro Sato; Shidara, Hisashi; Matsuda, Koji; Nakamura, Taro; Mito, Taro; Matsumoto, Yukihisa; Oka, Kotaro; Ogawa, Hiroto

    2013-12-01

    The cricket (Gryllus bimaculatus) is a hemimetabolous insect that is emerging as a model organism for the study of neural and molecular mechanisms of behavioral traits. However, research strategies have been limited by a lack of genetic manipulation techniques that target the nervous system of the cricket. The development of a new method for efficient gene delivery into cricket brains, using in vivo electroporation, is described here. Plasmid DNA, which contained an enhanced green fluorescent protein (eGFP) gene, under the control of a G. bimaculatus actin (Gb'-act) promoter, was injected into adult cricket brains. Injection was followed by electroporation at a sufficient voltage. Expression of eGFP was observed within the brain tissue. Localized gene expression, targeted to specific regions of the brain, was also achieved using a combination of local DNA injection and fine arrangement of the electroporation electrodes. Further studies using this technique will lead to a better understanding of the neural and molecular mechanisms that underlie cricket behaviors. Copyright © 2013 Elsevier Ltd. All rights reserved.

  12. Complex modulation of androgen responsive gene expression by methoxyacetic acid

    PubMed Central

    2011-01-01

    Background Optimal androgen signaling is critical for testicular development and spermatogenesis. Methoxyacetic acid (MAA), the primary active metabolite of the industrial chemical ethylene glycol monomethyl ether, disrupts spermatogenesis and causes testicular atrophy. Transcriptional trans-activation studies have indicated that MAA can enhance androgen receptor activity, however, whether MAA actually impacts the expression of androgen-responsive genes in vivo, and which genes might be affected is not known. Methods A mouse TM3 Leydig cell line that stably expresses androgen receptor (TM3-AR) was prepared and analyzed by transcriptional profiling to identify target gene interactions between MAA and testosterone on a global scale. Results MAA is shown to have widespread effects on androgen-responsive genes, affecting processes ranging from apoptosis to ion transport, cell adhesion, phosphorylation and transcription, with MAA able to enhance, as well as antagonize, androgenic responses. Moreover, testosterone is shown to exert both positive and negative effects on MAA gene responses. Motif analysis indicated that binding sites for FOX, HOX, LEF/TCF, STAT5 and MEF2 family transcription factors are among the most highly enriched in genes regulated by testosterone and MAA. Notably, 65 FOXO targets were repressed by testosterone or showed repression enhanced by MAA with testosterone; these include 16 genes associated with developmental processes, six of which are Hox genes. Conclusions These findings highlight the complex interactions between testosterone and MAA, and provide insight into the effects of MAA exposure on androgen-dependent processes in a Leydig cell model. PMID:21453523

  13. Argonautes promote male fertility and provide a paternal memory of germline gene expression in C. elegans

    PubMed Central

    Conine, Colin C.; Moresco, James J.; Gu, Weifeng; Shirayama, Masaki; Conte, Darryl; Yates, John R.; Mello, Craig C.

    2014-01-01

    SUMMARY During each life cycle germ cells preserve and pass on both genetic and epigenetic information. In C. elegans, the ALG-3/4 Argonaute proteins are expressed during male gametogenesis and promote male fertility. Here we show that the CSR-1 Argonaute functions with ALG-3/4 to positively regulate target genes required for spermiogenesis. Our findings suggest that ALG-3/4 functions during spermatogenesis to amplify a small-RNA signal that represents an epigenetic memory of male-specific gene expression. CSR-1, which is abundant in mature sperm, appears to transmit this memory to offspring. Surprisingly, in addition to small RNAs targeting male-specific genes, we show that males also harbor an extensive repertoire of CSR-1 small RNAs targeting oogenesis-specific mRNAs. Together these findings suggest that C. elegans sperm transmit not only the genome but also epigenetic binary signals in the form of Argonaute/small-RNA complexes that constitute a memory of gene expression in preceding generations. PMID:24360276

  14. Expression of Shigella flexneri ipaB Gene in Tobacco.

    PubMed

    Ohadi, Mandana; Rasouli, Rahimeh; Darzi-Eslam, Elham; Jafari, Anis; Ehsani, Parastoo

    2013-04-01

    Shigellosis is a leading cause of diarrhea in many developing countries and although the disease can be controlled and managed with antibiotics, the constant emergence of resistant species requiring ever newer antibacterial drugs make development of an effective vaccine necessary. The bacteria are highly contagious and since immunity to Shigella is serotype-specific a multi-serotype vaccine is required for adequate protection. Proteins encoded by Shigella invasion plasmid, which are part of the Type Three Secretion System (TTSS) of this bacteria, are good candidate as vaccine targets since they are both immunogenic and conserved between different Shigella species. The advent of molecular farming, which is a low cost system, has opened up new venues for production of recombinant proteins. In view of the difficulties encountered in expressing IpaB in Escherichia coli (E. coli), the feasibility of the expression of this protein in tobacco has been investigated. The ipaB gene was cloned in place of the Hygromycin gene in pCambia1304 containing GFP as a reporter gene. The vector was then transferred into competent Agrobacterium tumefaciens (A. tumefaciens) strain LBA4404 which was used for agro-infiltration of Nicotiana tobaccum (N. tobaccum) leaves. Transformation was confirmed by expression of GFP. The gene was also cloned in pBAD/geneIII A and transformed E. coli host containing the construct was induced using different amounts of L-arabinose as inducer. Expression of IpaB gene by both hosts was determined by Western blotting using anti-IpaB monoclonal antibody. The data obtained showed that IpaB was expressed in plant leaves but expression in E. coli was not detectable. This study showed that N. tobaccum is capable of expressing this protein without its specific chaperon and in levels detectable by Western blotting.

  15. Global Gene Expression Change Induced by Major Thoracoabdominal Surgery.

    PubMed

    Allen, Casey J; Griswold, Anthony J; Schulman, Carl I; Sleeman, Danny; Levi, Joe U; Livingstone, Alan S; Proctor, Kenneth G

    2017-12-01

    To test the hypothesis that major thoracoabdominal surgery induces gene expression changes associated with adverse outcomes. Widely different traumatic injuries evoke surprisingly similar gene expression profiles, but there is limited information on whether the iatrogenic injury caused by major surgery is associated with similar patterns. With informed consent, blood samples were obtained from 50 patients before and after open transhiatal esophagectomy or pancreaticoduodenectomy. Twelve cases with complicated recoveries (death, infection, venous thromboembolism) were matched with 12 cases with uneventful recoveries. Global gene expression was assayed using human microarray chips. A 2-fold change with a corrected P < 0.05 was considered differentially expressed. In these 24 patients, 522 genes were differentially expressed after surgery; 248 (48%) were upregulated (innate immunity and inflammation) and 274 (52%) were downregulated [adaptive immunity (antigen presentation, T-cell function)]. Hierarchical clustering of the profile reliably predicted pre- and postoperative status. The within-patient change was 3.08 ± 0.91-fold. There was no measurable association with age, malignancy, procedure, surgery length, operative blood loss, or transfusion requirements, but was positively associated with postoperative infection (3.81 ± 0.97 vs 2.79 ± 0.73; P = 0.009) and hospital length of stay (r = 0.583, P = 0.003). Venous thromboembolism and mortality each occurred in one patient, thus no associations were possible. Major surgery induces a quantifiable pattern of gene expression change that is associated with adverse outcome. This could reflect early impaired adaptive immunity and suggests potential therapeutic targets to improve postoperative recovery.

  16. Subclinical Pregnancy Toxemia-Induced Gene Expression Changes in Ovine Placenta and Uterus

    PubMed Central

    Kasimanickam, Ramanathan K.

    2016-01-01

    The objective was to elucidate gene expression differences in uterus, caruncle, and cotyledon of ewes with subclinical pregnancy toxemia (SCPT) and healthy ewes, and to identify associated biological functions and pathways involved in pregnancy toxemia. On Day 136 (±1 day) post-breeding, ewes (n = 18) had body condition score (BCS; 1–5; 1, emaciated; 5, obese) assessed, and blood samples were collected for plasma glucose and β-hydroxybutyrate (BHBA) analyses. The ewes were euthanized, and tissue samples were collected from the gravid uterus and placentomes. Based on BCS (2.0 ± 0.02), glucose (2.4 ± 0.33), and BHBA (0.97 ± 0.06) concentrations, ewes (n = 10) were grouped as healthy (n = 5) and subclinical SCPT (n = 5) ewes. The mRNA expressions were determined by quantitative PCR method, and prediction of miRNA partners and target genes for the predicted miRNA were identified using miRDB (http://mirdb.org/miRDB/). Top ranked target genes were used to identify associated biological functions and pathways in response to SPCT using PANTHER. The angiogenesis genes VEGF and PlGF, and AdipoQ, AdipoR2, PPARG, LEP, IGF1, IGF2, IL1b, and TNFα mRNA expressions were lower in abundances, whereas hypoxia genes eNOS, HIF1a, and HIF 2a, and sFlt1 and KDR mRNA expressions were greater in abundances in uterus and placenta of SCPT ewes compared to healthy ewes (P < 0.05). The predicted miRNA and associated target genes contributed to several biological processes, including apoptosis, biological adhesion, biological regulation, cellular component biogenesis, cellular process, developmental process, immune system process, localization, metabolic process, multicellular organismal process, reproduction, and response to stimulus. The target genes were involved in several pathways including angiogenesis, cytoskeletal regulation, hypoxia response via HIF activation, interleukin signaling, ubiquitin proteasome, and VEGF signaling pathway. In

  17. PPAR agonists regulate brain gene expression: relationship to their effects on ethanol consumption.

    PubMed

    Ferguson, Laura B; Most, Dana; Blednov, Yuri A; Harris, R Adron

    2014-11-01

    Peroxisome proliferator-activated receptors (PPARs) are nuclear hormone receptors that act as ligand-activated transcription factors. Although prescribed for dyslipidemia and type-II diabetes, PPAR agonists also possess anti-addictive characteristics. PPAR agonists decrease ethanol consumption and reduce withdrawal severity and susceptibility to stress-induced relapse in rodents. However, the cellular and molecular mechanisms facilitating these properties have yet to be investigated. We tested three PPAR agonists in a continuous access two-bottle choice (2BC) drinking paradigm and found that tesaglitazar (PPARα/γ; 1.5 mg/kg) and fenofibrate (PPARα; 150 mg/kg) decreased ethanol consumption in male C57BL/6J mice while bezafibrate (PPARα/γ/β; 75 mg/kg) did not. We hypothesized that changes in brain gene expression following fenofibrate and tesaglitazar treatment lead to reduced ethanol drinking. We studied unbiased genomic profiles in areas of the brain known to be important for ethanol dependence, the prefrontal cortex (PFC) and amygdala, and also profiled gene expression in liver. Genomic profiles from the non-effective bezafibrate treatment were used to filter out genes not associated with ethanol consumption. Because PPAR agonists are anti-inflammatory, they would be expected to target microglia and astrocytes. Surprisingly, PPAR agonists produced a strong neuronal signature in mouse brain, and fenofibrate and tesaglitazar (but not bezafibrate) targeted a subset of GABAergic interneurons in the amygdala. Weighted gene co-expression network analysis (WGCNA) revealed co-expression of treatment-significant genes. Functional annotation of these gene networks suggested that PPAR agonists might act via neuropeptide and dopaminergic signaling pathways in the amygdala. Our results reveal gene targets through which PPAR agonists can affect alcohol consumption behavior. Copyright © 2014 Elsevier Ltd. All rights reserved.

  18. Normal Collagen and Bone Production by Gene-targeted Human Osteogenesis Imperfecta iPSCs

    PubMed Central

    Deyle, David R; Khan, Iram F; Ren, Gaoying; Wang, Pei-Rong; Kho, Jordan; Schwarze, Ulrike; Russell, David W

    2012-01-01

    Osteogenesis imperfecta (OI) is caused by dominant mutations in the type I collagen genes. In principle, the skeletal abnormalities of OI could be treated by transplantation of patient-specific, bone-forming cells that no longer express the mutant gene. Here, we develop this approach by isolating mesenchymal cells from OI patients, inactivating their mutant collagen genes by adeno-associated virus (AAV)-mediated gene targeting, and deriving induced pluripotent stem cells (iPSCs) that were expanded and differentiated into mesenchymal stem cells (iMSCs). Gene-targeted iMSCs produced normal collagen and formed bone in vivo, but were less senescent and proliferated more than bone-derived MSCs. To generate iPSCs that would be more appropriate for clinical use, the reprogramming and selectable marker transgenes were removed by Cre recombinase. These results demonstrate that the combination of gene targeting and iPSC derivation can be used to produce potentially therapeutic cells from patients with genetic disease. PMID:22031238

  19. Inferring causal genomic alterations in breast cancer using gene expression data

    PubMed Central

    2011-01-01

    Background One of the primary objectives in cancer research is to identify causal genomic alterations, such as somatic copy number variation (CNV) and somatic mutations, during tumor development. Many valuable studies lack genomic data to detect CNV; therefore, methods that are able to infer CNVs from gene expression data would help maximize the value of these studies. Results We developed a framework for identifying recurrent regions of CNV and distinguishing the cancer driver genes from the passenger genes in the regions. By inferring CNV regions across many datasets we were able to identify 109 recurrent amplified/deleted CNV regions. Many of these regions are enriched for genes involved in many important processes associated with tumorigenesis and cancer progression. Genes in these recurrent CNV regions were then examined in the context of gene regulatory networks to prioritize putative cancer driver genes. The cancer driver genes uncovered by the framework include not only well-known oncogenes but also a number of novel cancer susceptibility genes validated via siRNA experiments. Conclusions To our knowledge, this is the first effort to systematically identify and validate drivers for expression based CNV regions in breast cancer. The framework where the wavelet analysis of copy number alteration based on expression coupled with the gene regulatory network analysis, provides a blueprint for leveraging genomic data to identify key regulatory components and gene targets. This integrative approach can be applied to many other large-scale gene expression studies and other novel types of cancer data such as next-generation sequencing based expression (RNA-Seq) as well as CNV data. PMID:21806811

  20. HAND2 Target Gene Regulatory Networks Control Atrioventricular Canal and Cardiac Valve Development.

    PubMed

    Laurent, Frédéric; Girdziusaite, Ausra; Gamart, Julie; Barozzi, Iros; Osterwalder, Marco; Akiyama, Jennifer A; Lincoln, Joy; Lopez-Rios, Javier; Visel, Axel; Zuniga, Aimée; Zeller, Rolf

    2017-05-23

    The HAND2 transcriptional regulator controls cardiac development, and we uncover additional essential functions in the endothelial to mesenchymal transition (EMT) underlying cardiac cushion development in the atrioventricular canal (AVC). In Hand2-deficient mouse embryos, the EMT underlying AVC cardiac cushion formation is disrupted, and we combined ChIP-seq of embryonic hearts with transcriptome analysis of wild-type and mutants AVCs to identify the functionally relevant HAND2 target genes. The HAND2 target gene regulatory network (GRN) includes most genes with known functions in EMT processes and AVC cardiac cushion formation. One of these is Snai1, an EMT master regulator whose expression is lost from Hand2-deficient AVCs. Re-expression of Snai1 in mutant AVC explants partially restores this EMT and mesenchymal cell migration. Furthermore, the HAND2-interacting enhancers in the Snai1 genomic landscape are active in embryonic hearts and other Snai1-expressing tissues. These results show that HAND2 directly regulates the molecular cascades initiating AVC cardiac valve development. Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.

  1. HAND2 Target Gene Regulatory Networks Control Atrioventricular Canal and Cardiac Valve Development

    DOE PAGES

    Laurent, Frédéric; Girdziusaite, Ausra; Gamart, Julie; ...

    2017-05-23

    The HAND2 transcriptional regulator controls cardiac development, and we uncover additional essential functions in the endothelial to mesenchymal transition (EMT) underlying cardiac cushion development in the atrioventricular canal (AVC). In Hand2-deficient mouse embryos, the EMT underlying AVC cardiac cushion formation is disrupted, and we combined ChIP-seq of embryonic hearts with transcriptome analysis of wild-type and mutants AVCs to identify the functionally relevant HAND2 target genes. The HAND2 target gene regulatory network (GRN) includes most genes with known functions in EMT processes and AVC cardiac cushion formation. One of these is Snai1, an EMT master regulator whose expression is lost frommore » Hand2-deficient AVCs. Re-expression of Snai1 in mutant AVC explants partially restores this EMT and mesenchymal cell migration. Furthermore, the HAND2-interacting enhancers in the Snai1 genomic landscape are active in embryonic hearts and other Snai1-expressing tissues. These results show that HAND2 directly regulates the molecular cascades initiating AVC cardiac valve development.« less

  2. HAND2 Target Gene Regulatory Networks Control Atrioventricular Canal and Cardiac Valve Development

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Laurent, Frédéric; Girdziusaite, Ausra; Gamart, Julie

    The HAND2 transcriptional regulator controls cardiac development, and we uncover additional essential functions in the endothelial to mesenchymal transition (EMT) underlying cardiac cushion development in the atrioventricular canal (AVC). In Hand2-deficient mouse embryos, the EMT underlying AVC cardiac cushion formation is disrupted, and we combined ChIP-seq of embryonic hearts with transcriptome analysis of wild-type and mutants AVCs to identify the functionally relevant HAND2 target genes. The HAND2 target gene regulatory network (GRN) includes most genes with known functions in EMT processes and AVC cardiac cushion formation. One of these is Snai1, an EMT master regulator whose expression is lost frommore » Hand2-deficient AVCs. Re-expression of Snai1 in mutant AVC explants partially restores this EMT and mesenchymal cell migration. Furthermore, the HAND2-interacting enhancers in the Snai1 genomic landscape are active in embryonic hearts and other Snai1-expressing tissues. These results show that HAND2 directly regulates the molecular cascades initiating AVC cardiac valve development.« less

  3. Generation of mammalian cells stably expressing multiple genes at predetermined levels.

    PubMed

    Liu, X; Constantinescu, S N; Sun, Y; Bogan, J S; Hirsch, D; Weinberg, R A; Lodish, H F

    2000-04-10

    Expression of cloned genes at desired levels in cultured mammalian cells is essential for studying protein function. Controlled levels of expression have been difficult to achieve, especially for cell lines with low transfection efficiency or when expression of multiple genes is required. An internal ribosomal entry site (IRES) has been incorporated into many types of expression vectors to allow simultaneous expression of two genes. However, there has been no systematic quantitative analysis of expression levels in individual cells of genes linked by an IRES, and thus the broad use of these vectors in functional analysis has been limited. We constructed a set of retroviral expression vectors containing an IRES followed by a quantitative selectable marker such as green fluorescent protein (GFP) or truncated cell surface proteins CD2 or CD4. The gene of interest is placed in a multiple cloning site 5' of the IRES sequence under the control of the retroviral long terminal repeat (LTR) promoter. These vectors exploit the approximately 100-fold differences in levels of expression of a retrovirus vector depending on its site of insertion in the host chromosome. We show that the level of expression of the gene downstream of the IRES and the expression level and functional activity of the gene cloned upstream of the IRES are highly correlated in stably infected target cells. This feature makes our vectors extremely useful for the rapid generation of stably transfected cell populations or clonal cell lines expressing specific amounts of a desired protein simply by fluorescent activated cell sorting (FACS) based on the level of expression of the gene downstream of the IRES. We show how these vectors can be used to generate cells expressing high levels of the erythropoietin receptor (EpoR) or a dominant negative Smad3 protein and to generate cells expressing two different cloned proteins, Ski and Smad4. Correlation of a biologic effect with the level of expression of the

  4. Targeted systemic gene therapy and molecular imaging of cancer contribution of the vascular-targeted AAVP vector.

    PubMed

    Hajitou, Amin

    2010-01-01

    Gene therapy and molecular-genetic imaging have faced a major problem: the lack of an efficient systemic gene delivery vector. Unquestionably, eukaryotic viruses have been the vectors of choice for gene delivery to mammalian cells; however, they have had limited success in systemic gene therapy. This is mainly due to undesired uptake by the liver and reticuloendothelial system, broad tropism for mammalian cells causing toxicity, and their immunogenicity. On the other hand, prokaryotic viruses such as bacteriophage (phage) have no tropism for mammalian cells, but can be engineered to deliver genes to these cells. However, phage-based vectors have inherently been considered poor vectors for mammalian cells. We have reported a new generation of vascular-targeted systemic hybrid prokaryotic-eukaryotic vectors as chimeras between an adeno-associated virus (AAV) and targeted bacteriophage (termed AAV/phage; AAVP). In this hybrid vector, the targeted bacteriophage serves as a shuttle to deliver the AAV transgene cassette inserted in an intergenomic region of the phage DNA genome. As a proof of concept, we assessed the in vivo efficacy of vector in animal models of cancer by displaying on the phage capsid the cyclic Arg-Gly-Asp (RGD-4C) ligand that binds to alphav integrin receptors specifically expressed on the angiogenic blood vessels of tumors. The ligand-directed vector was able to specifically deliver imaging and therapeutic transgenes to tumors in mice, rats, and dogs while sparing the normal organs. This chapter reviews some gene transfer strategies and the potential of the vascular-targeted AAVP vector for enhancing the effectiveness of existing systemic gene delivery and genetic-imaging technologies. Copyright (c) 2010 Elsevier Inc. All rights reserved.

  5. Vector design for liver specific expression of multiple interfering RNAs that target hepatitis B virus transcripts

    PubMed Central

    Snyder, Lindsey L.; Esser, Jonathan M.; Pachuk, Catherine J.; Steel, Laura F.

    2008-01-01

    RNA interference (RNAi) is a process that can target intracellular RNAs for degradation in a highly sequence specific manner, making it a powerful tool that is being pursued in both research and therapeutic applications. Hepatitis B virus (HBV) is a serious public health problem in need of better treatment options, and aspects of its life cycle make it an excellent target for RNAi-based therapeutics. We have designed a vector that expresses interfering RNAs that target HBV transcripts, including both viral RNA replicative intermediates and mRNAs encoding viral proteins. Our vector design incorporates many features of endogenous microRNA (miRNA) gene organization that are proving useful for the development of reagents for RNAi. In particular, our vector contains an RNA pol II driven gene cassette that leads to tissue specific expression and efficient processing of multiple interfering RNAs from a single transcript, without the co-expression of any protein product. This vector shows potent silencing of HBV targets in cell culture models of HBV infection. The vector design will be applicable to silencing of additional cellular or disease-related genes. PMID:18499277

  6. A multisite gateway-based toolkit for targeted gene expression and hairpin RNA silencing in tomato fruits.

    PubMed

    Estornell, Leandro Hueso; Orzáez, Diego; López-Peña, Lucas; Pineda, Benito; Antón, María Teresa; Moreno, Vicente; Granell, Antonio

    2009-04-01

    A collection of fruit promoters, reporter genes and protein tags has been constructed in a triple-gateway format, a recombination-based cloning system that facilitates the tandem assembly of three DNA fragments into plant expression vectors. The new pENFRUIT collection includes, among others, the classical tomato-ripening promoters E8 and 2A11 and a set of six new tomato promoters. The new promoter activities were characterized in both transient assays and stable transgenic plants. The range of expression of the new promoters comprises strong (PNH, PLI), medium (PLE, PFF, PHD) and weak (PSN) promoters driving gene expression preferentially in the fruit, and covering a wide range of tissues and developmental stages. Together, a total of 78 possible combinations for the expression of a gene of interest in the fruit, plus a set of five reporters for new promoter analysis, was made available in the current collection. Moreover, the pENFRUIT promoter collection is adaptable to hairpin RNA strategies aimed at tissue/organ-specific gene silencing with only an additional cloning step. The pENFRUIT toolkit broadens the spectrum of promoter activities available for fruit biotechnology and fundamental research, and bypasses technical difficulties of current ligase-dependent cloning techniques in the construction of fruit expression cassettes. The pENFRUIT vector collection is available for the research community in a plasmid repository, facilitating its accessibility.

  7. The myeloid-binding peptide adenoviral vector enables multi-organ vascular endothelial gene targeting.

    PubMed

    Lu, Zhi Hong; Kaliberov, Sergey; Zhang, Jingzhu; Muz, Barbara; Azab, Abdel K; Sohn, Rebecca E; Kaliberova, Lyudmila; Du, Yingqiu; Curiel, David T; Arbeit, Jeffrey M

    2014-08-01

    Vascular endothelial cells (ECs) are ideal gene therapy targets as they provide widespread tissue access and are the first contact surfaces following intravenous vector administration. Human recombinant adenovirus serotype 5 (Ad5) is the most frequently used gene transfer system because of its appreciable transgene payload capacity and lack of somatic mutation risk. However, standard Ad5 vectors predominantly transduce liver but not the vasculature following intravenous administration. We recently developed an Ad5 vector with a myeloid cell-binding peptide (MBP) incorporated into the knob-deleted, T4 fibritin chimeric fiber (Ad.MBP). This vector was shown to transduce pulmonary ECs presumably via a vector handoff mechanism. Here we tested the body-wide tropism of the Ad.MBP vector, its myeloid cell necessity, and vector-EC expression dose response. Using comprehensive multi-organ co-immunofluorescence analysis, we discovered that Ad.MBP produced widespread EC transduction in the lung, heart, kidney, skeletal muscle, pancreas, small bowel, and brain. Surprisingly, Ad.MBP retained hepatocyte tropism albeit at a reduced frequency compared with the standard Ad5. While binding specifically to myeloid cells ex vivo, multi-organ Ad.MBP expression was not dependent on circulating monocytes or macrophages. Ad.MBP dose de-escalation maintained full lung-targeting capacity but drastically reduced transgene expression in other organs. Swapping the EC-specific ROBO4 for the CMV promoter/enhancer abrogated hepatocyte expression but also reduced gene expression in other organs. Collectively, our multilevel targeting strategy could enable therapeutic biological production in previously inaccessible organs that pertain to the most debilitating or lethal human diseases.

  8. Neurobiology of autism gene products: towards pathogenesis and drug targets.

    PubMed

    Kleijer, Kristel T E; Schmeisser, Michael J; Krueger, Dilja D; Boeckers, Tobias M; Scheiffele, Peter; Bourgeron, Thomas; Brose, Nils; Burbach, J Peter H

    2014-03-01

    The genetic heterogeneity of autism spectrum disorders (ASDs) is enormous, and the neurobiology of proteins encoded by genes associated with ASD is very diverse. Revealing the mechanisms on which different neurobiological pathways in ASD pathogenesis converge may lead to the identification of drug targets. The main objective is firstly to outline the main molecular networks and neuronal mechanisms in which ASD gene products participate and secondly to answer the question how these converge. Finally, we aim to pinpoint drug targets within these mechanisms. Literature review of the neurobiological properties of ASD gene products with a special focus on the developmental consequences of genetic defects and the possibility to reverse these by genetic or pharmacological interventions. The regulation of activity-dependent protein synthesis appears central in the pathogenesis of ASD. Through sequential consequences for axodendritic function, neuronal disabilities arise expressed as behavioral abnormalities and autistic symptoms in ASD patients. Several known ASD gene products have their effect on this central process by affecting protein synthesis intrinsically, e.g., through enhancing the mammalian target of rapamycin (mTOR) signal transduction pathway or through impairing synaptic function in general. These are interrelated processes and can be targeted by compounds from various directions: inhibition of protein synthesis through Lovastatin, mTOR inhibition using rapamycin, or mGluR-related modulation of synaptic activity. ASD gene products may all feed into a central process of translational control that is important for adequate glutamatergic regulation of dendritic properties. This process can be modulated by available compounds but may also be targeted by yet unexplored routes.

  9. Methylation-Sensitive Expression of a DNA Demethylase Gene Serves As an Epigenetic Rheostat

    PubMed Central

    Williams, Ben P.; Pignatta, Daniela; Henikoff, Steven; Gehring, Mary

    2015-01-01

    Genomes must balance active suppression of transposable elements (TEs) with the need to maintain gene expression. In Arabidopsis, euchromatic TEs are targeted by RNA-directed DNA methylation (RdDM). Conversely, active DNA demethylation prevents accumulation of methylation at genes proximal to these TEs. It is unknown how a cellular balance between methylation and demethylation activities is achieved. Here we show that both RdDM and DNA demethylation are highly active at a TE proximal to the major DNA demethylase gene ROS1. Unexpectedly, and in contrast to most other genomic targets, expression of ROS1 is promoted by DNA methylation and antagonized by DNA demethylation. We demonstrate that inducing methylation in the ROS1 proximal region is sufficient to restore ROS1 expression in an RdDM mutant. Additionally, methylation-sensitive expression of ROS1 is conserved in other species, suggesting it is adaptive. We propose that the ROS1 locus functions as an epigenetic rheostat, tuning the level of demethylase activity in response to methylation alterations, thus ensuring epigenomic stability. PMID:25826366

  10. Integrated analysis of microRNAs, transcription factors and target genes expression discloses a specific molecular architecture of hyperdiploid multiple myeloma

    PubMed Central

    Caracciolo, Daniele; Agnelli, Luca; Neri, Antonino; Walker, Brian A.; Morgan, Gareth J.; Cannataro, Mario

    2015-01-01

    Multiple Myeloma (MM) is a malignancy characterized by the hyperdiploid (HD-MM) and the non-hyperdiploid (nHD-MM) subtypes. To shed light within the molecular architecture of these subtypes, we used a novel integromics approach. By annotated MM patient mRNA/microRNA (miRNA) datasets, we investigated mRNAs and miRNAs profiles with relation to changes in transcriptional regulators expression. We found that HD-MM displays specific gene and miRNA expression profiles, involving the Signal Transducer and Activator of Transcription (STAT)3 pathway as well as the Transforming Growth Factor–beta (TGFβ) and the transcription regulator Nuclear Protein-1 (NUPR1). Our data define specific molecular features of HD-MM that may translate in the identification of novel relevant druggable targets. PMID:26056083

  11. Mechanisms of crystalline silica-induced pulmonary toxicity revealed by global gene expression profiling

    PubMed Central

    Sellamuthu, Rajendran; Umbright, Christina; Li, Shengqiao; Kashon, Michael; Joseph, Pius

    2015-01-01

    A proper understanding of the mechanisms underlying crystalline silica-induced pulmonary toxicity has implications in the management and potential prevention of the adverse health effects associated with silica exposure including silicosis, cancer and several auto-immune diseases. Human lung type II epithelial cells and rat lungs exposed to crystalline silica were employed as experimental models to determine global gene expression changes in order to understand the molecular mechanisms underlying silica-induced pulmonary toxicity. The differential gene expression profile induced by silica correlated with its toxicity in the A549 cells. The biological processes perturbed by silica exposure in the A549 cells and rat lungs, as identified by the bioinformatics analysis of the differentially expressed genes, demonstrated significant similarity. Functional categorization of the differentially expressed genes identified cancer, cellular movement, cellular growth and proliferation, cell death, inflammatory response, cell cycle, cellular development, and genetic disorder as top ranking biological functions perturbed by silica exposure in A549 cells and rat lungs. Results of our study, in addition to confirming several previously identified molecular targets and mechanisms involved in silica toxicity, identified novel molecular targets and mechanisms potentially involved in silica-induced pulmonary toxicity. Further investigations, including those focused on the novel molecular targets and mechanisms identified in the current study may result in better management and, possibly, reduction and/or prevention of the potential adverse health effects associated with crystalline silica exposure. PMID:22087542

  12. In Vivo Bio-distribution and Efficient Tumor Targeting of Gelatin/Silica Nanoparticles for Gene Delivery

    NASA Astrophysics Data System (ADS)

    Zhao, Xueqin; Wang, Jun; Tao, SiJie; Ye, Ting; Kong, Xiangdong; Ren, Lei

    2016-04-01

    The non-viral gene delivery system is an attractive alternative to cancer therapy. The clinical success of non-viral gene delivery is hampered by transfection efficiency and tumor targeting, which can be individually overcome by addition of functional modules such as cell penetration or targeting. Here, we first engineered the multifunctional gelatin/silica (GS) nanovectors with separately controllable modules, including tumor-targeting aptamer AGRO100, membrane-destabilizing peptide HA2, and polyethylene glycol (PEG), and then studied their bio-distribution and in vivo transfection efficiencies by contrast resonance imaging (CRI). The results suggest that the sizes and zeta potentials of multifunctional gelatin/silica nanovectors were 203-217 nm and 2-8 mV, respectively. Functional GS-PEG nanoparticles mainly accumulated in the liver and tumor, with the lowest uptake by the heart and brain. Moreover, the synergistic effects of tumor-targeting aptamer AGRO100 and fusogenic peptide HA2 promoted the efficient cellular internalization in the tumor site. More importantly, the combined use of AGRO100 and PEG enhanced tumor gene expression specificity and effectively reduced toxicity in reticuloendothelial system (RES) organs after intravenous injection. Additionally, low accumulation of GS-PEG was observed in the heart tissues with high gene expression levels, which could provide opportunities for non-invasive gene therapy.

  13. Gene Expression Profiling of Benign and Malignant Pheochromocytoma

    PubMed Central

    BROUWERS, FREDERIEKE M.; ELKAHLOUN, ABDEL G.; MUNSON, PETER J.; EISENHOFER, GRAEME; BARB, JENNIFER; LINEHAN, W. MARSTON; LENDERS, JACQUES W.M.; DE KRIJGER, RONALD; MANNELLI, MASSIMO; UDELSMAN, ROBERT; OCAL, IDRIS T.; SHULKIN, BARRY L.; BORNSTEIN, STEFAN R.; BREZA, JAN; KSINANTOVA, LUCIA; PACAK, KAREL

    2016-01-01

    There are currently no reliable diagnostic and prognostic markers or effective treatments for malignant pheochromocytoma. This study used oligonucleotide microarrays to examine gene expression profiles in pheochromocytomas from 90 patients, including 20 with malignant tumors, the latter including metastases and primary tumors from which metastases developed. Other subgroups of tumors included those defined by tissue norepinephrine compared to epinephrine contents (i.e., noradrenergic versus adrenergic phenotypes), adrenal versus extra-adrenal locations, and presence of germline mutations of genes pre-disposing to the tumor. Correcting for the confounding influence of nora-drenergic versus adrenergic catecholamine phenotype by the analysis of variance revealed a larger and more accurate number of genes that discriminated benign from malignant pheochromocytomas than when the confounding influence of catecholamine phenotype was not considered. Seventy percent of these genes were underexpressed in malignant compared to benign tumors. Similarly, 89% of genes were underexpressed in malignant primary tumors compared to benign tumors, suggesting that malignant potential is largely characterized by a less-differentiated pattern of gene expression. The present database of differentially expressed genes provides a unique resource for mapping the pathways leading to malignancy and for establishing new targets for treatment and diagnostic and prognostic markers of malignant disease. The database may also be useful for examining mechanisms of tumorigenesis and genotype–phenotype relationships. Further progress on the basis of this database can be made from follow-up confirmatory studies, application of bioinformatics approaches for data mining and pathway analyses, testing in pheochromocytoma cell culture and animal model systems, and retrospective and prospective studies of diagnostic markers. PMID:17102123

  14. Molecular mechanisms of curcumin action: gene expression.

    PubMed

    Shishodia, Shishir

    2013-01-01

    Curcumin derived from the tropical plant Curcuma longa has a long history of use as a dietary agent, food preservative, and in traditional Asian medicine. It has been used for centuries to treat biliary disorders, anorexia, cough, diabetic wounds, hepatic disorders, rheumatism, and sinusitis. The preventive and therapeutic properties of curcumin are associated with its antioxidant, anti-inflammatory, and anticancer properties. Extensive research over several decades has attempted to identify the molecular mechanisms of curcumin action. Curcumin modulates numerous molecular targets by altering their gene expression, signaling pathways, or through direct interaction. Curcumin regulates the expression of inflammatory cytokines (e.g., TNF, IL-1), growth factors (e.g., VEGF, EGF, FGF), growth factor receptors (e.g., EGFR, HER-2, AR), enzymes (e.g., COX-2, LOX, MMP9, MAPK, mTOR, Akt), adhesion molecules (e.g., ELAM-1, ICAM-1, VCAM-1), apoptosis related proteins (e.g., Bcl-2, caspases, DR, Fas), and cell cycle proteins (e.g., cyclin D1). Curcumin modulates the activity of several transcription factors (e.g., NF-κB, AP-1, STAT) and their signaling pathways. Based on its ability to affect multiple targets, curcumin has the potential for the prevention and treatment of various diseases including cancers, arthritis, allergies, atherosclerosis, aging, neurodegenerative disease, hepatic disorders, obesity, diabetes, psoriasis, and autoimmune diseases. This review summarizes the molecular mechanisms of modulation of gene expression by curcumin. Copyright © 2012 International Union of Biochemistry and Molecular Biology, Inc.

  15. CRISPR/Cas9-mediated gene knockout is insensitive to target copy number but is dependent on guide RNA potency and Cas9/sgRNA threshold expression level

    PubMed Central

    Yuen, Garmen; Khan, Fehad J.; Gao, Shaojian; Stommel, Jayne M.; Batchelor, Eric; Wu, Xiaolin

    2017-01-01

    Abstract CRISPR/Cas9 is a powerful gene editing tool for gene knockout studies and functional genomic screens. Successful implementation of CRISPR often requires Cas9 to elicit efficient target knockout in a population of cells. In this study, we investigated the role of several key factors, including variation in target copy number, inherent potency of sgRNA guides, and expression level of Cas9 and sgRNA, in determining CRISPR knockout efficiency. Using isogenic, clonal cell lines with variable copy numbers of an EGFP transgene, we discovered that CRISPR knockout is relatively insensitive to target copy number, but is highly dependent on the potency of the sgRNA guide sequence. Kinetic analysis revealed that most target mutation occurs between 5 and 10 days following Cas9/sgRNA transduction, while sgRNAs with different potencies differ by their knockout time course and by their terminal-phase knockout efficiency. We showed that prolonged, low level expression of Cas9 and sgRNA often fails to elicit target mutation, particularly if the potency of the sgRNA is also low. Our findings provide new insights into the behavior of CRISPR/Cas9 in mammalian cells that could be used for future improvement of this platform. PMID:29036671

  16. CRISPR/Cas9-mediated gene knockout is insensitive to target copy number but is dependent on guide RNA potency and Cas9/sgRNA threshold expression level.

    PubMed

    Yuen, Garmen; Khan, Fehad J; Gao, Shaojian; Stommel, Jayne M; Batchelor, Eric; Wu, Xiaolin; Luo, Ji

    2017-11-16

    CRISPR/Cas9 is a powerful gene editing tool for gene knockout studies and functional genomic screens. Successful implementation of CRISPR often requires Cas9 to elicit efficient target knockout in a population of cells. In this study, we investigated the role of several key factors, including variation in target copy number, inherent potency of sgRNA guides, and expression level of Cas9 and sgRNA, in determining CRISPR knockout efficiency. Using isogenic, clonal cell lines with variable copy numbers of an EGFP transgene, we discovered that CRISPR knockout is relatively insensitive to target copy number, but is highly dependent on the potency of the sgRNA guide sequence. Kinetic analysis revealed that most target mutation occurs between 5 and 10 days following Cas9/sgRNA transduction, while sgRNAs with different potencies differ by their knockout time course and by their terminal-phase knockout efficiency. We showed that prolonged, low level expression of Cas9 and sgRNA often fails to elicit target mutation, particularly if the potency of the sgRNA is also low. Our findings provide new insights into the behavior of CRISPR/Cas9 in mammalian cells that could be used for future improvement of this platform. Published by Oxford University Press on behalf of Nucleic Acids Research 2017.

  17. Selection of suitable reference genes for gene expression studies in Staphylococcus capitis during growth under erythromycin stress.

    PubMed

    Cui, Bintao; Smooker, Peter M; Rouch, Duncan A; Deighton, Margaret A

    2016-08-01

    Accurate and reproducible measurement of gene transcription requires appropriate reference genes, which are stably expressed under different experimental conditions to provide normalization. Staphylococcus capitis is a human pathogen that produces biofilm under stress, such as imposed by antimicrobial agents. In this study, a set of five commonly used staphylococcal reference genes (gyrB, sodA, recA, tuf and rpoB) were systematically evaluated in two clinical isolates of Staphylococcus capitis (S. capitis subspecies urealyticus and capitis, respectively) under erythromycin stress in mid-log and stationary phases. Two public software programs (geNorm and NormFinder) and two manual calculation methods, reference residue normalization (RRN) and relative quantitative (RQ), were applied. The potential reference genes selected by the four algorithms were further validated by comparing the expression of a well-studied biofilm gene (icaA) with phenotypic biofilm formation in S. capitis under four different experimental conditions. The four methods differed considerably in their ability to predict the most suitable reference gene or gene combination for comparing icaA expression under different conditions. Under the conditions used here, the RQ method provided better selection of reference genes than the other three algorithms; however, this finding needs to be confirmed with a larger number of isolates. This study reinforces the need to assess the stability of reference genes for analysis of target gene expression under different conditions and the use of more than one algorithm in such studies. Although this work was conducted using a specific human pathogen, it emphasizes the importance of selecting suitable reference genes for accurate normalization of gene expression more generally.

  18. COX inhibitors directly alter gene expression: role in cancer prevention?

    PubMed Central

    Wang, Xingya; Baek, Seung Joon; Eling, Thomas

    2016-01-01

    Inflammation is an important contributor to the development and progression of human cancers. Inflammatory lipid metabolites, prostaglandins, formed from arachidonic acid by prostaglandin H synthases commonly called cyclooxygenases (COXs) bind to specific receptors that activate signaling pathways driving the development and progression of tumors. Inhibitors of prostaglandin formation, COX inhibitors, or nonsteroidal anti-inflammatory drugs (NSAIDs) are well documented as agents that inhibit tumor growth and with long-term use prevent tumor development. NSAIDs also alter gene expression independent of COX inhibition and these changes in gene expression also appear to contribute to the anti-tumorigenic activity of these drugs. Many NSAIDs, as illustrated by sulindac sulfide, alter gene expressions by altering the expression or phosphorylation status of the transcription factors specificity protein 1 and early growth response-1 with the balance between these two events resulting in increases or decreases in specific target genes. In this review, we have summarized and discussed the various genes altered by this mechanism after NSAID treatment and how these changes in expression relate to the anti-tumorigenic activity. A major focus of the review is on NSAID-activated gene (NAG-1) or growth differentiation factor 15. This unique member of the TGF-β superfamily is highly induced by NSAIDs and numerous drugs and chemicals with anti-tumorigenic activities. Investigations with a transgenic mouse expressing the human NAG-1 suggest it acts to suppress tumor development in several mouse models of cancer. The biochemistry and biology of NAG-1 were discussed as potential contributor to cancer prevention by COX inhibitors. PMID:22020924

  19. Region-specific changes in gene expression in rat brain after chronic treatment with levetiracetam or phenytoin

    PubMed Central

    Hassel, Bjørnar; Taubøll, Erik; Shaw, Renee; Gjerstad, Leif; Dingledine, Ray

    2014-01-01

    Summary Purpose It is commonly assumed that antiepileptic drugs (AEDs) act similarly in the various parts of the brain as long as their molecular targets are present. A few experimental studies on metabolic effects of vigabatrin, levetiracetam, valproate, and lamotrigine have shown that these drugs may act differently in different brain regions. We examined effects of chronic treatment with levetiracetam or phenytoin on mRNA levels to detect regional drug effects in a broad, nonbiased manner. Methods mRNA levels were monitored in three brain regions with oligonucleotide-based microarrays. Results Levetiracetam (150 mg/kg for 90 days) changed the expression of 65 genes in pons/medulla oblongata, two in hippocampus, and one in frontal cortex. Phenytoin (75 mg/kg), in contrast, changed the expression of only three genes in pons/medulla oblongata, but 64 genes in hippocampus, and 327 genes in frontal cortex. Very little overlap between regions or drug treatments was observed with respect to effects on gene expression. Discussion We conclude that chronic treatment with levetiracetam or phenytoin causes region-specific and highly differential effects on gene expression in the brain. Regional effects on gene expression could reflect regional differences in molecular targets of AEDs, and they could influence the clinical profiles of AEDs. PMID:20345932

  20. Microarray profiling of gene expression in human adipocytes in response to anthocyanins.

    PubMed

    Tsuda, Takanori; Ueno, Yuki; Yoshikawa, Toshikazu; Kojo, Hitoshi; Osawa, Toshihiko

    2006-04-14

    Adipocyte dysfunction is strongly associated with the development of obesity and insulin resistance. It is accepted that the regulation of adipocytokine secretion or the adipocyte specific gene expression is one of the most important targets for the prevention of obesity and amelioration of insulin sensitivity. Recently, we demonstrated that anthocyanins, which are pigments widespread in the plant kingdom, have the potency of anti-obesity in mice and the enhancement adipocytokine secretion and its gene expression in adipocytes. In this study, we have shown the gene expression profile in human adipocytes treated with anthocyanins (cyanidin 3-glucoside; C3G or cyanidin; Cy). The human adipocytes were treated with 100 microM C3G, Cy or vehicle for 24 h. The total RNA from the adipocytes was isolated and carried out GeneChip microarray analysis. Based on the gene expression profile, we demonstrated the significant changes of adipocytokine expression (up-regulation of adiponectin and down-regulation of plasminogen activator inhibitor-1 and interleukin-6). Some of lipid metabolism related genes (uncoupling protein2, acylCoA oxidase1 and perilipin) also significantly induced in both common the C3G or Cy treatment groups. These studies have provided an overview of the gene expression profiles in human adipocytes treated with anthocyanins and demonstrated that anthocyanins can regulate adipocytokine gene expression to ameliorate adipocyte function related with obesity and diabetes that merit further investigation.

  1. Investigation of candidate genes for osteoarthritis based on gene expression profiles.

    PubMed

    Dong, Shuanghai; Xia, Tian; Wang, Lei; Zhao, Qinghua; Tian, Jiwei

    2016-12-01

    To explore the mechanism of osteoarthritis (OA) and provide valid biological information for further investigation. Gene expression profile of GSE46750 was downloaded from Gene Expression Omnibus database. The Linear Models for Microarray Data (limma) package (Bioconductor project, http://www.bioconductor.org/packages/release/bioc/html/limma.html) was used to identify differentially expressed genes (DEGs) in inflamed OA samples. Gene Ontology function enrichment analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways enrichment analysis of DEGs were performed based on Database for Annotation, Visualization and Integrated Discovery data, and protein-protein interaction (PPI) network was constructed based on the Search Tool for the Retrieval of Interacting Genes/Proteins database. Regulatory network was screened based on Encyclopedia of DNA Elements. Molecular Complex Detection was used for sub-network screening. Two sub-networks with highest node degree were integrated with transcriptional regulatory network and KEGG functional enrichment analysis was processed for 2 modules. In total, 401 up- and 196 down-regulated DEGs were obtained. Up-regulated DEGs were involved in inflammatory response, while down-regulated DEGs were involved in cell cycle. PPI network with 2392 protein interactions was constructed. Moreover, 10 genes including Interleukin 6 (IL6) and Aurora B kinase (AURKB) were found to be outstanding in PPI network. There are 214 up- and 8 down-regulated transcription factor (TF)-target pairs in the TF regulatory network. Module 1 had TFs including SPI1, PRDM1, and FOS, while module 2 contained FOSL1. The nodes in module 1 were enriched in chemokine signaling pathway, while the nodes in module 2 were mainly enriched in cell cycle. The screened DEGs including IL6, AGT, and AURKB might be potential biomarkers for gene therapy for OA by being regulated by TFs such as FOS and SPI1, and participating in the cell cycle and cytokine-cytokine receptor

  2. Identification of target genes of synovial sarcoma-associated fusion oncoprotein using human pluripotent stem cells

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Hayakawa, Kazuo; Department of Cell Growth and Differentiation, Center for iPS Cell Research and Application, Kyoto University, Kyoto; Department of Orthopaedic Surgery, Graduate School of Medical Sciences, Nagoya City University, Nagoya

    2013-03-22

    Highlights: ► We tried to identify targets of synovial sarcoma (SS)-associated SYT–SSX fusion gene. ► We established pluripotent stem cell (PSC) lines with inducible SYT–SSX gene. ► SYT–SSX responsive genes were identified by the induction of SYT–SSX in PSC. ► SS-related genes were selected from database by in silico analyses. ► 51 genes were finally identified among SS-related genes as targets of SYT–SSX in PSC. -- Abstract: Synovial sarcoma (SS) is a malignant soft tissue tumor harboring chromosomal translocation t(X; 18)(p11.2; q11.2), which produces SS-specific fusion gene, SYT–SSX. Although precise function of SYT–SSX remains to be investigated, accumulating evidences suggestmore » its role in gene regulation via epigenetic mechanisms, and the product of SYT–SSX target genes may serve as biomarkers of SS. Lack of knowledge about the cell-of-origin of SS, however, has placed obstacle in the way of target identification. Here we report a novel approach to identify SYT–SSX2 target genes using human pluripotent stem cells (hPSCs) containing a doxycycline-inducible SYT–SSX2 gene. SYT–SSX2 was efficiently induced both at mRNA and protein levels within three hours after doxycycline administration, while no morphological change of hPSCs was observed until 24 h. Serial microarray analyses identified genes of which the expression level changed more than twofold within 24 h. Surprisingly, the majority (297/312, 95.2%) were up-regulated genes and a result inconsistent with the current concept of SYT–SSX as a transcriptional repressor. Comparing these genes with SS-related genes which were selected by a series of in silico analyses, 49 and 2 genes were finally identified as candidates of up- and down-regulated target of SYT–SSX, respectively. Association of these genes with SYT–SSX in SS cells was confirmed by knockdown experiments. Expression profiles of SS-related genes in hPSCs and human mesenchymal stem cells (hMSCs) were

  3. Differential gene expression patterns between smokers and non‐smokers: cause or consequence?

    PubMed Central

    Jansen, Rick; Brooks, Andy; Willemsen, Gonneke; van Grootheest, Gerard; de Geus, Eco; Smit, Jan H.; Penninx, Brenda W.; Boomsma, Dorret I.

    2015-01-01

    Abstract The molecular mechanisms causing smoking‐induced health decline are largely unknown. To elucidate the molecular pathways involved in cause and consequences of smoking behavior, we conducted a genome‐wide gene expression study in peripheral blood samples targeting 18 238 genes. Data of 743 smokers, 1686 never smokers and 890 ex‐smokers were available from two population‐based cohorts from the Netherlands. In addition, data of 56 monozygotic twin pairs discordant for ever smoking were used. One hundred thirty‐two genes were differentially expressed between current smokers and never smokers (P < 1.2 × 10−6, Bonferroni correction). The most significant genes were G protein‐coupled receptor 15 (P < 1 × 10−150) and leucine‐rich repeat neuronal 3 (P < 1 × 10−44). The smoking‐related genes were enriched for immune system, blood coagulation, natural killer cell and cancer pathways. By taking the data of ex‐smokers into account, expression of these 132 genes was classified into reversible (94 genes), slowly reversible (31 genes), irreversible (6 genes) or inconclusive (1 gene). Expression of 6 of the 132 genes (three reversible and three slowly reversible) was confirmed to be reactive to smoking as they were differentially expressed in monozygotic pairs discordant for smoking. Cis‐expression quantitative trait loci for GPR56 and RARRES3 (downregulated in smokers) were associated with increased number of cigarettes smoked per day in a large genome‐wide association meta‐analysis, suggesting a causative effect of GPR56 and RARRES3 expression on smoking behavior. In conclusion, differential gene expression patterns in smokers are extensive and cluster in several underlying disease pathways. Gene expression differences seem mainly direct consequences of smoking, and largely reversible after smoking cessation. However, we also identified DNA variants that may influence smoking behavior via the mediating gene

  4. Molecular-genetic imaging based on reporter gene expression.

    PubMed

    Kang, Joo Hyun; Chung, June-Key

    2008-06-01

    Molecular imaging includes proteomic, metabolic, cellular biologic process, and genetic imaging. In a narrow sense, molecular imaging means genetic imaging and can be called molecular-genetic imaging. Imaging reporter genes play a leading role in molecular-genetic imaging. There are 3 major methods of molecular-genetic imaging, based on optical, MRI, and nuclear medicine modalities. For each of these modalities, various reporter genes and probes have been developed, and these have resulted in successful transitions from bench to bedside applications. Each of these imaging modalities has its unique advantages and disadvantages. Fluorescent and bioluminescent optical imaging modalities are simple, less expensive, more convenient, and more user friendly than other imaging modalities. Another advantage, especially of bioluminescence imaging, is its ability to detect low levels of gene expression. MRI has the advantage of high spatial resolution, whereas nuclear medicine methods are highly sensitive and allow data from small-animal imaging studies to be translated to clinical practice. Moreover, multimodality imaging reporter genes will allow us to choose the imaging technologies that are most appropriate for the biologic problem at hand and facilitate the clinical application of reporter gene technologies. Reporter genes can be used to visualize the levels of expression of particular exogenous and endogenous genes and several intracellular biologic phenomena, including specific signal transduction pathways, nuclear receptor activities, and protein-protein interactions. This technique provides a straightforward means of monitoring tumor mass and can visualize the in vivo distributions of target cells, such as immune cells and stem cells. Molecular imaging has gradually evolved into an important tool for drug discovery and development, and transgenic mice with an imaging reporter gene can be useful during drug and stem cell therapy development. Moreover, instrumentation

  5. Knock-in strategy at 3'-end of Crx gene by CRISPR/Cas9 system shows the gene expression profiles during human photoreceptor differentiation.

    PubMed

    Homma, Kohei; Usui, Sumiko; Kaneda, Makoto

    2017-03-01

    Fluorescent reporter gene knock-in induced pluripotent stem cell (iPSC) lines have been used to evaluate the efficiency of differentiation into specific cell lineages. Here, we report a knock-in strategy for the generation of human iPSC reporter lines in which a 2A peptide sequence and a red fluorescent protein (E2-Crimson) gene were inserted at the termination codon of the cone-rod homeobox (Crx) gene, a photoreceptor-specific transcriptional factor gene. The knock-in iPSC lines were differentiated into fluorescence-expressing cells in 3D retinal differentiation culture, and the fluorescent cells also expressed Crx specifically in the nucleus. We found that the fluorescence intensity was positively correlated with the expression levels of Crx mRNA and that fluorescent cells expressed rod photoreceptor-specific genes in the later stage of differentiation. Finally, we treated the fluorescent cells with DAPT, a Notch inhibitor, and found that DAPT-enhanced retinal differentiation was associated with up-regulation of Crx, Otx2 and NeuroD1, and down-regulation of Hes5 and Ngn2. These suggest that this knock-in strategy at the 3'-end of the target gene, combined with the 2A peptide linked to fluorescent proteins, offers a useful tool for labeling specific cell lineages or monitoring expression of any marker genes without affecting the function of the target gene. © 2017 Molecular Biology Society of Japan and John Wiley & Sons Australia, Ltd.

  6. Development of an Expression Vector to Overexpress or Downregulate Genes in Curvularia protuberata.

    PubMed

    Liu, Chengke; Cleckler, Blake; Morsy, Mustafa

    2018-05-05

    Curvularia protuberata , an endophytic fungus in the Ascomycota, provides plants with thermotolerance only when it carries a mycovirus known as Curvularia thermotolerance virus (CThTV), and forms a three-way symbiotic relationship among these organisms. Under heat stress, several genes are expressed differently between virus-free C. protuberata (VF) and C. protuberata carrying CThTV (AN). We developed an expression vector, pM2Z-fun, carrying a zeocin resistance gene driven by the ToxA promoter, to study gene functions in C. protuberata to better understand this three-way symbiosis. Using this new 3.7-kb vector, five genes that are differentially expressed in C. protuberata —including genes involved in the trehalose, melanin, and catalase biosynthesis pathways—were successfully overexpressed or downregulated in VF or AN C. protuberata strains, respectively. The VF overexpression lines showed higher metabolite and enzyme activity than in the control VF strain. Furthermore, downregulation of expression of the same genes in the AN strain resulted in lower metabolite and enzyme activity than in the control AN strain. The newly generated expression vector, pM2Z-fun, has been successfully used to express target genes in C. protuberata and will be useful in further functional expression studies in other Ascomycota fungi.

  7. Study of NGEP expression in androgen sensitive prostate cancer cells: A potential target for immunotherapy

    PubMed Central

    Mohsenzadegan, Monireh; Tajik, Nader; Madjd, Zahra; Shekarabi, Mehdi; Farajollahi, Mohammad M

    2015-01-01

    Background: Prostate cancer is one of the leading causes of cancer deaths among men. New gene expressed in prostate (NGEP), is a prostate-specific gene expressed only in normal prostate and prostate cancer tissue. Because of its selective expression in prostate cancer cell surface, NGEP is a potential immunotherapeutic target. To target the NGEP in prostate cancer, it is essential to investigate its expression in prostate cancer cells. Methods: In the present study, we investigated NGEP expression in LNCaP and DU145 cells by real time and RT-PCR, flow cytometric and immunocytochemical analyses. Results: Real time and RT-PCR analyses of NGEP expression showed that NGEP was expressed in the LNCaP cells but not in DU145 cells. The detection of NGEP protein by flow cytometric and immunocytochemistry analyses indicated that NGEP protein was weakly expressed only in LNCaP cell membrane. Conclusion: Our results demonstrate that LNCaP cell line is more suitable than DU145 for NGEP expression studies; however, its low-level expression is a limiting issue. NGEP expression may be increased by androgen supplementation of LNCaP cell culture medium. PMID:26000254

  8. Glioma IL13Rα2 Is Associated with Mesenchymal Signature Gene Expression and Poor Patient Prognosis

    PubMed Central

    Starr, Renate; Deng, Xutao; Badie, Behnam; Yuan, Yate-Ching; Forman, Stephen J.; Barish, Michael E.

    2013-01-01

    A major challenge for successful immunotherapy against glioma is the identification and characterization of validated targets. We have taken a bioinformatics approach towards understanding the biological context of IL-13 receptor α2 (IL13Rα2) expression in brain tumors, and its functional significance for patient survival. Querying multiple gene expression databases, we show that IL13Rα2 expression increases with glioma malignancy grade, and expression for high-grade tumors is bimodal, with approximately 58% of WHO grade IV gliomas over-expressing this receptor. By several measures, IL13Rα2 expression in patient samples and low-passage primary glioma lines most consistently correlates with the expression of signature genes defining mesenchymal subclass tumors and negatively correlates with proneural signature genes as defined by two studies. Positive associations were also noted with proliferative signature genes, whereas no consistent associations were found with either classical or neural signature genes. Probing the potential functional consequences of this mesenchymal association through IPA analysis suggests that IL13Rα2 expression is associated with activation of proinflammatory and immune pathways characteristic of mesenchymal subclass tumors. In addition, survival analyses indicate that IL13Rα2 over-expression is associated with poor patient prognosis, a single gene correlation ranking IL13Rα2 in the top ~1% of total gene expression probes with regard to survival association with WHO IV gliomas. This study better defines the functional consequences of IL13Rα2 expression by demonstrating association with mesenchymal signature gene expression and poor patient prognosis. It thus highlights the utility of IL13Rα2 as a therapeutic target, and helps define patient populations most likely to respond to immunotherapy in present and future clinical trials. PMID:24204956

  9. Glioma IL13Rα2 is associated with mesenchymal signature gene expression and poor patient prognosis.

    PubMed

    Brown, Christine E; Warden, Charles D; Starr, Renate; Deng, Xutao; Badie, Behnam; Yuan, Yate-Ching; Forman, Stephen J; Barish, Michael E

    2013-01-01

    A major challenge for successful immunotherapy against glioma is the identification and characterization of validated targets. We have taken a bioinformatics approach towards understanding the biological context of IL-13 receptor α2 (IL13Rα2) expression in brain tumors, and its functional significance for patient survival. Querying multiple gene expression databases, we show that IL13Rα2 expression increases with glioma malignancy grade, and expression for high-grade tumors is bimodal, with approximately 58% of WHO grade IV gliomas over-expressing this receptor. By several measures, IL13Rα2 expression in patient samples and low-passage primary glioma lines most consistently correlates with the expression of signature genes defining mesenchymal subclass tumors and negatively correlates with proneural signature genes as defined by two studies. Positive associations were also noted with proliferative signature genes, whereas no consistent associations were found with either classical or neural signature genes. Probing the potential functional consequences of this mesenchymal association through IPA analysis suggests that IL13Rα2 expression is associated with activation of proinflammatory and immune pathways characteristic of mesenchymal subclass tumors. In addition, survival analyses indicate that IL13Rα2 over-expression is associated with poor patient prognosis, a single gene correlation ranking IL13Rα2 in the top ~1% of total gene expression probes with regard to survival association with WHO IV gliomas. This study better defines the functional consequences of IL13Rα2 expression by demonstrating association with mesenchymal signature gene expression and poor patient prognosis. It thus highlights the utility of IL13Rα2 as a therapeutic target, and helps define patient populations most likely to respond to immunotherapy in present and future clinical trials.

  10. Combining mouse mammary gland gene expression and comparative mapping for the identification of candidate genes for QTL of milk production traits in cattle

    PubMed Central

    Ron, Micha; Israeli, Galit; Seroussi, Eyal; Weller, Joel I; Gregg, Jeffrey P; Shani, Moshe; Medrano, Juan F

    2007-01-01

    Background Many studies have found segregating quantitative trait loci (QTL) for milk production traits in different dairy cattle populations. However, even for relatively large effects with a saturated marker map the confidence interval for QTL location by linkage analysis spans tens of map units, or hundreds of genes. Combining mapping and arraying has been suggested as an approach to identify candidate genes. Thus, gene expression analysis in the mammary gland of genes positioned in the confidence interval of the QTL can bridge the gap between fine mapping and quantitative trait nucleotide (QTN) determination. Results We hybridized Affymetrix microarray (MG-U74v2), containing 12,488 murine probes, with RNA derived from mammary gland of virgin, pregnant, lactating and involuting C57BL/6J mice in a total of nine biological replicates. We combined microarray data from two additional studies that used the same design in mice with a total of 75 biological replicates. The same filtering and normalization was applied to each microarray data using GeneSpring software. Analysis of variance identified 249 differentially expressed probe sets common to the three experiments along the four developmental stages of puberty, pregnancy, lactation and involution. 212 genes were assigned to their bovine map positions through comparative mapping, and thus form a list of candidate genes for previously identified QTLs for milk production traits. A total of 82 of the genes showed mammary gland-specific expression with at least 3-fold expression over the median representing all tissues tested in GeneAtlas. Conclusion This work presents a web tool for candidate genes for QTL (cgQTL) that allows navigation between the map of bovine milk production QTL, potential candidate genes and their level of expression in mammary gland arrays and in GeneAtlas. Three out of four confirmed genes that affect QTL in livestock (ABCG2, DGAT1, GDF8, IGF2) were over expressed in the target organ. Thus, cg

  11. HisB as novel selection marker for gene targeting approaches in Aspergillus niger.

    PubMed

    Fiedler, Markus R M; Gensheimer, Tarek; Kubisch, Christin; Meyer, Vera

    2017-03-08

    For Aspergillus niger, a broad set of auxotrophic and dominant resistance markers is available. However, only few offer targeted modification of a gene of interest into or at a genomic locus of choice, which hampers functional genomics studies. We thus aimed to extend the available set by generating a histidine auxotrophic strain with a characterized hisB locus for targeted gene integration and deletion in A. niger. A histidine-auxotrophic strain was established via disruption of the A. niger hisB gene by using the counterselectable pyrG marker. After curing, a hisB - , pyrG - strain was obtained, which served as recipient strain for further studies. We show here that both hisB orthologs from A. nidulans and A. niger can be used to reestablish histidine prototrophy in this recipient strain. Whereas the hisB gene from A. nidulans was suitable for efficient gene targeting at different loci in A. niger, the hisB gene from A. niger allowed efficient integration of a Tet-on driven luciferase reporter construct at the endogenous non-functional hisB locus. Subsequent analysis of the luciferase activity revealed that the hisB locus is tight under non-inducing conditions and allows even higher luciferase expression levels compared to the pyrG integration locus. Taken together, we provide here an alternative selection marker for A. niger, hisB, which allows efficient homologous integration rates as well as high expression levels which compare favorably to the well-established pyrG selection marker.

  12. Influence of in vivo growth on human glioma cell line gene expression: Convergent profiles under orthotopic conditions

    PubMed Central

    Camphausen, Kevin; Purow, Benjamin; Sproull, Mary; Scott, Tamalee; Ozawa, Tomoko; Deen, Dennis F.; Tofilon, Philip J.

    2005-01-01

    Defining the molecules that regulate tumor cell survival is an essential prerequisite for the development of targeted approaches to cancer treatment. Whereas many studies aimed at identifying such targets use human tumor cells grown in vitro or as s.c. xenografts, it is unclear whether such experimental models replicate the phenotype of the in situ tumor cell. To begin addressing this issue, we have used microarray analysis to define the gene expression profile of two human glioma cell lines (U251 and U87) when grown in vitro and in vivo as s.c. or as intracerebral (i.c.) xenografts. For each cell line, the gene expression profile generated from tissue culture was significantly different from that generated from the s.c. tumor, which was significantly different from those grown i.c. The disparity between the i.c gene expression profiles and those generated from s.c. xenografts suggests that whereas an in vivo growth environment modulates gene expression, orthotopic growth conditions induce a different set of modifications. In this study the U251 and U87 gene expression profiles generated under the three growth conditions were also compared. As expected, the profiles of the two glioma cell lines were significantly different when grown as monolayer cultures. However, the glioma cell lines had similar gene expression profiles when grown i.c. These results suggest that tumor cell gene expression, and thus phenotype, as defined in vitro is affected not only by in vivo growth but also by orthotopic growth, which may have implications regarding the identification of relevant targets for cancer therapy. PMID:15928080

  13. [Differentially expressed genes of cell signal transduction associated with benzene poisoning by cDNA microarray].

    PubMed

    Wang, Hong; Bi, Yongyi; Tao, Ning; Wang, Chunhong

    2005-08-01

    To detect the differential expression of cell signal transduction genes associated with benzene poisoning, and to explore the pathogenic mechanisms of blood system damage induced by benzene. Peripheral white blood cell gene expression profile of 7 benzene poisoning patients, including one aplastic anemia, was determined by cDNA microarray. Seven chips from normal workers were served as controls. Cluster analysis of gene expression profile was performed. Among the 4265 target genes, 176 genes associated with cell signal transduction were differentially expressed. 35 up-regulated genes including PTPRC, STAT4, IFITM1 etc were found in at least 6 pieces of microarray; 45 down-regulated genes including ARHB, PPP3CB, CDC37 etc were found in at least 5 pieces of microarray. cDNA microarray technology is an effective technique for screening the differentially expressed genes of cell signal transduction. Disorder in cell signal transduction may play certain role in the pathogenic mechanism of benzene poisoning.

  14. Single cell gene expression profiling in Alzheimer's disease.

    PubMed

    Ginsberg, Stephen D; Che, Shaoli; Counts, Scott E; Mufson, Elliott J

    2006-07-01

    Development and implementation of microarray techniques to quantify expression levels of dozens to hundreds to thousands of transcripts simultaneously within select tissue samples from normal control subjects and neurodegenerative diseased brains has enabled scientists to create molecular fingerprints of vulnerable neuronal populations in Alzheimer's disease (AD) and related disorders. A goal is to sample gene expression from homogeneous cell types within a defined region without potential contamination by expression profiles of adjacent neuronal subpopulations and nonneuronal cells. The precise resolution afforded by single cell and population cell RNA analysis in combination with microarrays and real-time quantitative polymerase chain reaction (qPCR)-based analyses allows for relative gene expression level comparisons across cell types under different experimental conditions and disease progression. The ability to analyze single cells is an important distinction from global and regional assessments of mRNA expression and can be applied to optimally prepared tissues from animal models of neurodegeneration as well as postmortem human brain tissues. Gene expression analysis in postmortem AD brain regions including the hippocampal formation and neocortex reveals selectively vulnerable cell types share putative pathogenetic alterations in common classes of transcripts, for example, markers of glutamatergic neurotransmission, synaptic-related markers, protein phosphatases and kinases, and neurotrophins/neurotrophin receptors. Expression profiles of vulnerable regions and neurons may reveal important clues toward the understanding of the molecular pathogenesis of various neurological diseases and aid in identifying rational targets toward pharmacotherapeutic interventions for progressive, late-onset neurodegenerative disorders such as mild cognitive impairment (MCI) and AD.

  15. Gene Expression Changes in the Motor Cortex Mediating Motor Skill Learning

    PubMed Central

    Cheung, Vincent C. K.; DeBoer, Caroline; Hanson, Elizabeth; Tunesi, Marta; D'Onofrio, Mara; Arisi, Ivan; Brandi, Rossella; Cattaneo, Antonino; Goosens, Ki A.

    2013-01-01

    The primary motor cortex (M1) supports motor skill learning, yet little is known about the genes that contribute to motor cortical plasticity. Such knowledge could identify candidate molecules whose targeting might enable a new understanding of motor cortical functions, and provide new drug targets for the treatment of diseases which impair motor function, such as ischemic stroke. Here, we assess changes in the motor-cortical transcriptome across different stages of motor skill acquisition. Adult rats were trained on a gradually acquired appetitive reach and grasp task that required different strategies for successful pellet retrieval, or a sham version of the task in which the rats received pellet reward without needing to develop the reach and grasp skill. Tissue was harvested from the forelimb motor-cortical area either before training commenced, prior to the initial rise in task performance, or at peak performance. Differential classes of gene expression were observed at the time point immediately preceding motor task improvement. Functional clustering revealed that gene expression changes were related to the synapse, development, intracellular signaling, and the fibroblast growth factor (FGF) family, with many modulated genes known to regulate synaptic plasticity, synaptogenesis, and cytoskeletal dynamics. The modulated expression of synaptic genes likely reflects ongoing network reorganization from commencement of training till the point of task improvement, suggesting that motor performance improves only after sufficient modifications in the cortical circuitry have accumulated. The regulated FGF-related genes may together contribute to M1 remodeling through their roles in synaptic growth and maturation. PMID:23637843

  16. ck2-dependent phosphorylation of progesterone receptors (PR) on Ser81 regulates PR-B isoform-specific target gene expression in breast cancer cells.

    PubMed

    Hagan, Christy R; Regan, Tarah M; Dressing, Gwen E; Lange, Carol A

    2011-06-01

    Progesterone receptors (PR) are critical mediators of mammary gland development and contribute to breast cancer progression. Progestin-induced rapid activation of cytoplasmic protein kinases leads to selective regulation of growth-promoting genes by phospho-PR species. Herein, we show that phosphorylation of PR Ser81 is ck2 dependent and progestin regulated in intact cells but also occurs in the absence of PR ligands when cells enter the G(1)/S phase of the cell cycle. T47D breast cancer cells stably expressing a PR-B mutant receptor that cannot be phosphorylated at Ser79/81 (S79/81A) formed fewer soft agar colonies. Regulation of selected genes by PR-B, but not PR-A, also required Ser79/81 phosphorylation for basal and/or progestin-regulated (BIRC3, HSD11β2, and HbEGF) expression. Additionally, wild-type (wt) PR-B, but not S79/81A mutant PR, was robustly recruited to a progesterone response element (PRE)-containing transcriptional enhancer region of BIRC3; abundant ck2 also associated with this region in cells expressing wt but not S79/81A PR. We conclude that phospho-Ser81 PR provides a platform for ck2 recruitment and regulation of selected PR-B target genes. Understanding how ligand-independent PRs function in the context of high levels of kinase activities characteristic of breast cancer is critical to understanding the basis of tumor-specific changes in gene expression and will speed the development of highly selective treatments.

  17. The effect of boron deficiency on gene expression and boron compartmentalization in sugarbeet

    USDA-ARS?s Scientific Manuscript database

    NIP5, BOR1, NIP6, and WRKY6 genes were investigated for their role in boron deficiency in sugar beet, each with a proposed role in boron use in model plant species. All genes showed evidence of polymorphism in fragment size and gene expression in the target genomic DNA and cDNA libraries, with no co...

  18. Intrinsic gene expression subsets of diffuse cutaneous systemic sclerosis are stable in serial skin biopsies

    PubMed Central

    Pendergrass, Sarah A.; Lemaire, Raphael; Francis, Ian; Mahoney, J. Matthew; Lafyatis, Robert; Whitfield, Michael L.

    2012-01-01

    Skin biopsy gene expression was analyzed by DNA microarray from 13 dSSc patients enrolled in an open label study of rituximab, 9 dSSc patients not treated with rituximab, and 9 healthy controls. These data recapitulate the patient ‘intrinsic’ gene expression subsets described previously including proliferation, inflammatory, and normal-like groups. Serial skin biopsies showed consistent and non-progressing gene expression over time, and importantly, the patients in the inflammatory subset do not move to the fibroproliferative subset, and vice versa. We were unable to detect significant differences in gene expression before and after rituximab treatment, consistent with an apparent lack of clinical response. Serial biopsies from each patient stayed within the same gene expression subset regardless of treatment regimen or the time point at which they were taken. Collectively, these data emphasize the heterogeneous nature of SSc and demonstrate that the intrinsic subsets are an inherent, reproducible and stable feature of the disease that is independent of disease duration. Moreover, these data have fundamental importance for the future development of personalized therapy for SSc; drugs targeting inflammation are likely to benefit those patients with an inflammatory signature, whereas drugs targeting fibrosis are likely to benefit those with a fibroproliferative signature. PMID:22318389

  19. Identification and validation of superior reference gene for gene expression normalization via RT-qPCR in staminate and pistillate flowers of Jatropha curcas - A biodiesel plant.

    PubMed

    Karuppaiya, Palaniyandi; Yan, Xiao-Xue; Liao, Wang; Wu, Jun; Chen, Fang; Tang, Lin

    2017-01-01

    Physic nut (Jatropha curcas L) seed oil is a natural resource for the alternative production of fossil fuel. Seed oil production is mainly depended on seed yield, which was restricted by the low ratio of staminate flowers to pistillate flowers. Further, the mechanism of physic nut flower sex differentiation has not been fully understood yet. Quantitative Real Time-Polymerase Chain Reaction is a reliable and widely used technique to quantify the gene expression pattern in biological samples. However, for accuracy of qRT-PCR, appropriate reference gene is highly desirable to quantify the target gene level. Hence, the present study was aimed to identify the stable reference genes in staminate and pistillate flowers of J. curcas. In this study, 10 candidate reference genes were selected and evaluated for their expression stability in staminate and pistillate flowers, and their stability was validated by five different algorithms (ΔCt, BestKeeper, NormFinder, GeNorm and RefFinder). Resulting, TUB and EF found to be the two most stably expressed reference for staminate flower; while GAPDH1 and EF found to be the most stably expressed reference gene for pistillate flowers. Finally, RT-qPCR assays of target gene AGAMOUS using the identified most stable reference genes confirmed the reliability of selected reference genes in different stages of flower development. AGAMOUS gene expression levels at different stages were further proved by gene copy number analysis. Therefore, the present study provides guidance for selecting appropriate reference genes for analyzing the expression pattern of floral developmental genes in staminate and pistillate flowers of J. curcas.

  20. The human RHOX gene cluster: target genes and functional analysis of gene variants in infertile men.

    PubMed

    Borgmann, Jennifer; Tüttelmann, Frank; Dworniczak, Bernd; Röpke, Albrecht; Song, Hye-Won; Kliesch, Sabine; Wilkinson, Miles F; Laurentino, Sandra; Gromoll, Jörg

    2016-11-15

    The X-linked reproductive homeobox (RHOX) gene cluster encodes transcription factors preferentially expressed in reproductive tissues. This gene cluster has important roles in male fertility based on phenotypic defects of Rhox-mutant mice and the finding that aberrant RHOX promoter methylation is strongly associated with abnormal human sperm parameters. However, little is known about the molecular mechanism of RHOX function in humans. Using gene expression profiling, we identified genes regulated by members of the human RHOX gene cluster. Some genes were uniquely regulated by RHOXF1 or RHOXF2/2B, while others were regulated by both of these transcription factors. Several of these regulated genes encode proteins involved in processes relevant to spermatogenesis; e.g. stress protection and cell survival. One of the target genes of RHOXF2/2B is RHOXF1, suggesting cross-regulation to enhance transcriptional responses. The potential role of RHOX in human infertility was addressed by sequencing all RHOX exons in a group of 250 patients with severe oligozoospermia. This revealed two mutations in RHOXF1 (c.515G > A and c.522C > T) and four in RHOXF2/2B (-73C > G, c.202G > A, c.411C > T and c.679G > A), of which only one (c.202G > A) was found in a control group of men with normal sperm concentration. Functional analysis demonstrated that c.202G > A and c.679G > A significantly impaired the ability of RHOXF2/2B to regulate downstream genes. Molecular modelling suggested that these mutations alter RHOXF2/F2B protein conformation. By combining clinical data with in vitro functional analysis, we demonstrate how the X-linked RHOX gene cluster may function in normal human spermatogenesis and we provide evidence that it is impaired in human male fertility.

  1. Gene silencing in Escherichia coli using antisense RNAs expressed from doxycycline-inducible vectors.

    PubMed

    Nakashima, N; Tamura, T

    2013-06-01

    Here, we report on the construction of doxycycline (tetracycline analogue)-inducible vectors that express antisense RNAs in Escherichia coli. Using these vectors, the expression of genes of interest can be silenced conditionally. The expression of antisense RNAs from the vectors was more tightly regulated than the previously constructed isopropyl-β-D-galactopyranoside-inducible vectors. Furthermore, expression levels of antisense RNAs were enhanced by combining the doxycycline-inducible promoter with the T7 promoter-T7 RNA polymerase system; the T7 RNA polymerase gene, under control of the doxycycline-inducible promoter, was integrated into the lacZ locus of the genome without leaving any antibiotic marker. These vectors are useful for investigating gene functions or altering cell phenotypes for biotechnological and industrial applications. A gene silencing method using antisense RNAs in Escherichia coli is described, which facilitates the investigation of bacterial gene function. In particular, the method is suitable for comprehensive analyses or phenotypic analyses of genes essential for growth. Here, we describe expansion of vector variations for expressing antisense RNAs, allowing choice of a vector appropriate for the target genes or experimental purpose. © 2013 The Society for Applied Microbiology.

  2. Comprehensive evaluation of candidate reference genes for gene expression studies in Lysiphlebia japonica (Hymenoptera: Aphidiidae) using RT-qPCR.

    PubMed

    Gao, Xue-Ke; Zhang, Shuai; Luo, Jun-Yu; Wang, Chun-Yi; Lü, Li-Min; Zhang, Li-Juan; Zhu, Xiang-Zhen; Wang, Li; Lu, Hui; Cui, Jin-Jie

    2017-12-30

    Lysiphlebia japonica (Ashmead) is a predominant parasitoid of cotton-melon aphids in the fields of northern China with a proven ability to effectively control cotton aphid populations in early summer. For accurate normalization of gene expression in L. japonica using quantitative reverse transcriptase-polymerase chain reaction (RT-qPCR), reference genes with stable gene expression patterns are essential. However, no appropriate reference genes is L. japonica have been investigated to date. In the present study, 12 selected housekeeping genes from L. japonica were cloned. We evaluated the stability of these genes under various experimental treatments by RT-qPCR using four independent (geNorm, NormFinder, BestKeeper and Delta Ct) and one comparative (RefFinder) algorithm. We identified genes showing the most stable levels of expression: DIMT, 18S rRNA, and RPL13 during different stages; AK, RPL13, and TBP among sexes; EF1A, PPI, and RPL27 in different tissues, and EF1A, RPL13, and PPI in adults fed on different diets. Moreover, the expression profile of a target gene (odorant receptor 1, OR1) studied during the developmental stages confirms the reliability of the chosen selected reference genes. This study provides for the first time a comprehensive list of suitable reference genes for gene expression studies in L. japonica and will benefit subsequent genomics and functional genomics research on this natural enemy. Copyright © 2017. Published by Elsevier B.V.

  3. Effects of insulin and exercise training on FGF21, its receptors and target genes in obesity and type 2 diabetes.

    PubMed

    Kruse, Rikke; Vienberg, Sara G; Vind, Birgitte F; Andersen, Birgitte; Højlund, Kurt

    2017-10-01

    Pharmacological doses of FGF21 improve glucose tolerance, lipid metabolism and energy expenditure in rodents. Induced expression and secretion of FGF21 from muscle may increase browning of white adipose tissue (WAT) in a myokine-like manner. Recent studies have reported that insulin and exercise increase FGF21 in plasma. Obesity and type 2 diabetes are potentially FGF21-resistant states, but to what extent FGF21 responses to insulin and exercise training are preserved, and whether FGF21, its receptors and target genes are altered, remains to be established. The effects of insulin during euglycaemic-hyperinsulinaemic clamps and 10 week endurance training on serum FGF21 were examined in individuals with type 2 diabetes and in glucose tolerant overweight/obese and lean individuals. Gene expression of FGF21, its receptors and target genes in muscle and WAT biopsies was evaluated by quantitative real-time PCR (qPCR). Insulin increased serum and muscle FGF21 independent of overweight/obesity or type 2 diabetes, and there were no effects associated with exercise training. The insulin-induced increases in serum FGF21 and muscle FGF21 expression correlated tightly (p < 0.001). In WAT, overweight/obesity with and without type 2 diabetes led to reduced expression of KLB, but increased FGFR1c expression. However, the expression of most FGF21 target genes was unaltered except for reduced CIDEA expression in individuals with type 2 diabetes. Insulin-induced expression of muscle FGF21 correlates strongly with a rise in serum FGF21, and this response appears intact in overweight/obesity and type 2 diabetes. FGF21 resistance may involve reduced KLB expression in WAT. However, increased FGFR1c expression or other mechanisms seem to ensure adequate expression of most FGF21 target genes in WAT.

  4. Gene Expression Profiling of Gastric Cancer

    PubMed Central

    Marimuthu, Arivusudar; Jacob, Harrys K.C.; Jakharia, Aniruddha; Subbannayya, Yashwanth; Keerthikumar, Shivakumar; Kashyap, Manoj Kumar; Goel, Renu; Balakrishnan, Lavanya; Dwivedi, Sutopa; Pathare, Swapnali; Dikshit, Jyoti Bajpai; Maharudraiah, Jagadeesha; Singh, Sujay; Sameer Kumar, Ghantasala S; Vijayakumar, M.; Veerendra Kumar, Kariyanakatte Veeraiah; Premalatha, Chennagiri Shrinivasamurthy; Tata, Pramila; Hariharan, Ramesh; Roa, Juan Carlos; Prasad, T.S.K; Chaerkady, Raghothama; Kumar, Rekha Vijay; Pandey, Akhilesh

    2015-01-01

    Gastric cancer is the second leading cause of cancer death worldwide, both in men and women. A genomewide gene expression analysis was carried out to identify differentially expressed genes in gastric adenocarcinoma tissues as compared to adjacent normal tissues. We used Agilent’s whole human genome oligonucleotide microarray platform representing ~41,000 genes to carry out gene expression analysis. Two-color microarray analysis was employed to directly compare the expression of genes between tumor and normal tissues. Through this approach, we identified several previously known candidate genes along with a number of novel candidate genes in gastric cancer. Testican-1 (SPOCK1) was one of the novel molecules that was 10-fold upregulated in tumors. Using tissue microarrays, we validated the expression of testican-1 by immunohistochemical staining. It was overexpressed in 56% (160/282) of the cases tested. Pathway analysis led to the identification of several networks in which SPOCK1 was among the topmost networks of interacting genes. By gene enrichment analysis, we identified several genes involved in cell adhesion and cell proliferation to be significantly upregulated while those corresponding to metabolic pathways were significantly downregulated. The differentially expressed genes identified in this study are candidate biomarkers for gastric adenoacarcinoma. PMID:27030788

  5. Unmasking Upstream Gene Expression Regulators with miRNA-corrected mRNA Data

    PubMed Central

    Bollmann, Stephanie; Bu, Dengpan; Wang, Jiaqi; Bionaz, Massimo

    2015-01-01

    Expressed micro-RNA (miRNA) affects messenger RNA (mRNA) abundance, hindering the accuracy of upstream regulator analysis. Our objective was to provide an algorithm to correct such bias. Large mRNA and miRNA analyses were performed on RNA extracted from bovine liver and mammary tissue. Using four levels of target scores from TargetScan (all miRNA:mRNA target gene pairs or only the top 25%, 50%, or 75%). Using four levels of target scores from TargetScan (all miRNA:mRNA target gene pairs or only the top 25%, 50%, or 75%) and four levels of the magnitude of miRNA effect (ME) on mRNA expression (30%, 50%, 75%, and 83% mRNA reduction), we generated 17 different datasets (including the original dataset). For each dataset, we performed upstream regulator analysis using two bioinformatics tools. We detected an increased effect on the upstream regulator analysis with larger miRNA:mRNA pair bins and higher ME. The miRNA correction allowed identification of several upstream regulators not present in the analysis of the original dataset. Thus, the proposed algorithm improved the prediction of upstream regulators. PMID:27279737

  6. Gene expression complex networks: synthesis, identification, and analysis.

    PubMed

    Lopes, Fabrício M; Cesar, Roberto M; Costa, Luciano Da F

    2011-10-01

    Thanks to recent advances in molecular biology, allied to an ever increasing amount of experimental data, the functional state of thousands of genes can now be extracted simultaneously by using methods such as cDNA microarrays and RNA-Seq. Particularly important related investigations are the modeling and identification of gene regulatory networks from expression data sets. Such a knowledge is fundamental for many applications, such as disease treatment, therapeutic intervention strategies and drugs design, as well as for planning high-throughput new experiments. Methods have been developed for gene networks modeling and identification from expression profiles. However, an important open problem regards how to validate such approaches and its results. This work presents an objective approach for validation of gene network modeling and identification which comprises the following three main aspects: (1) Artificial Gene Networks (AGNs) model generation through theoretical models of complex networks, which is used to simulate temporal expression data; (2) a computational method for gene network identification from the simulated data, which is founded on a feature selection approach where a target gene is fixed and the expression profile is observed for all other genes in order to identify a relevant subset of predictors; and (3) validation of the identified AGN-based network through comparison with the original network. The proposed framework allows several types of AGNs to be generated and used in order to simulate temporal expression data. The results of the network identification method can then be compared to the original network in order to estimate its properties and accuracy. Some of the most important theoretical models of complex networks have been assessed: the uniformly-random Erdös-Rényi (ER), the small-world Watts-Strogatz (WS), the scale-free Barabási-Albert (BA), and geographical networks (GG). The experimental results indicate that the inference

  7. Enhanced Whitefly Resistance in Transgenic Tobacco Plants Expressing Double Stranded RNA of v-ATPase A Gene

    PubMed Central

    Thakur, Nidhi; Upadhyay, Santosh Kumar; Verma, Praveen C.; Chandrashekar, Krishnappa; Tuli, Rakesh; Singh, Pradhyumna K.

    2014-01-01

    Background Expression of double strand RNA (dsRNA) designed against important insect genes in transgenic plants have been shown to give protection against pests through RNA interference (RNAi), thus opening the way for a new generation of insect-resistant crops. We have earlier compared the efficacy of dsRNAs/siRNAs, against a number of target genes, for interference in growth of whitefly (Bemisia tabaci) upon oral feeding. The v-ATPase subunit A (v-ATPaseA) coding gene was identified as a crucial target. We now report the effectiveness of transgenic tobacco plants expressing siRNA to silence v-ATPaseA gene expression for the control of whitefly infestation. Methodology/Principal Findings Transgenic tobacco lines were developed for the expression of long dsRNA precursor to make siRNA and knock down the v-ATPaseA mRNA in whitefly. Molecular analysis and insecticidal properties of the transgenic plants established the formation of siRNA targeting the whitefly v-ATPaseA, in the leaves. The transcript level of v-ATPaseA in whiteflies was reduced up to 62% after feeding on the transgenic plants. Heavy infestation of whiteflies on the control plants caused significant loss of sugar content which led to the drooping of leaves. The transgenic plants did not show drooping effect. Conclusions/Significance Host plant derived pest resistance was achieved against whiteflies by genetic transformation of tobacco which generated siRNA against the whitefly v-ATPaseA gene. Transgenic tobacco lines expressing dsRNA of v-ATPaseA, delivered sufficient siRNA to whiteflies feeding on them, mounting a significant silencing response, leading to their mortality. The transcript level of the target gene was reduced in whiteflies feeding on transgenic plants. The strategy can be taken up for genetic engineering of plants to control whiteflies in field crops. PMID:24595215

  8. Tumor targeting RGD conjugated bio-reducible polymer for VEGF siRNA expressing plasmid delivery

    PubMed Central

    Kim, Hyun Ah; Nam, Kihoon; Kim, Sung Wan

    2014-01-01

    Targeted delivery of therapeutic genes to the tumor site is critical for successful and safe cancer gene therapy. The arginine grafted bio-reducible poly (cystamine bisacrylamide-diaminohexane, CBA-DAH) polymer (ABP) conjugated poly (amido amine) (PAMAM), PAM-ABP (PA) was designed previously as an efficient gene delivery carrier. To achieve high efficacy in cancer selective delivery, we developed the tumor targeting bio-reducible polymer, PA-PEG1k-RGD, by conjugating cyclic RGDfC (RGD) peptides, which bind αvβ3/5 integrins, to the PAM-ABP using polyethylene glycol (PEG,1kDa) as a spacer. Physical characterization showed nanocomplex formation with bio-reducible properties between PA-PEG1k-RGD and plasmid DNA (pDNA). In transfection assays, PA-PEG1k-RGD showed significantly higher transfection efficiency in comparison with PAM-ABP or PA-PEG1k-RGD in αvβ3/5 positive MCF7 breast cancer and PANC-1 pancreatic cancer cells. The targeting ability of PA-PEG1k-RGD was further established using a competition assay. To confirm the therapeutic effect, the VEGF siRNA expressing plasmid was constructed and then delivered into cancer cells using PA-PEG1k-RGD. PA-PEG1k-RGD showed 20-59% higher cellular uptake rate into MCF7 and PANC-1 than that of non-targeted polymers. In addition, MCF7 and PANC-1 cancer cells transfected with PA-PEG1k-RGD/pshVEGF complexes had significantly decreased VEGF gene expression (51-71%) and cancer cell viability (35-43%) compared with control. These results demonstrate that a tumor targeting bio-reducible polymer with an anti-angiogenic therapeutic gene could be used for efficient and safe cancer gene therapy. PMID:24894645

  9. [Detection of stable expression of human interlukin-2 gene in transfected keratinocytes].

    PubMed

    Liao, W; Liu, Y; Ye, L

    1999-09-01

    To investigate the stable expression and secretion of human interlukin-2 gene in transfected keratinocytes. Keratinocytes were transfected with lipofectamine and selected by G418. Then the samples were analyzed with the techniques of DNA dot blot, RNA dot blot, hybridization in situ, immunohistochemistry, Western blot and MTT. The positive signals were observed in transfected keratinocytes by DNA dot blot, RNA dot blot, hybridization in situ and immunohistochemistry. With Western blot analysis, a specific band exhibiting a molecular weight of 15,000 was detected in transfected keratinocytes, which was in acordance with that of IL-2. The expression of IL-2 can maintain for up to 1 month. The amounts of IL-2 in the supernatants of two and four passages transfected keratinocytes were 27.7 U/ml and 15.0 U/ml, respectively. Keratinocytes have the potential for stable gene expression and secretion of active transgene products. Thus, it is possible to use keratinocytes as a target cell for gene transfection, gene expression and even gene therapy.

  10. Detection of growth hormone doping by gene expression profiling of peripheral blood.

    PubMed

    Mitchell, Christopher J; Nelson, Anne E; Cowley, Mark J; Kaplan, Warren; Stone, Glenn; Sutton, Selina K; Lau, Amie; Lee, Carol M Y; Ho, Ken K Y

    2009-12-01

    GH abuse is a significant problem in many sports, and there is currently no robust test that allows detection of doping beyond a short window after administration. Our objective was to evaluate gene expression profiling in peripheral blood leukocytes in-vivo as a test for GH doping in humans. Seven men and thirteen women were administered GH, 2 mg/d sc for 8 wk. Blood was collected at baseline and at 8 wk. RNA was extracted from the white cell fraction. Microarray analysis was undertaken using Agilent 44K G4112F arrays using a two-color design. Quantitative RT-PCR using TaqMan gene expression assays was performed for validation of selected differentially expressed genes. GH induced an approximately 2-fold increase in circulating IGF-I that was maintained throughout the 8 wk of the study. GH induced significant changes in gene expression with 353 in women and 41 in men detected with a false discovery rate of less than 5%. None of the differentially expressed genes were common between men and women. The maximal changes were a doubling for up-regulated or halving for down-regulated genes, similar in magnitude to the variation between individuals. Quantitative RT-PCR for seven target genes showed good concordance between microarray and quantitative PCR data in women but not in men. Gene expression analysis of peripheral blood leukocytes is unlikely to be a viable approach for the detection of GH doping.

  11. PDPR Gene Expression Correlates with Exercise-Training Insulin Sensitivity Changes

    PubMed Central

    Barberio, Matthew D.; Huffman, Kim M.; Giri, Mamta; Hoffman, Eric P.; Kraus, William E.; Hubal, Monica J.

    2016-01-01

    Purpose Whole body insulin sensitivity (Si) typically improves following aerobic exercise training; however, individual responses can be highly variable. The purpose of this study was to use global gene expression to identify skeletal muscle genes that correlate with exercise-induced Si changes. Methods Longitudinal cohorts from the Studies of Targeted Risk Reduction Intervention through Defined Exercise (STRRIDE) were utilized as Discovery (Affymetrix) and Confirmation (Illumina) of vastus lateralis gene expression profiles. Discovery (n=39; 21 men) and Confirmation (n=42; 19 men) cohorts were matched for age (52 ± 8 vs. 51 ± 10 yr), BMI (30.4 ± 2.8 vs. 29.7 ± 2.8 kg*m-2), and VO2max (30.4 ± 2.8 vs. 29.7 ± 2.8 mL/kg/min). Si was determined via intravenous glucose tolerance test pre- and post-training. Pearson product-moment correlation coefficients determined relationships between a) baseline and b) training-induced changes in gene expression and %ΔSi after training. Results Expression of 2454 (Discovery) and 1778 genes (Confirmation) at baseline were significantly (P<0.05) correlated to %ΔSi; 112 genes overlapped. Pathway analyses identified Ca2+-signaling-related transcripts in this 112-gene list. Expression changes of 1384 (Discovery) and 1288 genes (Confirmation) following training were significantly (P<0.05) correlated to % ΔSi; 33 genes overlapped, representing contractile apparatus of skeletal and smooth muscle genes. Pyruvate dehydrogenase phosphatase regulatory subunit (PDPR) expression at baseline (p=0.01, r=0.41) and post-training (p=0.01, r=0.43) were both correlated with %ΔSi. Conclusion Exercise-induced adaptations in skeletal muscle Si are related to baseline levels of Ca+2-regulating transcripts, which may prime the muscle for adaptation. Relationships between %ΔSi and PDPR, a regulatory subunit of the pyruvate dehydrogenase complex, indicate that the Si response is strongly related to key steps in metabolic regulation. PMID:27846149

  12. Visual gene-network analysis reveals the cancer gene co-expression in human endometrial cancer

    PubMed Central

    2014-01-01

    Background Endometrial cancers (ECs) are the most common form of gynecologic malignancy. Recent studies have reported that ECs reveal distinct markers for molecular pathogenesis, which in turn is linked to the various histological types of ECs. To understand further the molecular events contributing to ECs and endometrial tumorigenesis in general, a more precise identification of cancer-associated molecules and signaling networks would be useful for the detection and monitoring of malignancy, improving clinical cancer therapy, and personalization of treatments. Results ECs-specific gene co-expression networks were constructed by differential expression analysis and weighted gene co-expression network analysis (WGCNA). Important pathways and putative cancer hub genes contribution to tumorigenesis of ECs were identified. An elastic-net regularized classification model was built using the cancer hub gene signatures to predict the phenotypic characteristics of ECs. The 19 cancer hub gene signatures had high predictive power to distinguish among three key principal features of ECs: grade, type, and stage. Intriguingly, these hub gene networks seem to contribute to ECs progression and malignancy via cell-cycle regulation, antigen processing and the citric acid (TCA) cycle. Conclusions The results of this study provide a powerful biomarker discovery platform to better understand the progression of ECs and to uncover potential therapeutic targets in the treatment of ECs. This information might lead to improved monitoring of ECs and resulting improvement of treatment of ECs, the 4th most common of cancer in women. PMID:24758163

  13. T-cell lymphomas associated gene expression signature: Bioinformatics analysis based on gene expression Omnibus.

    PubMed

    Zhou, Lei-Lei; Xu, Xiao-Yue; Ni, Jie; Zhao, Xia; Zhou, Jian-Wei; Feng, Ji-Feng

    2018-06-01

    Due to the low incidence and the heterogeneity of subtypes, the biological process of T-cell lymphomas is largely unknown. Although many genes have been detected in T-cell lymphomas, the role of these genes in biological process of T-cell lymphomas was not further analyzed. Two qualified datasets were downloaded from Gene Expression Omnibus database. The biological functions of differentially expressed genes were evaluated by gene ontology enrichment and KEGG pathway analysis. The network for intersection genes was constructed by the cytoscape v3.0 software. Kaplan-Meier survival curves and log-rank test were employed to assess the association between differentially expressed genes and clinical characters. The intersection mRNAs were proved to be associated with fundamental processes of T-cell lymphoma cells. These intersection mRNAs were involved in the activation of some cancer-related pathways, including PI3K/AKT, Ras, JAK-STAT, and NF-kappa B signaling pathway. PDGFRA, CXCL12, and CCL19 were the most significant central genes in the signal-net analysis. The results of survival analysis are not entirely credible. Our findings uncovered aberrantly expressed genes and a complex RNA signal network in T-cell lymphomas and indicated cancer-related pathways involved in disease initiation and progression, providing a new insight for biotargeted therapy in T-cell lymphomas. © 2018 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  14. Using thermal energy produced by irradiation of Mn-Zn ferrite magnetic nanoparticles (MZF-NPs) for heat-inducible gene expression.

    PubMed

    Tang, Qiu-sha; Zhang, Dong-sheng; Cong, Xiao-ming; Wan, Mei-ling; Jin, Li-qiang

    2008-06-01

    One of the main advantages of gene therapy over traditional therapy is the potential to target the expression of therapeutic genes in desired cells or tissues. To achieve targeted gene expression, we developed a novel heat-inducible gene expression system in which thermal energy generated by Mn-Zn ferrite magnetic nanoparticles (MZF-NPs) under an alternating magnetic field (AMF) was used to activate gene expression. MZF-NPs, obtained by co-precipitation method, were firstly surface modified with cation poly(ethylenimine) (PEI). Then thermodynamic test of various doses of MZF-NPs was preformed in vivo and in vitro. PEI-MZF-NPs showed good DNA binding ability and high transfection efficiency. In AMF, they could rise to a steady temperature. To analyze the heat-induced gene expression under an AMF, we combined P1730OR vector transfection with hyperthermia produced by irradiation of MZF-NPs. By using LacZ gene as a reporter gene and Hsp70 as a promoter, it was demonstrated that expression of a heterogeneous gene could be elevated to 10 to 500-fold over background by moderate hyperthermia (added 12.24 or 25.81 mg MZF-NPs to growth medium) in tissue cultured cells. When injected with 2.6 or 4.6 mg MZF-NPs, the temperature of tumor-bearing nude mice could rise to 39.5 or 42.8 degrees C, respectively, and the beta-gal concentration could increase up to 3.8 or 8.1 mU/mg proteins accordingly 1 day after hyperthermia treatment. Our results therefore supported hyperthermia produced by irradiation of MZF-NPs under an AMF as a feasible approach for targeted heat-induced gene expression. This novel system made use of the relative low Curie point of MZF-NPs to control the in vivo hyperthermia temperature and therefore acquired safe and effective heat-inducible transgene expression.

  15. Fatty acid composition and desaturase gene expression in flax (Linum usitatissimum L.).

    PubMed

    Thambugala, Dinushika; Cloutier, Sylvie

    2014-11-01

    Little is known about the relationship between expression levels of fatty acid desaturase genes during seed development and fatty acid (FA) composition in flax. In the present study, we looked at promoter structural variations of six FA desaturase genes and their relative expression throughout seed development. Computational analysis of the nucleotide sequences of the sad1, sad2, fad2a, fad2b, fad3a and fad3b promoters showed several basic transcriptional elements including CAAT and TATA boxes, and several putative target-binding sites for transcription factors, which have been reported to be involved in the regulation of lipid metabolism. Using semi-quantitative reverse transcriptase PCR, the expression patterns throughout seed development of the six FA desaturase genes were measured in six flax genotypes that differed for FA composition but that carried the same desaturase isoforms. FA composition data were determined by phenotyping the field grown genotypes over four years in two environments. All six genes displayed a bell-shaped pattern of expression peaking at 20 or 24 days after anthesis. Sad2 was the most highly expressed. The expression of all six desaturase genes did not differ significantly between genotypes (P = 0.1400), hence there were no correlations between FA desaturase gene expression and variations in FA composition in relatively low, intermediate and high linolenic acid genotypes expressing identical isoforms for all six desaturases. These results provide further clues towards understanding the genetic factors responsible for FA composition in flax.

  16. BDNF gene delivery mediated by neuron-targeted nanoparticles is neuroprotective in peripheral nerve injury.

    PubMed

    Lopes, Cátia D F; Gonçalves, Nádia P; Gomes, Carla P; Saraiva, Maria J; Pêgo, Ana P

    2017-03-01

    Neuron-targeted gene delivery is a promising strategy to treat peripheral neuropathies. Here we propose the use of polymeric nanoparticles based on thiolated trimethyl chitosan (TMCSH) to mediate targeted gene delivery to peripheral neurons upon a peripheral and minimally invasive intramuscular administration. Nanoparticles were grafted with the non-toxic carboxylic fragment of the tetanus neurotoxin (HC) to allow neuron targeting and were explored to deliver a plasmid DNA encoding for the brain-derived neurotrophic factor (BDNF) in a peripheral nerve injury model. The TMCSH-HC/BDNF nanoparticle treatment promoted the release and significant expression of BDNF in neural tissues, which resulted in an enhanced functional recovery after injury as compared to control treatments (vehicle and non-targeted nanoparticles), associated with an improvement in key pro-regenerative events, namely, the increased expression of neurofilament and growth-associated protein GAP-43 in the injured nerves. Moreover, the targeted nanoparticle treatment was correlated with a significantly higher density of myelinated axons in the distal stump of injured nerves, as well as with preservation of unmyelinated axon density as compared with controls and a protective role in injury-denervated muscles, preventing them from denervation. These results highlight the potential of TMCSH-HC nanoparticles as non-viral gene carriers to deliver therapeutic genes into the peripheral neurons and thus, pave the way for their use as an effective therapeutic intervention for peripheral neuropathies. Copyright © 2016 Elsevier Ltd. All rights reserved.

  17. Identification of differentially expressed genes in Monochamus alternatus digested with azadirachtin.

    PubMed

    Lin, Tong; Liu, Qisi; Chen, Jingxiang

    2016-09-15

    The pine sawyer beetle Monochamus alternatus Hope, a major forest insect pest, is the primary vector of the destructive forest pest pine wood nematode, Bursaphelenchus xylophilus. Azadirachtin, an active compound of neem, is biologically interesting because it represents a group of important, successful botanical pesticides. We provide insight into the molecular effects of azadirachtin on M. alternatus at the transcriptional level to provide clues about possible molecular-level targets and to establish a link between azadirachtin and insect global responses. We found that 920 and 9894 unique genes were significantly up- and down-regulated, respectively. We obtained expression patterns of the differentially expressed genes (DEGs), identifying 4247, 3488 and 7613 sequences that involved cellular components, molecular functions and biological processes, respectively, and showed that the DEGs were distributed among 50 Gene Ontology categories. The Encyclopedia of Genes and Genomes pathway enrichment analysis indicated that the DEGs were enriched in 50 pathways. Detailed gene profile knowledge of the interaction of azadirachtin with M. alternatus should facilitate the development of more effective azadirachtin-based products against M. alternatus and other target Coleoptera. These results further enhance the value of azadirachtin as a potential insecticide of biological origin, as well as for other biological applications.

  18. Identification of differentially expressed genes in Monochamus alternatus digested with azadirachtin

    PubMed Central

    Lin, Tong; Liu, Qisi; Chen, Jingxiang

    2016-01-01

    The pine sawyer beetle Monochamus alternatus Hope, a major forest insect pest, is the primary vector of the destructive forest pest pine wood nematode, Bursaphelenchus xylophilus. Azadirachtin, an active compound of neem, is biologically interesting because it represents a group of important, successful botanical pesticides. We provide insight into the molecular effects of azadirachtin on M. alternatus at the transcriptional level to provide clues about possible molecular-level targets and to establish a link between azadirachtin and insect global responses. We found that 920 and 9894 unique genes were significantly up- and down-regulated, respectively. We obtained expression patterns of the differentially expressed genes (DEGs), identifying 4247, 3488 and 7613 sequences that involved cellular components, molecular functions and biological processes, respectively, and showed that the DEGs were distributed among 50 Gene Ontology categories. The Encyclopedia of Genes and Genomes pathway enrichment analysis indicated that the DEGs were enriched in 50 pathways. Detailed gene profile knowledge of the interaction of azadirachtin with M. alternatus should facilitate the development of more effective azadirachtin-based products against M. alternatus and other target Coleoptera. These results further enhance the value of azadirachtin as a potential insecticide of biological origin, as well as for other biological applications. PMID:27629396

  19. Fungal Gene Expression on Demand: an Inducible, Tunable, and Metabolism-Independent Expression System for Aspergillus niger▿†

    PubMed Central

    Meyer, Vera; Wanka, Franziska; van Gent, Janneke; Arentshorst, Mark; van den Hondel, Cees A. M. J. J.; Ram, Arthur F. J.

    2011-01-01

    Filamentous fungi are the cause of serious human and plant diseases but are also exploited in biotechnology as production platforms. Comparative genomics has documented their genetic diversity, and functional genomics and systems biology approaches are under way to understand the functions and interaction of fungal genes and proteins. In these approaches, gene functions are usually inferred from deletion or overexpression mutants. However, studies at these extreme points give only limited information. Moreover, many overexpression studies use metabolism-dependent promoters, often causing pleiotropic effects and thus limitations in their significance. We therefore established and systematically evaluated a tunable expression system for Aspergillus niger that is independent of carbon and nitrogen metabolism and silent under noninduced conditions. The system consists of two expression modules jointly targeted to a defined genomic locus. One module ensures constitutive expression of the tetracycline-dependent transactivator rtTA2S-M2, and one module harbors the rtTA2S-M2-dependent promoter that controls expression of the gene of interest (the Tet-on system). We show here that the system is tight, responds within minutes after inducer addition, and allows fine-tuning based on the inducer concentration or gene copy number up to expression levels higher than the expression levels of the gpdA promoter. We also validate the Tet-on system for the generation of conditional overexpression mutants and demonstrate its power when combined with a gene deletion approach. Finally, we show that the system is especially suitable when the functions of essential genes must be examined. PMID:21378046

  20. The biofilm-specific antibiotic resistance gene ndvB is important for expression of ethanol oxidation genes in Pseudomonas aeruginosa biofilms.

    PubMed

    Beaudoin, Trevor; Zhang, Li; Hinz, Aaron J; Parr, Christopher J; Mah, Thien-Fah

    2012-06-01

    Bacteria growing in biofilms are responsible for a large number of persistent infections and are often more resistant to antibiotics than are free-floating bacteria. In a previous study, we identified a Pseudomonas aeruginosa gene, ndvB, which is important for the formation of periplasmic glucans. We established that these glucans function in biofilm-specific antibiotic resistance by sequestering antibiotic molecules away from their cellular targets. In this study, we investigate another function of ndvB in biofilm-specific antibiotic resistance. DNA microarray analysis identified 24 genes that were responsive to the presence of ndvB. A subset of 20 genes, including 8 ethanol oxidation genes (ercS', erbR, exaA, exaB, eraR, pqqB, pqqC, and pqqE), was highly expressed in wild-type biofilm cells but not in ΔndvB biofilms, while 4 genes displayed the reciprocal expression pattern. Using quantitative real-time PCR, we confirmed the ndvB-dependent expression of the ethanol oxidation genes and additionally demonstrated that these genes were more highly expressed in biofilms than in planktonic cultures. Expression of erbR in ΔndvB biofilms was restored after the treatment of the biofilm with periplasmic extracts derived from wild-type biofilm cells. Inactivation of ethanol oxidation genes increased the sensitivity of biofilms to tobramycin. Together, these results reveal that ndvB affects the expression of multiple genes in biofilms and that ethanol oxidation genes are linked to biofilm-specific antibiotic resistance.

  1. Two White Spot Syndrome Virus MicroRNAs Target the Dorsal Gene To Promote Virus Infection in Marsupenaeus japonicus Shrimp

    PubMed Central

    Ren, Qian; Huang, Xin; Cui, Yalei; Sun, Jiejie; Wang, Wen

    2017-01-01

    ABSTRACT In eukaryotes, microRNAs (miRNAs) serve as regulators of many biological processes, including virus infection. An miRNA can generally target diverse genes during virus-host interactions. However, the regulation of gene expression by multiple miRNAs has not yet been extensively explored during virus infection. This study found that the Spaztle (Spz)-Toll-Dorsal-antilipopolysaccharide factor (ALF) signaling pathway plays a very important role in antiviral immunity against invasion of white spot syndrome virus (WSSV) in shrimp (Marsupenaeus japonicus). Dorsal, the central gene in the Toll pathway, was targeted by two viral miRNAs (WSSV-miR-N13 and WSSV-miR-N23) during WSSV infection. The regulation of Dorsal expression by viral miRNAs suppressed the Spz-Toll-Dorsal-ALF signaling pathway in shrimp in vivo, leading to virus infection. Our study contributes novel insights into the viral miRNA-mediated Toll signaling pathway during the virus-host interaction. IMPORTANCE An miRNA can target diverse genes during virus-host interactions. However, the regulation of gene expression by multiple miRNAs during virus infection has not yet been extensively explored. The results of this study indicated that the shrimp Dorsal gene, the central gene in the Toll pathway, was targeted by two viral miRNAs during infection with white spot syndrome virus. Regulation of Dorsal expression by viral miRNAs suppressed the Spz-Toll-Dorsal-ALF signaling pathway in shrimp in vivo, leading to virus infection. Our study provides new insight into the viral miRNA-mediated Toll signaling pathway in virus-host interactions. PMID:28179524

  2. Stability of Reference Gene Expression After Porcine Sapelovirus Infection in Porcine Intestinal Epithelial Cells.

    PubMed

    Huang, Yong; Chen, Yabing; Sun, Huan; Lan, Daoliang

    2016-01-01

    Intestinal epithelial cells, which serve as the first physical barrier to protect intestinal tract from external antigens, have an important role in the local innate immunity. Screening of reference genes that have stable expression levels after viral infection in porcine intestinal epithelial cells is critical for ensuring the reliability of the expression analysis on anti-infection genes in porcine intestinal epithelial cells. In this study, nine common reference genes in pigs, including ACTB, B2M, GAPDH, HMBS, SDHA, HPRT1, TBP, YWHAZ, and RPL32, were chosen as the candidate reference genes. Porcine sapelovirus (PSV) was used as a model virus to infect porcine intestinal epithelial cell line (IPEC-J2). The expression stability of the nine genes was assessed by the geNorm, NormFinder, and BestKeeper software. Moreover, RefFinder program was used to evaluate the analytical results of above three softwares, and a relative expression experiment of selected target gene was used to verify the analysis results. The comprehensive results indicated that the gene combination of TBP and RPL32 has the most stable expression, which could be considered as an appropriate reference gene for research on gene expression after PSV infection in IPEC-J2cells. The results provided essential data for expression analysis of anti-infection genes in porcine intestinal epithelial cells.

  3. Blood-Derived Smooth Muscle Cells as a Target for Gene Delivery

    PubMed Central

    Yang, Zhe; Shao, Hongwei; Tan, Yaohong; Eton, Darwin; Yu, Hong

    2008-01-01

    Objective To examine the feasibility of using blood-derived smooth muscle cells (BD-SMCs) as a target for to deliver therapeutic proteins. Materials and Methods Mononuclear cells (MNC) were isolated from peripheral blood. The outgrowth colonies from MNC culture were differentiated into BD-SMCs in media containing platelet-derived growth factor BB. Phenotypic characterization of BD-SMCs was assessed by immunocytochemistry. Cell proliferation, gene transfer efficiency with a retroviral vector, apoptosis, and the biological activity of the transduced gene product from the BD-SMCs were evaluated in vitro and in vivo in comparison with vascular derived SMC (VSMCs). Results BD-SMCs stained positive for SMC markers. No significant difference was observed between BD-SMCs and VSMCs in cell proliferation, migration, adhesiveness, and gene transfer efficiency. After BD-SMCs were transduced with a retroviral vector carrying the secreted alkaline phosphatase gene (SEAP), 174 ± 50 μg biologically active SEAP was produced per 106 cells over 24 hrs. After injecting 5×106 cells expressing SEAP intravenously into rabbits, SEAP concentration increased significantly in the circulation from 0.14 ± 0.04 μg/ml to 2.34 ± 0.16 μg/ml 3 days after cell injection (P<0.01, n=3). Circulating levels of SEAP decreased to 1.76 μg /ml one week later and remained at this level up to 8 weeks, then declined to pre-cell injection level at 12 weeks. VSMC in vivo gene expression data were equivalent. Conclusion BD-SMCs have similar characteristics to mature VSMCs, and can be used as a novel target for gene transfer to deliver a therapeutic protein. Clinical relevance Cell-based therapy strategies offer the potential to correct a wide spectrum of inherited and acquired human diseases. Translation to a clinical trial will require a detailed pre-clinical study to understand the characteristics of the isolated cells. BD-SMC are practical and effective targets for ex vivo genetic engineering. They are

  4. Non-coplanar polychlorinated biphenyls (PCBs) are direct agonists for the human pregnane-X receptor and constitutive androstane receptor, and activate target gene expression in a tissue-specific manner

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Al-Salman, Fadheela; Plant, Nick, E-mail: N.Plant@Surrey.ac.uk

    The polychlorinated biphenyl group possesses high environmental persistence, leading to bioaccumulation and a number of adverse effects in mammals. Whilst coplanar PCBs elicit their toxic effects through agonism of the aryl hydrocarbon receptor; however, non-coplanar PCBs are not ligands for AhR, but may be ligands for members of the nuclear receptor family of proteins. To better understand the biological actions of non-coplanar PCBs, we have undertaken a systematic analysis of their ability to activate PXR and CAR-mediated effects. Cells were exposed to a range of non-coplanar PCBs (99, 138, 153, 180 and 194), or the coplanar PCB77: Direct activation ofmore » PXR and CAR was measured using a mammalian receptor activation assay in human liver cells, with rifampicin and CITCO used as positive controls ligands for PXR and CAR, respectively; activation of target gene expression was examined using reporter gene plasmids for CYP3A4 and MDR1 transfected into liver, intestine and lung cell lines. Several of the non-coplanar PCBs directly activated PXR and CAR, whilst the coplanar PCB77 did not. Non-coplanar PCBs were also able to activate PXR/CAR target gene expression in a substitution- and tissue-specific manner. Non-coplanar PCBs act as direct activators for the nuclear receptors PXR and CAR, and are able to elicit transcriptional activation of target genes in a substitution- and tissue-dependent manner. Chronic activation of PXR/CAR is linked to adverse effects and must be included in any risk assessment of PCBs. -- Highlights: ► Several Non-coplanar PCBs are able to directly activate both PXR and CAR in vitro. ► PCB153 is the most potent direct activator of PXR and CAR nuclear receptors. ► Non-coplanar PCB activation of CYP3A4/MDR1 reporter genes is structure-dependent. ► Non-coplanar PCB activate CYP3A4/MDR1 reporter genes in a tissue-dependent. ► PCB153 is the most potent activator of PXR/CAR target gene in all tissues.« less

  5. Comparison of gene expression responses to hypoxia in viviparous (Xiphophorus) and oviparous (Oryzias) fishes using a medaka microarray.

    PubMed

    Boswell, Mikki G; Wells, Melissa C; Kirk, Lyndsey M; Ju, Zhenlin; Zhang, Ziping; Booth, Rachell E; Walter, Ronald B

    2009-03-01

    Gene expression profiling using DNA microarray technology is a useful tool for assessing gene transcript level responses after an organism is exposed to environmental stress. Herein, we detail results from studies using an 8 k medaka (Oryzias latipes) microarray to assess modulated gene expression patterns upon hypoxia exposure of the live-bearing aquaria fish, Xiphophorus maculatus. To assess the reproducibility and reliability of using the medaka array in cross-genus hybridization, a two-factor ANOVA analysis of gene expression was employed. The data show the tissue source of the RNA used for array hybridization contributed more to the observed response of modulated gene targets than did the species source of the RNA. In addition, hierarchical clustering via heat map analyses of groupings of tissues and species (Xiphophorus and medaka) suggests that hypoxia induced similar responses in the same tissues from these two diverse aquatic model organisms. Our Xiphophorus results indicate 206 brain, 37 liver, and 925 gill gene targets exhibit hypoxia induced expression changes. Analysis of the Xiphophorus data to determine those features exhibiting a significant (p<0.05)+/-3 fold change produced only two gene targets within brain tissue and 80 features within gill tissue. Of these 82 characterized features, 39 were identified via homology searching (cut-off E-value of 1 x 10(-5)) and placed into one or more biological process gene ontology groups. Among these 39 genes, metabolic energy changes and manipulation was the most affected biological pathway (13 genes).

  6. Dmrta1 regulates proneural gene expression downstream of Pax6 in the mammalian telencephalon.

    PubMed

    Kikkawa, Takako; Obayashi, Takeshi; Takahashi, Masanori; Fukuzaki-Dohi, Urara; Numayama-Tsuruta, Keiko; Osumi, Noriko

    2013-08-01

    The transcription factor Pax6 balances cell proliferation and neuronal differentiation in the mammalian developing neocortex by regulating the expression of target genes. Using microarray analysis, we observed the down-regulation of Dmrta1 (doublesex and mab-3-related transcription factor-like family A1) in the telencephalon of Pax6 homozygous mutant rats (rSey(2) /rSey(2) ). Dmrta1 expression was restricted to the neural stem/progenitor cells of the dorsal telencephalon. Overexpression of Dmrta1 induced the expression of the proneural gene Neurogenin2 (Neurog2) and conversely repressed Ascl1 (Mash1), a proneural gene expressed in the ventral telencephalon. We found that another Dmrt family molecule, Dmrt3, induced Neurog2 expression in the dorsal telencephalon. Our novel findings suggest that dual regulation of proneural genes mediated by Pax6 and Dmrt family members is crucial for cortical neurogenesis. © 2013 The Authors Genes to Cells © 2013 by the Molecular Biology Society of Japan and Wiley Publishing Asia Pty Ltd.

  7. Identification of key target genes and pathways in laryngeal carcinoma

    PubMed Central

    Liu, Feng; Du, Jintao; Liu, Jun; Wen, Bei

    2016-01-01

    The purpose of the present study was to screen the key genes associated with laryngeal carcinoma and to investigate the molecular mechanism of laryngeal carcinoma progression. The gene expression profile of GSE10935 [Gene Expression Omnibus (GEO) accession number], including 12 specimens from laryngeal papillomas and 12 specimens from normal laryngeal epithelia controls, was downloaded from the GEO database. Differentially expressed genes (DEGs) were screened in laryngeal papillomas compared with normal controls using Limma package in R language, followed by Gene Ontology (GO) enrichment analysis and pathway enrichment analysis. Furthermore, the protein-protein interaction (PPI) network of DEGs was constructed using Cytoscape software and modules were analyzed using MCODE plugin from the PPI network. Furthermore, significant biological pathway regions (sub-pathway) were identified by using iSubpathwayMiner analysis. A total of 67 DEGs were identified, including 27 up-regulated genes and 40 down-regulated genes and they were involved in different GO terms and pathways. PPI network analysis revealed that Ras association (RalGDS/AF-6) domain family member 1 (RASSF1) was a hub protein. The sub-pathway analysis identified 9 significantly enriched sub-pathways, including glycolysis/gluconeogenesis and nitrogen metabolism. Genes such as phosphoglycerate kinase 1 (PGK1), carbonic anhydrase II (CA2), and carbonic anhydrase XII (CA12) whose node degrees were >10 were identified in the disease risk sub-pathway. Genes in the sub-pathway, such as RASSF1, PGK1, CA2 and CA12 were presumed to serve critical roles in laryngeal carcinoma. The present study identified DEGs and their sub-pathways in the disease, which may serve as potential targets for treatment of laryngeal carcinoma. PMID:27446427

  8. Molecular evolution and expression of oxygen transport genes in livebearing fishes (Poeciliidae) from hydrogen sulfide rich springs.

    PubMed

    Barts, Nicholas; Greenway, Ryan; Passow, Courtney N; Arias-Rodriguez, Lenin; Kelley, Joanna L; Tobler, Michael

    2018-04-01

    Hydrogen sulfide (H 2 S) is a natural toxicant in some aquatic environments that has diverse molecular targets. It binds to oxygen transport proteins, rendering them non-functional by reducing oxygen-binding affinity. Hence, organisms permanently inhabiting H 2 S-rich environments are predicted to exhibit adaptive modifications to compensate for the reduced capacity to transport oxygen. We investigated 10 lineages of fish of the family Poeciliidae that have colonized freshwater springs rich in H 2 S-along with related lineages from non-sulfidic environments-to test hypotheses about the expression and evolution of oxygen transport genes in a phylogenetic context. We predicted shifts in the expression of and signatures of positive selection on oxygen transport genes upon colonization of H 2 S-rich habitats. Our analyses indicated significant shifts in gene expression for multiple hemoglobin genes in lineages that have colonized H 2 S-rich environments, and three hemoglobin genes exhibited relaxed selection in sulfidic compared to non-sulfidic lineages. However, neither changes in gene expression nor signatures of selection were consistent among all lineages in H 2 S-rich environments. Oxygen transport genes may consequently be predictable targets of selection during adaptation to sulfidic environments, but changes in gene expression and molecular evolution of oxygen transport genes in H 2 S-rich environments are not necessarily repeatable across replicated lineages.

  9. Diversity and Gene Expression of Phosphatase Genes Provide Insight into Soil Phosphorus Dynamics in a New Zealand Managed Grassland

    NASA Astrophysics Data System (ADS)

    Dunfield, K. E.; Gaiero, J. R.; Condron, L.

    2017-12-01

    Healthy and diverse communities of soil organisms influence key soil ecosystem services such as carbon sequestration, water quality protection, climate regulation and nutrient cycling. Microbially driven mineralization of organic phosphorus is an important contributor to plant available inorganic orthophosphates. In acidic soils, microbes produce non-specific acid phosphatases (NSAPs) which act on common forms of organic phosphorus (P). Our current understanding of P turnover in soils has been limited by lack of research tools capable of targeting these genes. Thus, we developed a set of oligonucleotide PCR primers that targeted bacteria with the genetic potential for acid phosphatase production. A long term randomized-block pasture trial was sampled following 22 years of continued aerial biomass removal and retention. Primers were used to target genes encoding alkaline phosphatase (phoD) and the three classes (CAAP, CBAP, CCAP) of non-specific acid phosphatases. PCR amplicons targeting total genes and gene transcripts were sequenced using Illumina MiSeq to understand the diversity of the bacterial phosphatase producing communities. In general, the majority of operational taxonomic units (OTUs) were shared across both treatments and across metagenomes and transcriptomes. However, analysis of DNA OTUs revealed significantly different communities driven by treatment differences (P < 0.05). Transcript expression was highest in the removed biomass treatment which corresponded the reduced Olsen P levels (15 vs. 36 mg kg-1 in retained treatment). Acid phosphatase activity was measured in all samples, and found to be highest in the biomass retained treatment (16.8 vs. 11.4 µmol g-1 dry soil h-1), likely elevated due to plant-derived enzymes; however, was still correlated to bacterial gene abundances. Overall, the phosphatase producing microbial communities responded to the effect of consistent P limitation as expected, through alteration in the composition of the

  10. Expression of short hairpin RNAs using the compact architecture of retroviral microRNA genes.

    PubMed

    Burke, James M; Kincaid, Rodney P; Aloisio, Francesca; Welch, Nicole; Sullivan, Christopher S

    2017-09-29

    Short hairpin RNAs (shRNAs) are effective in generating stable repression of gene expression. RNA polymerase III (RNAP III) type III promoters (U6 or H1) are typically used to drive shRNA expression. While useful for some knockdown applications, the robust expression of U6/H1-driven shRNAs can induce toxicity and generate heterogeneous small RNAs with undesirable off-target effects. Additionally, typical U6/H1 promoters encompass the majority of the ∼270 base pairs (bp) of vector space required for shRNA expression. This can limit the efficacy and/or number of delivery vector options, particularly when delivery of multiple gene/shRNA combinations is required. Here, we develop a compact shRNA (cshRNA) expression system based on retroviral microRNA (miRNA) gene architecture that uses RNAP III type II promoters. We demonstrate that cshRNAs coded from as little as 100 bps of total coding space can precisely generate small interfering RNAs (siRNAs) that are active in the RNA-induced silencing complex (RISC). We provide an algorithm with a user-friendly interface to design cshRNAs for desired target genes. This cshRNA expression system reduces the coding space required for shRNA expression by >2-fold as compared to the typical U6/H1 promoters, which may facilitate therapeutic RNAi applications where delivery vector space is limiting. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

  11. Modulation of ColE1-like Plasmid Replication for Recombinant Gene Expression

    PubMed Central

    Camps, Manel

    2010-01-01

    ColE1-like plasmids constitute the most popular vectors for recombinant protein expression. ColE1 plasmid replication is tightly controlled by an antisense RNA mechanism that is highly dynamic, tuning plasmid metabolic burden to the physiological state of the host. Plasmid homeostasis is upset upon induction of recombinant protein expression because of non-physiological levels of expression and because of the frequently biased amino acid composition of recombinant proteins. Disregulation of plasmid replication is the main cause of collapse of plasmid-based expression systems because of a simultaneous increase in the metabolic burden (due to increased average copy number) and in the probability of generation of plasmid-free cells (due to increased copy number variation). Interference between regulatory elements of co-resident plasmids causes comparable effects on plasmid stability (plasmid incompatibility). Modulating plasmid copy number for recombinant gene expression aims at achieving a high gene dosage while preserving the stability of the expression system. Here I present strategies targeting plasmid replication for optimizing recombinant gene expression. Specifically, I review approaches aimed at modulating the antisense regulatory system (as well as their implications for plasmid incompatibility) and innovative strategies involving modulation of host factors, of R-loop formation, and of the timing of recombinant gene expression. PMID:20218961

  12. The Genomic Impact of DNA CpG Methylation on Gene Expression; Relationships in Prostate Cancer.

    PubMed

    Long, Mark D; Smiraglia, Dominic J; Campbell, Moray J

    2017-02-14

    The process of DNA CpG methylation has been extensively investigated for over 50 years and revealed associations between changing methylation status of CpG islands and gene expression. As a result, DNA CpG methylation is implicated in the control of gene expression in developmental and homeostasis processes, as well as being a cancer-driver mechanism. The development of genome-wide technologies and sophisticated statistical analytical approaches has ushered in an era of widespread analyses, for example in the cancer arena, of the relationships between altered DNA CpG methylation, gene expression, and tumor status. The remarkable increase in the volume of such genomic data, for example, through investigators from the Cancer Genome Atlas (TCGA), has allowed dissection of the relationships between DNA CpG methylation density and distribution, gene expression, and tumor outcome. In this manner, it is now possible to test that the genome-wide correlations are measurable between changes in DNA CpG methylation and gene expression. Perhaps surprisingly is that these associations can only be detected for hundreds, but not thousands, of genes, and the direction of the correlations are both positive and negative. This, perhaps, suggests that CpG methylation events in cancer systems can act as disease drivers but the effects are possibly more restricted than suspected. Additionally, the positive and negative correlations suggest direct and indirect events and an incomplete understanding. Within the prostate cancer TCGA cohort, we examined the relationships between expression of genes that control DNA methylation, known targets of DNA methylation and tumor status. This revealed that genes that control the synthesis of S -adenosyl-l-methionine (SAM) associate with altered expression of DNA methylation targets in a subset of aggressive tumors.

  13. Identification and Analyses of AUX-IAA target genes controlling multiple pathways in developing fiber cells of Gossypium hirsutum L

    PubMed Central

    Nigam, Deepti; Sawant, Samir V

    2013-01-01

    Technological development led to an increased interest in systems biological approaches in plants to characterize developmental mechanism and candidate genes relevant to specific tissue or cell morphology. AUX-IAA proteins are important plant-specific putative transcription factors. There are several reports on physiological response of this family in Arabidopsis but in cotton fiber the transcriptional network through which AUX-IAA regulated its target genes is still unknown. in-silico modelling of cotton fiber development specific gene expression data (108 microarrays and 22,737 genes) using Algorithm for the Reconstruction of Accurate Cellular Networks (ARACNe) reveals 3690 putative AUX-IAA target genes of which 139 genes were known to be AUX-IAA co-regulated within Arabidopsis. Further AUX-IAA targeted gene regulatory network (GRN) had substantial impact on the transcriptional dynamics of cotton fiber, as showed by, altered TF networks, and Gene Ontology (GO) biological processes and metabolic pathway associated with its target genes. Analysis of the AUX-IAA-correlated gene network reveals multiple functions for AUX-IAA target genes such as unidimensional cell growth, cellular nitrogen compound metabolic process, nucleosome organization, DNA-protein complex and process related to cell wall. These candidate networks/pathways have a variety of profound impacts on such cellular functions as stress response, cell proliferation, and cell differentiation. While these functions are fairly broad, their underlying TF networks may provide a global view of AUX-IAA regulated gene expression and a GRN that guides future studies in understanding role of AUX-IAA box protein and its targets regulating fiber development. PMID:24497725

  14. Hepatic expression of spermatogenic genes and their transiently remarkable downregulations in Wistar-Kyoto rats in response to lead-nitrate administration: strain-difference in the gene expression patterns.

    PubMed

    Nemoto, Kiyomitsu; Ito, Sei; Yoshida, Chiaki; Miyata, Misaki; Kojima, Misaki; Degawa, Masakuni

    2011-06-01

    Administration of lead ion (Pb) to rats and mice affects hepatic functions such as the induction of hepatic cell proliferation and upregulation of cholesterol biosynthesis. To identify the genes for which expression changes in response to Pb-administration, we analyzed hepatic gene expression patterns in stroke-prone spontaneously hypertensive rat (SHRSP), its normotensive control, Wistar-Kyoto rat (WKY), and Spraque-Dawley (SD) rat strains, 3, 6, and 12 hr later after single i.v. injection of lead nitrate (LN) at a dose of 100 µmol using a DNA microarray technique. The data analysis demonstrated that the expression of a great number of genes was transiently and remarkably downregulated 3 hr after LN-injection, and then recovered to control levels only in LN-injected WKY. These normal hepatic expression levels in WKY and SHRSP were much higher than those in SD rats. Furthermore, most of these genes were ones thought to be expressed specifically in the spermatids and/or testes; i.e. genes encoding protamin 1, transition protein 1, and transition protein 2. These findings suggest that the regulation system common to expression of all of these genes could be a target site of Pb-toxic action, at least, in the liver of WKY, and that this system might be similar to the system essential for spermatogenesis, especially spermiogenesis, in the testis. In addition, it appears that clarifying the cause of the difference between the systems of WKY and SHRSP might aid in identifying the pathologic genes in SHRSP. Finally, it will be an important to clarify how the products of the genes related to spermatogenesis, including spermiogenesis, are functional in the livers of WKY and SHRSP.

  15. Genomic identification, characterization and differential expression analysis of SBP-box gene family in Brassica napus.

    PubMed

    Cheng, Hongtao; Hao, Mengyu; Wang, Wenxiang; Mei, Desheng; Tong, Chaobo; Wang, Hui; Liu, Jia; Fu, Li; Hu, Qiong

    2016-09-08

    SBP-box genes belong to one of the largest families of transcription factors. Though members of this family have been characterized to be important regulators of diverse biological processes, information of SBP-box genes in the third most important oilseed crop Brassica napus is largely undefined. In the present study, by whole genome bioinformatics analysis and transcriptional profiling, 58 putative members of SBP-box gene family in oilseed rape (Brassica napus L.) were identified and their expression pattern in different tissues as well as possible interaction with miRNAs were analyzed. In addition, B. napus lines with contrasting branch angle were used for investigating the involvement of SBP-box genes in plant architecture regulation. Detailed gene information, including genomic organization, structural feature, conserved domain and phylogenetic relationship of the genes were systematically characterized. By phylogenetic analysis, BnaSBP proteins were classified into eight distinct groups representing the clear orthologous relationships to their family members in Arabidopsis and rice. Expression analysis in twelve tissues including vegetative and reproductive organs showed different expression patterns among the SBP-box genes and a number of the genes exhibit tissue specific expression, indicating their diverse functions involved in the developmental process. Forty-four SBP-box genes were ascertained to contain the putative miR156 binding site, with 30 and 14 of the genes targeted by miR156 at the coding and 3'UTR region, respectively. Relative expression level of miR156 is varied across tissues. Different expression pattern of some BnaSBP genes and the negative correlation of transcription levels between miR156 and its target BnaSBP gene were observed in lines with different branch angle. Taken together, this study represents the first systematic analysis of the SBP-box gene family in Brassica napus. The data presented here provides base foundation for

  16. Human microRNA target analysis and gene ontology clustering by GOmir, a novel stand-alone application

    PubMed Central

    Roubelakis, Maria G; Zotos, Pantelis; Papachristoudis, Georgios; Michalopoulos, Ioannis; Pappa, Kalliopi I; Anagnou, Nicholas P; Kossida, Sophia

    2009-01-01

    Background microRNAs (miRNAs) are single-stranded RNA molecules of about 20–23 nucleotides length found in a wide variety of organisms. miRNAs regulate gene expression, by interacting with target mRNAs at specific sites in order to induce cleavage of the message or inhibit translation. Predicting or verifying mRNA targets of specific miRNAs is a difficult process of great importance. Results GOmir is a novel stand-alone application consisting of two separate tools: JTarget and TAGGO. JTarget integrates miRNA target prediction and functional analysis by combining the predicted target genes from TargetScan, miRanda, RNAhybrid and PicTar computational tools as well as the experimentally supported targets from TarBase and also providing a full gene description and functional analysis for each target gene. On the other hand, TAGGO application is designed to automatically group gene ontology annotations, taking advantage of the Gene Ontology (GO), in order to extract the main attributes of sets of proteins. GOmir represents a new tool incorporating two separate Java applications integrated into one stand-alone Java application. Conclusion GOmir (by using up to five different databases) introduces miRNA predicted targets accompanied by (a) full gene description, (b) functional analysis and (c) detailed gene ontology clustering. Additionally, a reverse search initiated by a potential target can also be conducted. GOmir can freely be downloaded BRFAA. PMID:19534746

  17. Human microRNA target analysis and gene ontology clustering by GOmir, a novel stand-alone application.

    PubMed

    Roubelakis, Maria G; Zotos, Pantelis; Papachristoudis, Georgios; Michalopoulos, Ioannis; Pappa, Kalliopi I; Anagnou, Nicholas P; Kossida, Sophia

    2009-06-16

    microRNAs (miRNAs) are single-stranded RNA molecules of about 20-23 nucleotides length found in a wide variety of organisms. miRNAs regulate gene expression, by interacting with target mRNAs at specific sites in order to induce cleavage of the message or inhibit translation. Predicting or verifying mRNA targets of specific miRNAs is a difficult process of great importance. GOmir is a novel stand-alone application consisting of two separate tools: JTarget and TAGGO. JTarget integrates miRNA target prediction and functional analysis by combining the predicted target genes from TargetScan, miRanda, RNAhybrid and PicTar computational tools as well as the experimentally supported targets from TarBase and also providing a full gene description and functional analysis for each target gene. On the other hand, TAGGO application is designed to automatically group gene ontology annotations, taking advantage of the Gene Ontology (GO), in order to extract the main attributes of sets of proteins. GOmir represents a new tool incorporating two separate Java applications integrated into one stand-alone Java application. GOmir (by using up to five different databases) introduces miRNA predicted targets accompanied by (a) full gene description, (b) functional analysis and (c) detailed gene ontology clustering. Additionally, a reverse search initiated by a potential target can also be conducted. GOmir can freely be downloaded BRFAA.

  18. Monoallelic Gene Expression in Mammals.

    PubMed

    Chess, Andrew

    2016-11-23

    Monoallelic expression not due to cis-regulatory sequence polymorphism poses an intriguing problem in epigenetics because it requires the unequal treatment of two segments of DNA that are present in the same nucleus and that can indeed have absolutely identical sequences. Here, I focus on a few recent developments in the field of monoallelic expression that are of particular interest and raise interesting questions for future work. One development is regarding analyses of imprinted genes, in which recent work suggests the possibility that intriguing networks of imprinted genes exist and are important for genetic and physiological studies. Another issue that has been raised in recent years by a number of publications is the question of how skewed allelic expression should be for it to be designated as monoallelic expression and, further, what methods are appropriate or inappropriate for analyzing genomic data to examine allele-specific expression. Perhaps the most exciting recent development in mammalian monoallelic expression is a clever and carefully executed analysis of genetic diversity of autosomal genes subject to random monoallelic expression (RMAE), which provides compelling evidence for distinct evolutionary forces acting on random monoallelically expressed genes.

  19. Sho-saiko-to, a traditional herbal medicine, regulates gene expression and biological function by way of microRNAs in primary mouse hepatocytes

    PubMed Central

    2014-01-01

    Background Sho-saiko-to (SST) (also known as so-shi-ho-tang or xiao-chai-hu-tang) has been widely prescribed for chronic liver diseases in traditional Oriental medicine. Despite the substantial amount of clinical evidence for SST, its molecular mechanism has not been clearly identified at a genome-wide level. Methods By using a microarray, we analyzed the temporal changes of messenger RNA (mRNA) and microRNA expression in primary mouse hepatocytes after SST treatment. The pattern of genes regulated by SST was identified by using time-series microarray analysis. The biological function of genes was measured by pathway analysis. For the identification of the exact targets of the microRNAs, a permutation-based correlation method was implemented in which the temporal expression of mRNAs and microRNAs were integrated. The similarity of the promoter structure between temporally regulated genes was measured by analyzing the transcription factor binding sites in the promoter region. Results The SST-regulated gene expression had two major patterns: (1) a temporally up-regulated pattern (463 genes) and (2) a temporally down-regulated pattern (177 genes). The integration of the genes and microRNA demonstrated that 155 genes could be the targets of microRNAs from the temporally up-regulated pattern and 19 genes could be the targets of microRNAs from the temporally down-regulated pattern. The temporally up-regulated pattern by SST was associated with signaling pathways such as the cell cycle pathway, whereas the temporally down-regulated pattern included drug metabolism-related pathways and immune-related pathways. All these pathways could be possibly associated with liver regenerative activity of SST. Genes targeted by microRNA were moreover associated with different biological pathways from the genes not targeted by microRNA. An analysis of promoter similarity indicated that co-expressed genes after SST treatment were clustered into subgroups, depending on the temporal

  20. Sho-saiko-to, a traditional herbal medicine, regulates gene expression and biological function by way of microRNAs in primary mouse hepatocytes.

    PubMed

    Song, Kwang Hoon; Kim, Yun Hee; Kim, Bu-Yeo

    2014-01-11

    Sho-saiko-to (SST) (also known as so-shi-ho-tang or xiao-chai-hu-tang) has been widely prescribed for chronic liver diseases in traditional Oriental medicine. Despite the substantial amount of clinical evidence for SST, its molecular mechanism has not been clearly identified at a genome-wide level. By using a microarray, we analyzed the temporal changes of messenger RNA (mRNA) and microRNA expression in primary mouse hepatocytes after SST treatment. The pattern of genes regulated by SST was identified by using time-series microarray analysis. The biological function of genes was measured by pathway analysis. For the identification of the exact targets of the microRNAs, a permutation-based correlation method was implemented in which the temporal expression of mRNAs and microRNAs were integrated. The similarity of the promoter structure between temporally regulated genes was measured by analyzing the transcription factor binding sites in the promoter region. The SST-regulated gene expression had two major patterns: (1) a temporally up-regulated pattern (463 genes) and (2) a temporally down-regulated pattern (177 genes). The integration of the genes and microRNA demonstrated that 155 genes could be the targets of microRNAs from the temporally up-regulated pattern and 19 genes could be the targets of microRNAs from the temporally down-regulated pattern. The temporally up-regulated pattern by SST was associated with signaling pathways such as the cell cycle pathway, whereas the temporally down-regulated pattern included drug metabolism-related pathways and immune-related pathways. All these pathways could be possibly associated with liver regenerative activity of SST. Genes targeted by microRNA were moreover associated with different biological pathways from the genes not targeted by microRNA. An analysis of promoter similarity indicated that co-expressed genes after SST treatment were clustered into subgroups, depending on the temporal expression patterns. We are the

  1. Integrated microarray and ChIP analysis identifies multiple Foxa2 dependent target genes in the notochord.

    PubMed

    Tamplin, Owen J; Cox, Brian J; Rossant, Janet

    2011-12-15

    The node and notochord are key tissues required for patterning of the vertebrate body plan. Understanding the gene regulatory network that drives their formation and function is therefore important. Foxa2 is a key transcription factor at the top of this genetic hierarchy and finding its targets will help us to better understand node and notochord development. We performed an extensive microarray-based gene expression screen using sorted embryonic notochord cells to identify early notochord-enriched genes. We validated their specificity to the node and notochord by whole mount in situ hybridization. This provides the largest available resource of notochord-expressed genes, and therefore candidate Foxa2 target genes in the notochord. Using existing Foxa2 ChIP-seq data from adult liver, we were able to identify a set of genes expressed in the notochord that had associated regions of Foxa2-bound chromatin. Given that Foxa2 is a pioneer transcription factor, we reasoned that these sites might represent notochord-specific enhancers. Candidate Foxa2-bound regions were tested for notochord specific enhancer function in a zebrafish reporter assay and 7 novel notochord enhancers were identified. Importantly, sequence conservation or predictive models could not have readily identified these regions. Mutation of putative Foxa2 binding elements in two of these novel enhancers abrogated reporter expression and confirmed their Foxa2 dependence. The combination of highly specific gene expression profiling and genome-wide ChIP analysis is a powerful means of understanding developmental pathways, even for small cell populations such as the notochord. Copyright © 2011 Elsevier Inc. All rights reserved.

  2. Identification and validation of superior reference gene for gene expression normalization via RT-qPCR in staminate and pistillate flowers of Jatropha curcas – A biodiesel plant

    PubMed Central

    Karuppaiya, Palaniyandi; Yan, Xiao-Xue; Liao, Wang; Chen, Fang; Tang, Lin

    2017-01-01

    Physic nut (Jatropha curcas L) seed oil is a natural resource for the alternative production of fossil fuel. Seed oil production is mainly depended on seed yield, which was restricted by the low ratio of staminate flowers to pistillate flowers. Further, the mechanism of physic nut flower sex differentiation has not been fully understood yet. Quantitative Real Time—Polymerase Chain Reaction is a reliable and widely used technique to quantify the gene expression pattern in biological samples. However, for accuracy of qRT-PCR, appropriate reference gene is highly desirable to quantify the target gene level. Hence, the present study was aimed to identify the stable reference genes in staminate and pistillate flowers of J. curcas. In this study, 10 candidate reference genes were selected and evaluated for their expression stability in staminate and pistillate flowers, and their stability was validated by five different algorithms (ΔCt, BestKeeper, NormFinder, GeNorm and RefFinder). Resulting, TUB and EF found to be the two most stably expressed reference for staminate flower; while GAPDH1 and EF found to be the most stably expressed reference gene for pistillate flowers. Finally, RT-qPCR assays of target gene AGAMOUS using the identified most stable reference genes confirmed the reliability of selected reference genes in different stages of flower development. AGAMOUS gene expression levels at different stages were further proved by gene copy number analysis. Therefore, the present study provides guidance for selecting appropriate reference genes for analyzing the expression pattern of floral developmental genes in staminate and pistillate flowers of J. curcas. PMID:28234941

  3. Identification, Expression Analysis, and Target Prediction of Flax Genotroph MicroRNAs Under Normal and Nutrient Stress Conditions

    PubMed Central

    Melnikova, Nataliya V.; Dmitriev, Alexey A.; Belenikin, Maxim S.; Koroban, Nadezhda V.; Speranskaya, Anna S.; Krinitsina, Anastasia A.; Krasnov, George S.; Lakunina, Valentina A.; Snezhkina, Anastasiya V.; Sadritdinova, Asiya F.; Kishlyan, Natalya V.; Rozhmina, Tatiana A.; Klimina, Kseniya M.; Amosova, Alexandra V.; Zelenin, Alexander V.; Muravenko, Olga V.; Bolsheva, Nadezhda L.; Kudryavtseva, Anna V.

    2016-01-01

    Cultivated flax (Linum usitatissimum L.) is an important plant valuable for industry. Some flax lines can undergo heritable phenotypic and genotypic changes (LIS-1 insertion being the most common) in response to nutrient stress and are called plastic lines. Offspring of plastic lines, which stably inherit the changes, are called genotrophs. MicroRNAs (miRNAs) are involved in a crucial regulatory mechanism of gene expression. They have previously been assumed to take part in nutrient stress response and can, therefore, participate in genotroph formation. In the present study, we performed high-throughput sequencing of small RNAs (sRNAs) extracted from flax plants grown under normal, phosphate deficient and nutrient excess conditions to identify miRNAs and evaluate their expression. Our analysis revealed expression of 96 conserved miRNAs from 21 families in flax. Moreover, 475 novel potential miRNAs were identified for the first time, and their targets were predicted. However, none of the identified miRNAs were transcribed from LIS-1. Expression of seven miRNAs (miR168, miR169, miR395, miR398, miR399, miR408, and lus-miR-N1) with up- or down-regulation under nutrient stress (on the basis of high-throughput sequencing data) was evaluated on extended sampling using qPCR. Reference gene search identified ETIF3H and ETIF3E genes as most suitable for this purpose. Down-regulation of novel potential lus-miR-N1 and up-regulation of conserved miR399 were revealed under the phosphate deficient conditions. In addition, the negative correlation of expression of lus-miR-N1 and its predicted target, ubiquitin-activating enzyme E1 gene, as well as, miR399 and its predicted target, ubiquitin-conjugating enzyme E2 gene, was observed. Thus, in our study, miRNAs expressed in flax plastic lines and genotrophs were identified and their expression and expression of their targets was evaluated using high-throughput sequencing and qPCR for the first time. These data provide new insights

  4. Selection of Reliable Reference Genes for Gene Expression Studies on Rhododendron molle G. Don.

    PubMed

    Xiao, Zheng; Sun, Xiaobo; Liu, Xiaoqing; Li, Chang; He, Lisi; Chen, Shangping; Su, Jiale

    2016-01-01

    The quantitative real-time polymerase chain reaction (qRT-PCR) approach has become a widely used method to analyze expression patterns of target genes. The selection of an optimal reference gene is a prerequisite for the accurate normalization of gene expression in qRT-PCR. The present study constitutes the first systematic evaluation of potential reference genes in Rhododendron molle G. Don. Eleven candidate reference genes in different tissues and flowers at different developmental stages of R. molle were assessed using the following three software packages: GeNorm, NormFinder, and BestKeeper. The results showed that EF1- α (elongation factor 1-alpha), 18S (18s ribosomal RNA), and RPL3 (ribosomal protein L3) were the most stable reference genes in developing rhododendron flowers and, thus, in all of the tested samples, while tublin ( TUB ) was the least stable. ACT5 (actin), RPL3 , 18S , and EF1- α were found to be the top four choices for different tissues, whereas TUB was not found to favor qRT-PCR normalization in these tissues. Three stable reference genes are recommended for the normalization of qRT-PCR data in R. molle . Furthermore, the expression profiles of RmPSY (phytoene synthase) and RmPDS (phytoene dehydrogenase) were assessed using EF1- α, 18S , ACT5 , RPL3 , and their combination as internals. Similar trends were found, but these trends varied when the least stable reference gene TUB was used. The results further prove that it is necessary to validate the stability of reference genes prior to their use for normalization under different experimental conditions. This study provides useful information for reliable qRT-PCR data normalization in gene studies of R. molle .

  5. Peroxisome Proliferator-Activated Receptor γ Target Gene Encoding a Novel Angiopoietin-Related Protein Associated with Adipose Differentiation

    PubMed Central

    Yoon, J. Cliff; Chickering, Troy W.; Rosen, Evan D.; Dussault, Barry; Qin, Yubin; Soukas, Alexander; Friedman, Jeffrey M.; Holmes, William E.; Spiegelman, Bruce M.

    2000-01-01

    The nuclear receptor peroxisome proliferator-activated receptor γ regulates adipose differentiation and systemic insulin signaling via ligand-dependent transcriptional activation of target genes. However, the identities of the biologically relevant target genes are largely unknown. Here we describe the isolation and characterization of a novel target gene induced by PPARγ ligands, termed PGAR (for PPARγ angiopoietin related), which encodes a novel member of the angiopoietin family of secreted proteins. The transcriptional induction of PGAR follows a rapid time course typical of immediate-early genes and occurs in the absence of protein synthesis. The expression of PGAR is predominantly localized to adipose tissues and placenta and is consistently elevated in genetic models of obesity. Hormone-dependent adipocyte differentiation coincides with a dramatic early induction of the PGAR transcript. Alterations in nutrition and leptin administration are found to modulate the PGAR expression in vivo. Taken together, these data suggest a possible role for PGAR in the regulation of systemic lipid metabolism or glucose homeostasis. PMID:10866690

  6. Altered Gene Expressions and Cytogenetic Repair Efficiency in Cells with Suppressed Expression of XPA after Proton Exposure

    NASA Technical Reports Server (NTRS)

    Zhang, Ye; Rohde, Larry H.; Gridley, Daila S.; Mehta, Satish K.; Pierson, Duane L.; Wu, Honglu

    2009-01-01

    Cellular responses to damages from ionizing radiation (IR) exposure are influenced not only by the genes involved in DNA double strand break (DSB) repair, but also by non- DSB repair genes. We demonstrated previously that suppressed expression of several non-DSB repair genes, such as XPA, elevated IR-induced cytogenetic damages. In the present study, we exposed human fibroblasts that were treated with control or XPA targeting siRNA to 250 MeV protons (0 to 4 Gy), and analyzed chromosome aberrations and expressions of genes involved in DNA repair. As expected, after proton irradiation, cells with suppressed expression of XPA showed a significantly elevated frequency of chromosome aberrations compared with control siRNA treated (CS) cells. Protons caused more severe DNA damages in XPA knock-down cells, as 36% cells contained multiple aberrations compared to 25% in CS cells after 4Gy proton irradiation. Comparison of gene expressions using the real-time PCR array technique revealed that expressions of p53 and its regulated genes in irradiated XPA suppressed cells were altered similarly as in CS cells, suggesting that the impairment of IR induced DNA repair in XPA suppressed cells is p53-independent. Except for XPA, which was more than 2 fold down regulated in XPA suppressed cells, several other DNA damage sensing and repair genes (GTSE1, RBBP8, RAD51, UNG and XRCC2) were shown a more than 1.5 fold difference between XPA knock-down cells and CS cells after proton exposure. The possible involvement of these genes in the impairment of DNA repair in XPA suppressed cells will be further investigated.

  7. A splice junction-targeted CRISPR approach (spJCRISPR) reveals human FOXO3B to be a protein-coding gene.

    PubMed

    Santo, Evan E; Paik, Jihye

    2018-06-17

    The rapid development of CRISPR technology is revolutionizing molecular approaches to the dissection of complex biological phenomena. Here we describe an alternative generally applicable implementation of the CRISPR-Cas9 system that allows for selective knockdown of extremely homologous genes. This strategy employs the lentiviral delivery of paired sgRNAs and nickase Cas9 (Cas9D10A) to achieve targeted deletion of splice junctions. This general strategy offers several advantages over standard single-guide exon-targeting CRISPR-Cas9 such as greatly reduced off-target effects, more restricted genomic editing, routine disruption of target gene mRNA expression and the ability to differentiate between closely related genes. Here we demonstrate the utility of this strategy by achieving selective knockdown of the highly homologous human genes FOXO3A and suspected pseudogene FOXO3B. We find the spJCRISPR strategy to efficiently and selectively disrupt FOXO3A and FOXO3B mRNA and protein expression; thus revealing that the human FOXO3B locus encodes a bona fide human gene. Unlike FOXO3A, we find the FOXO3B protein to be cytosolically localized in both the presence and absence of active Akt. The ability to selectively target and efficiently disrupt the expression of the closely-related FOXO3A and FOXO3B genes demonstrates the efficacy of the spJCRISPR approach. Copyright © 2018. Published by Elsevier B.V.

  8. Selection of reference genes for gene expression studies in virus-infected monocots using quantitative real-time PCR.

    PubMed

    Zhang, Kun; Niu, Shaofang; Di, Dianping; Shi, Lindan; Liu, Deshui; Cao, Xiuling; Miao, Hongqin; Wang, Xianbing; Han, Chenggui; Yu, Jialin; Li, Dawei; Zhang, Yongliang

    2013-10-10

    Both genome-wide transcriptomic surveys of the mRNA expression profiles and virus-induced gene silencing-based molecular studies of target gene during virus-plant interaction involve the precise estimation of the transcript abundance. Quantitative real-time PCR (qPCR) is the most widely adopted technique for mRNA quantification. In order to obtain reliable quantification of transcripts, identification of the best reference genes forms the basis of the preliminary work. Nevertheless, the stability of internal controls in virus-infected monocots needs to be fully explored. In this work, the suitability of ten housekeeping genes (ACT, EF1α, FBOX, GAPDH, GTPB, PP2A, SAND, TUBβ, UBC18 and UK) for potential use as reference genes in qPCR were investigated in five different monocot plants (Brachypodium, barley, sorghum, wheat and maize) under infection with different viruses including Barley stripe mosaic virus (BSMV), Brome mosaic virus (BMV), Rice black-streaked dwarf virus (RBSDV) and Sugarcane mosaic virus (SCMV). By using three different algorithms, the most appropriate reference genes or their combinations were identified for different experimental sets and their effectiveness for the normalisation of expression studies were further validated by quantitative analysis of a well-studied PR-1 gene. These results facilitate the selection of desirable reference genes for more accurate gene expression studies in virus-infected monocots. Copyright © 2013 Elsevier B.V. All rights reserved.

  9. Lung tumor diagnosis and subtype discovery by gene expression profiling.

    PubMed

    Wang, Lu-yong; Tu, Zhuowen

    2006-01-01

    The optimal treatment of patients with complex diseases, such as cancers, depends on the accurate diagnosis by using a combination of clinical and histopathological data. In many scenarios, it becomes tremendously difficult because of the limitations in clinical presentation and histopathology. To accurate diagnose complex diseases, the molecular classification based on gene or protein expression profiles are indispensable for modern medicine. Moreover, many heterogeneous diseases consist of various potential subtypes in molecular basis and differ remarkably in their response to therapies. It is critical to accurate predict subgroup on disease gene expression profiles. More fundamental knowledge of the molecular basis and classification of disease could aid in the prediction of patient outcome, the informed selection of therapies, and identification of novel molecular targets for therapy. In this paper, we propose a new disease diagnostic method, probabilistic boosting tree (PB tree) method, on gene expression profiles of lung tumors. It enables accurate disease classification and subtype discovery in disease. It automatically constructs a tree in which each node combines a number of weak classifiers into a strong classifier. Also, subtype discovery is naturally embedded in the learning process. Our algorithm achieves excellent diagnostic performance, and meanwhile it is capable of detecting the disease subtype based on gene expression profile.

  10. Gene expression analysis uncovers novel Hedgehog interacting protein (HHIP) effects in human bronchial epithelial cells

    PubMed Central

    Zhou, Xiaobo; Qiu, Weiliang; Sathirapongsasuti, J. Fah.; Cho, Michael H.; Mancini, John D.; Lao, Taotao; Thibault, Derek M.; Litonjua, Gus; Bakke, Per S.; Gulsvik, Amund; Lomas, David A.; Beaty, Terri H.; Hersh, Craig P.; Anderson, Christopher; Geigenmuller, Ute; Raby, Benjamin A.; Rennard, Stephen I.; Perrella, Mark A.; Choi, Augustine M.K.; Quackenbush, John; Silverman, Edwin K.

    2013-01-01

    Hedgehog Interacting Protein (HHIP) was implicated in chronic obstructive pulmonary disease (COPD) by genome-wide association studies (GWAS). However, it remains unclear how HHIP contributes to COPD pathogenesis. To identify genes regulated by HHIP, we performed gene expression microarray analysis in a human bronchial epithelial cell line (Beas-2B) stably infected with HHIP shRNAs. HHIP silencing led to differential expression of 296 genes; enrichment for variants nominally associated with COPD was found. Eighteen of the differentially expressed genes were validated by real-time PCR in Beas-2B cells. Seven of 11 validated genes tested in human COPD and control lung tissues demonstrated significant gene expression differences. Functional annotation indicated enrichment for extracellular matrix and cell growth genes. Network modeling demonstrated that the extracellular matrix and cell proliferation genes influenced by HHIP tended to be interconnected. Thus, we identified potential HHIP targets in human bronchial epithelial cells that may contribute to COPD pathogenesis. PMID:23459001

  11. Predicting essential genes for identifying potential drug targets in Aspergillus fumigatus.

    PubMed

    Lu, Yao; Deng, Jingyuan; Rhodes, Judith C; Lu, Hui; Lu, Long Jason

    2014-06-01

    Aspergillus fumigatus (Af) is a ubiquitous and opportunistic pathogen capable of causing acute, invasive pulmonary disease in susceptible hosts. Despite current therapeutic options, mortality associated with invasive Af infections remains unacceptably high, increasing 357% since 1980. Therefore, there is an urgent need for the development of novel therapeutic strategies, including more efficacious drugs acting on new targets. Thus, as noted in a recent review, "the identification of essential genes in fungi represents a crucial step in the development of new antifungal drugs". Expanding the target space by rapidly identifying new essential genes has thus been described as "the most important task of genomics-based target validation". In previous research, we were the first to show that essential gene annotation can be reliably transferred between distantly related four Prokaryotic species. In this study, we extend our machine learning approach to the much more complex Eukaryotic fungal species. A compendium of essential genes is predicted in Af by transferring known essential gene annotations from another filamentous fungus Neurospora crassa. This approach predicts essential genes by integrating diverse types of intrinsic and context-dependent genomic features encoded in microbial genomes. The predicted essential datasets contained 1674 genes. We validated our results by comparing our predictions with known essential genes in Af, comparing our predictions with those predicted by homology mapping, and conducting conditional expressed alleles. We applied several layers of filters and selected a set of potential drug targets from the predicted essential genes. Finally, we have conducted wet lab knockout experiments to verify our predictions, which further validates the accuracy and wide applicability of the machine learning approach. The approach presented here significantly extended our ability to predict essential genes beyond orthologs and made it possible to

  12. Expansion of the CRISPR-Cas9 genome targeting space through the use of H1 promoter-expressed guide RNAs.

    PubMed

    Ranganathan, Vinod; Wahlin, Karl; Maruotti, Julien; Zack, Donald J

    2014-08-08

    The repurposed CRISPR-Cas9 system has recently emerged as a revolutionary genome-editing tool. Here we report a modification in the expression of the guide RNA (gRNA) required for targeting that greatly expands the targetable genome. gRNA expression through the commonly used U6 promoter requires a guanosine nucleotide to initiate transcription, thus constraining genomic-targeting sites to GN19NGG. We demonstrate the ability to modify endogenous genes using H1 promoter-expressed gRNAs, which can be used to target both AN19NGG and GN19NGG genomic sites. AN19NGG sites occur ~15% more frequently than GN19NGG sites in the human genome and the increase in targeting space is also enriched at human genes and disease loci. Together, our results enhance the versatility of the CRISPR technology by more than doubling the number of targetable sites within the human genome and other eukaryotic species.

  13. Hindsight regulates photoreceptor axon targeting through transcriptional control of jitterbug/Filamin and multiple genes involved in axon guidance in Drosophila.

    PubMed

    Oliva, Carlos; Molina-Fernandez, Claudia; Maureira, Miguel; Candia, Noemi; López, Estefanía; Hassan, Bassem; Aerts, Stein; Cánovas, José; Olguín, Patricio; Sierralta, Jimena

    2015-09-01

    During axon targeting, a stereotyped pattern of connectivity is achieved by the integration of intrinsic genetic programs and the response to extrinsic long and short-range directional cues. How this coordination occurs is the subject of intense study. Transcription factors play a central role due to their ability to regulate the expression of multiple genes required to sense and respond to these cues during development. Here we show that the transcription factor HNT regulates layer-specific photoreceptor axon targeting in Drosophila through transcriptional control of jbug/Filamin and multiple genes involved in axon guidance and cytoskeleton organization.Using a microarray analysis we identified 235 genes whose expression levels were changed by HNT overexpression in the eye primordia. We analyzed nine candidate genes involved in cytoskeleton regulation and axon guidance, six of which displayed significantly altered gene expression levels in hnt mutant retinas. Functional analysis confirmed the role of OTK/PTK7 in photoreceptor axon targeting and uncovered Tiggrin, an integrin ligand, and Jbug/Filamin, a conserved actin- binding protein, as new factors that participate of photoreceptor axon targeting. Moreover, we provided in silico and molecular evidence that supports jbug/Filamin as a direct transcriptional target of HNT and that HNT acts partially through Jbug/Filamin in vivo to regulate axon guidance. Our work broadens the understanding of how HNT regulates the coordinated expression of a group of genes to achieve the correct connectivity pattern in the Drosophila visual system. © 2015 Wiley Periodicals, Inc. Develop Neurobiol 75: 1018-1032, 2015. © 2015 Wiley Periodicals, Inc.

  14. Functional visualization and disruption of targeted genes using CRISPR/Cas9-mediated eGFP reporter integration in zebrafish.

    PubMed

    Ota, Satoshi; Taimatsu, Kiyohito; Yanagi, Kanoko; Namiki, Tomohiro; Ohga, Rie; Higashijima, Shin-Ichi; Kawahara, Atsuo

    2016-10-11

    The CRISPR/Cas9 complex, which is composed of a guide RNA (gRNA) and the Cas9 nuclease, is useful for carrying out genome modifications in various organisms. Recently, the CRISPR/Cas9-mediated locus-specific integration of a reporter, which contains the Mbait sequence targeted using Mbait-gRNA, the hsp70 promoter and the eGFP gene, has allowed the visualization of the target gene expression. However, it has not been ascertained whether the reporter integrations at both targeted alleles cause loss-of-function phenotypes in zebrafish. In this study, we have inserted the Mbait-hs-eGFP reporter into the pax2a gene because the disruption of pax2a causes the loss of the midbrain-hindbrain boundary (MHB) in zebrafish. In the heterozygous Tg[pax2a-hs:eGFP] embryos, MHB formed normally and the eGFP expression recapitulated the endogenous pax2a expression, including the MHB. We observed the loss of the MHB in homozygous Tg[pax2a-hs:eGFP] embryos. Furthermore, we succeeded in integrating the Mbait-hs-eGFP reporter into an uncharacterized gene epdr1. The eGFP expression in heterozygous Tg[epdr1-hs:eGFP] embryos overlapped the epdr1 expression, whereas the distribution of eGFP-positive cells was disorganized in the MHB of homozygous Tg[epdr1-hs:eGFP] embryos. We propose that the locus-specific integration of the Mbait-hs-eGFP reporter is a powerful method to investigate both gene expression profiles and loss-of-function phenotypes.

  15. Functional visualization and disruption of targeted genes using CRISPR/Cas9-mediated eGFP reporter integration in zebrafish

    PubMed Central

    Ota, Satoshi; Taimatsu, Kiyohito; Yanagi, Kanoko; Namiki, Tomohiro; Ohga, Rie; Higashijima, Shin-ichi; Kawahara, Atsuo

    2016-01-01

    The CRISPR/Cas9 complex, which is composed of a guide RNA (gRNA) and the Cas9 nuclease, is useful for carrying out genome modifications in various organisms. Recently, the CRISPR/Cas9-mediated locus-specific integration of a reporter, which contains the Mbait sequence targeted using Mbait-gRNA, the hsp70 promoter and the eGFP gene, has allowed the visualization of the target gene expression. However, it has not been ascertained whether the reporter integrations at both targeted alleles cause loss-of-function phenotypes in zebrafish. In this study, we have inserted the Mbait-hs-eGFP reporter into the pax2a gene because the disruption of pax2a causes the loss of the midbrain-hindbrain boundary (MHB) in zebrafish. In the heterozygous Tg[pax2a-hs:eGFP] embryos, MHB formed normally and the eGFP expression recapitulated the endogenous pax2a expression, including the MHB. We observed the loss of the MHB in homozygous Tg[pax2a-hs:eGFP] embryos. Furthermore, we succeeded in integrating the Mbait-hs-eGFP reporter into an uncharacterized gene epdr1. The eGFP expression in heterozygous Tg[epdr1-hs:eGFP] embryos overlapped the epdr1 expression, whereas the distribution of eGFP-positive cells was disorganized in the MHB of homozygous Tg[epdr1-hs:eGFP] embryos. We propose that the locus-specific integration of the Mbait-hs-eGFP reporter is a powerful method to investigate both gene expression profiles and loss-of-function phenotypes. PMID:27725766

  16. New gene targets for glucagon-like peptide-1 during embryonic development and in undifferentiated pluripotent cells.

    PubMed

    Sanz, Carmen; Blázquez, Enrique

    2011-09-01

    In humans, glucagon-like peptide (GLP-1) functions during adult life as an incretin hormone with anorexigenic and antidiabetogenic properties. Also, the therapeutic potential of GLP-1 in preventing the adipocyte hyperplasia associated with obesity and in bolstering the maintenance of human mesenchymal stem cell (hMSC) stores by promoting the proliferation and cytoprotection of hMSC seems to be relevant. Since these observations suggest a role for GLP-1 during developmental processes, the aim of the present work was to characterize GLP-1 in early development as well as its gene targets in mouse embryonic stem (mES) cells. Mouse embryos E6, E8, and E10.5 and pluripotent mES were used for the inmunodetection of GLP-1 and GLP-1 receptor. Quantitative real-time PCR was used to determine the expression levels of GLP-1R in several tissues from E12.5 mouse embryos. Additionally, GLP-1 gene targets were studied in mES by multiple gene expression analyses. GLP-1 and its receptors were identified in mES and during embryonic development. In pluripotent mES, GLP-1 modified the expression of endodermal, ectodermal, and mesodermal gene markers as well as sonic hedgehog, noggin, members of the fibroblast and hepatic growth factor families, and others involved in pancreatic development. Additionally, GLP-1 promoted the expression of the antiapoptotic gene bcl2 and at the same time reduced proapoptotic caspase genes. Our results indicate that apart from the effects and therapeutic benefits of GLP-1 in adulthood, it may have additional gene targets in mES cells during embryonic life. Furthermore, the pathophysiological implications of GLP-1 imbalance in adulthood may have a counterpart during development.

  17. Expressing genes do not forget their LINEs: transposable elements and gene expression

    PubMed Central

    Kines, Kristine J.; Belancio, Victoria P.

    2012-01-01

    1. ABSTRACT Historically the accumulated mass of mammalian transposable elements (TEs), particularly those located within gene boundaries, was viewed as a genetic burden potentially detrimental to the genomic landscape. This notion has been strengthened by the discovery that transposable sequences can alter the architecture of the transcriptome, not only through insertion, but also long after the integration process is completed. Insertions previously considered harmless are now known to impact the expression of host genes via modification of the transcript quality or quantity, transcriptional interference, or by the control of pathways that affect the mRNA life-cycle. Conversely, several examples of the evolutionary advantageous impact of TEs on the host gene structure that diversified the cellular transcriptome are reported. TE-induced changes in gene expression can be tissue-or disease-specific, raising the possibility that the impact of TE sequences may vary during development, among normal cell types, and between normal and disease-affected tissues. The understanding of the rules and abundance of TE-interference with gene expression is in its infancy, and its contribution to human disease and/or evolution remains largely unexplored. PMID:22201807

  18. Meta-type analysis of dopaminergic effects on gene expression in the neuroendocrine brain of female goldfish.

    PubMed

    Popesku, Jason T; Martyniuk, Christopher J; Trudeau, Vance L

    2012-01-01

    Dopamine (DA) is a major neurotransmitter important for neuroendocrine control and recent studies have described genomic signaling pathways activated and inhibited by DA agonists and antagonists in the goldfish brain. Here we perform a meta-type analysis using microarray datasets from experiments conducted with female goldfish to characterize the gene expression responses that underlie dopaminergic signaling. Sexually mature, pre-spawning [gonadosomatic index (GSI) = 4.5 ± 1.3%] or sexually regressing (GSI = 3 ± 0.4%) female goldfish (15-40 g) injected intraperitoneally with either SKF 38393, LY 171555, SCH 23390, sulpiride, or a combination of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine and α-methyl-p-tyrosine. Microarray meta-type analysis identified 268 genes in the telencephalon and hypothalamus as having reciprocal (i.e., opposite between agonism and antagonism/depletion) fold change responses, suggesting that these transcripts are likely targets for DA-mediated regulation. Noteworthy genes included ependymin, vimentin, and aromatase, genes that support the significance of DA in neuronal plasticity and tissue remodeling. Sub-network enrichment analysis (SNEA) was used to identify common gene regulators and binding proteins associated with the differentially expressed genes mediated by DA. SNEA analysis identified gene expression targets that were related to three major categories that included cell signaling (STAT3, SP1, SMAD, Jun/Fos), immune response (IL-6, IL-1β, TNFs, cytokine, NF-κB), and cell proliferation and growth (IGF1, TGFβ1). These gene networks are also known to be associated with neurodegenerative disorders such as Parkinsons' disease, well-known to be associated with loss of dopaminergic neurons. This study identifies genes and networks that underlie DA signaling in the vertebrate CNS and provides targets that may be key neuroendocrine regulators. The results provide a foundation for future work on dopaminergic

  19. Genome-wide profiling of gene expression in the epididymis of alpha-chlorohydrin-induced infertile rats using an oligonucleotide microarray

    PubMed Central

    2010-01-01

    Background As one of the chlorinated antifertility compounds, alpha-chlorohydrin (ACH) can inhibit glyceraldehyde-3-phosphate dehydrogenase (G3PDH) activity in epididymal sperm and affect sperm energy metabolism, maturation and fertilization, eventually leading to male infertility. Further studies demonstrated that the inhibitory effect of ACH on G3PDH is not only confined to epididymal sperm but also to the epididymis. Moreover, little investigation on gene expression changes in the epididymis after ACH treatment has been conducted. Therefore, gene expression studies may indicate new epididymal targets related to sperm maturation and fertility through the analysis of ACH-treated infertile animals. Methods Rats were treated with ACH for ten consecutive days, and then each male rat copulated with two female rats in proestrus. Then sperm maturation and other fertility parameters were analyzed. Furthermore, we identified epididymal-specific genes that are associated with fertility between control and ACH groups using an Affymetrix Rat 230 2.0 oligo-microarray. Finally, we performed RT-PCR analysis for several differentially expressed genes to validate the alteration in gene expression observed by oligonucleotide microarray. Results Among all the differentially expressed genes, we analyzed and screened the down-regulated genes associated with metabolism processes, which are considered the major targets of ACH action. Simultaneously, the genes that were up-regulated by chlorohydrin were detected. The genes that negatively regulate sperm maturation and fertility include apoptosis and immune-related genes and have not been reported previously. The overall results of PCR analysis for selected genes were consistent with the array data. Conclusions In this study, we have described the genome-wide profiles of gene expression in the epididymides of infertile rats induced by ACH, which could become potential epididymal specific targets for male contraception and infertility

  20. Genome-wide profiling of gene expression in the epididymis of alpha-chlorohydrin-induced infertile rats using an oligonucleotide microarray.

    PubMed

    Xie, Shuwu; Zhu, Yan; Ma, Li; Lu, Yingying; Zhou, Jieyun; Gui, Youlun; Cao, Lin

    2010-04-22

    As one of the chlorinated antifertility compounds, alpha-chlorohydrin (ACH) can inhibit glyceraldehyde-3-phosphate dehydrogenase (G3PDH) activity in epididymal sperm and affect sperm energy metabolism, maturation and fertilization, eventually leading to male infertility. Further studies demonstrated that the inhibitory effect of ACH on G3PDH is not only confined to epididymal sperm but also to the epididymis. Moreover, little investigation on gene expression changes in the epididymis after ACH treatment has been conducted. Therefore, gene expression studies may indicate new epididymal targets related to sperm maturation and fertility through the analysis of ACH-treated infertile animals. Rats were treated with ACH for ten consecutive days, and then each male rat copulated with two female rats in proestrus. Then sperm maturation and other fertility parameters were analyzed. Furthermore, we identified epididymal-specific genes that are associated with fertility between control and ACH groups using an Affymetrix Rat 230 2.0 oligo-microarray. Finally, we performed RT-PCR analysis for several differentially expressed genes to validate the alteration in gene expression observed by oligonucleotide microarray. Among all the differentially expressed genes, we analyzed and screened the down-regulated genes associated with metabolism processes, which are considered the major targets of ACH action. Simultaneously, the genes that were up-regulated by chlorohydrin were detected. The genes that negatively regulate sperm maturation and fertility include apoptosis and immune-related genes and have not been reported previously. The overall results of PCR analysis for selected genes were consistent with the array data. In this study, we have described the genome-wide profiles of gene expression in the epididymides of infertile rats induced by ACH, which could become potential epididymal specific targets for male contraception and infertility treatment.

  1. Transcription in space--environmental vs. genetic effects on differential immune gene expression.

    PubMed

    Lenz, Tobias L

    2015-09-01

    Understanding how organisms adapt to their local environment is one of the key goals in molecular ecology. Adaptation can be achieved through qualitative changes in the coding sequence and/or quantitative changes in gene expression, where the optimal dosage of a gene's product in a given environment is being selected for. Differences in gene expression among populations inhabiting distinct environments can be suggestive of locally adapted gene regulation and have thus been studied in different species (Whitehead & Crawford ; Hodgins-Davis & Townsend ). However, in contrast to a gene's coding sequence, its expression level at a given point in time may depend on various factors, including the current environment. Although critical for understanding the extent of local adaptation, it is usually difficult to disentangle the heritable differences in gene regulation from environmental effects. In this issue of Molecular Ecology, Stutz et al. () describe an experiment in which they reciprocally transplanted three-spined sticklebacks (Gasterosteus aculeatus) between independent pairs of small and large lakes. Their experimental design allows them to attribute differences in gene expression among sticklebacks either to lake of origin or destination lake. Interestingly, they find that translocated sticklebacks show a pattern of gene expression more similar to individuals from the destination lake than to individuals from the lake of origin, suggesting that expression of the targeted genes is more strongly regulated by environmental effects than by genetics. The environmental effect by itself is not entirely surprising; however, the relative extent of it is. Especially when put in the context of local adaptation and population differentiation, as done here, these findings cast a new light onto the heritability of differential gene expression and specifically its relative importance during population divergence and ultimately ecological speciation. © 2015 John Wiley & Sons

  2. The yeast Hot1 transcription factor is critical for activating a single target gene, STL1

    PubMed Central

    Bai, Chen; Tesker, Masha; Engelberg, David

    2015-01-01

    Transcription factors are commonly activated by signal transduction cascades and induce expression of many genes. They therefore play critical roles in determining the cell's fate. The yeast Hog1 MAP kinase pathway is believed to control the transcription of hundreds of genes via several transcription factors. To identify the bona fide target genes of Hog1, we inducibly expressed the spontaneously active variant Hog1D170A+F318L in cells lacking the Hog1 activator Pbs2. This system allowed monitoring the effects of Hog1 by itself. Expression of Hog1D170A+F318L in pbs2∆ cells imposed induction of just 105 and suppression of only 26 transcripts by at least twofold. We looked for the Hog1-responsive element within the promoter of the most highly induced gene, STL1 (88-fold). A novel Hog1 responsive element (HoRE) was identified and shown to be the direct target of the transcription factor Hot1. Unexpectedly, we could not find this HoRE in any other yeast promoter. In addition, the only gene whose expression was abolished in hot1∆ cells was STL1. Thus Hot1 is essential for transcription of just one gene, STL1. Hot1 may represent a class of transcription factors that are essential for transcription of a very few genes or even just one. PMID:25904326

  3. Genome-wide localization and expression profiling establish Sp2 as a sequence-specific transcription factor regulating vitally important genes

    PubMed Central

    Terrados, Gloria; Finkernagel, Florian; Stielow, Bastian; Sadic, Dennis; Neubert, Juliane; Herdt, Olga; Krause, Michael; Scharfe, Maren; Jarek, Michael; Suske, Guntram

    2012-01-01

    The transcription factor Sp2 is essential for early mouse development and for proliferation of mouse embryonic fibroblasts in culture. Yet its mechanisms of action and its target genes are largely unknown. In this study, we have combined RNA interference, in vitro DNA binding, chromatin immunoprecipitation sequencing and global gene-expression profiling to investigate the role of Sp2 for cellular functions, to define target sites and to identify genes regulated by Sp2. We show that Sp2 is important for cellular proliferation that it binds to GC-boxes and occupies proximal promoters of genes essential for vital cellular processes including gene expression, replication, metabolism and signalling. Moreover, we identified important key target genes and cellular pathways that are directly regulated by Sp2. Most significantly, Sp2 binds and activates numerous sequence-specific transcription factor and co-activator genes, and represses the whole battery of cholesterol synthesis genes. Our results establish Sp2 as a sequence-specific regulator of vitally important genes. PMID:22684502

  4. Targeted and genome-scale methylomics reveals gene body signatures in human cell lines

    PubMed Central

    Ball, Madeleine Price; Li, Jin Billy; Gao, Yuan; Lee, Je-Hyuk; LeProust, Emily; Park, In-Hyun; Xie, Bin; Daley, George Q.; Church, George M.

    2012-01-01

    Cytosine methylation, an epigenetic modification of DNA, is a target of growing interest for developing high throughput profiling technologies. Here we introduce two new, complementary techniques for cytosine methylation profiling utilizing next generation sequencing technology: bisulfite padlock probes (BSPPs) and methyl sensitive cut counting (MSCC). In the first method, we designed a set of ~10,000 BSPPs distributed over the ENCODE pilot project regions to take advantage of existing expression and chromatin immunoprecipitation data. We observed a pattern of low promoter methylation coupled with high gene body methylation in highly expressed genes. Using the second method, MSCC, we gathered genome-scale data for 1.4 million HpaII sites and confirmed that gene body methylation in highly expressed genes is a consistent phenomenon over the entire genome. Our observations highlight the usefulness of techniques which are not inherently or intentionally biased in favor of only profiling particular subsets like CpG islands or promoter regions. PMID:19329998

  5. Analysis of bHLH coding genes using gene co-expression network approach.

    PubMed

    Srivastava, Swati; Sanchita; Singh, Garima; Singh, Noopur; Srivastava, Gaurava; Sharma, Ashok

    2016-07-01

    Network analysis provides a powerful framework for the interpretation of data. It uses novel reference network-based metrices for module evolution. These could be used to identify module of highly connected genes showing variation in co-expression network. In this study, a co-expression network-based approach was used for analyzing the genes from microarray data. Our approach consists of a simple but robust rank-based network construction. The publicly available gene expression data of Solanum tuberosum under cold and heat stresses were considered to create and analyze a gene co-expression network. The analysis provide highly co-expressed module of bHLH coding genes based on correlation values. Our approach was to analyze the variation of genes expression, according to the time period of stress through co-expression network approach. As the result, the seed genes were identified showing multiple connections with other genes in the same cluster. Seed genes were found to be vary in different time periods of stress. These analyzed seed genes may be utilized further as marker genes for developing the stress tolerant plant species.

  6. A Novel Method for Gene-Specific Enhancement of Protein Translation by Targeting 5’UTRs of Selected Tumor Suppressors

    PubMed Central

    Master, Adam; Wójcicka, Anna; Giżewska, Kamilla; Popławski, Piotr; Williams, Graham R.; Nauman, Alicja

    2016-01-01

    Background Translational control is a mechanism of protein synthesis regulation emerging as an important target for new therapeutics. Naturally occurring microRNAs and synthetic small inhibitory RNAs (siRNAs) are the most recognized regulatory molecules acting via RNA interference. Surprisingly, recent studies have shown that interfering RNAs may also activate gene transcription via the newly discovered phenomenon of small RNA-induced gene activation (RNAa). Thus far, the small activating RNAs (saRNAs) have only been demonstrated as promoter-specific transcriptional activators. Findings We demonstrate that oligonucleotide-based trans-acting factors can also specifically enhance gene expression at the level of protein translation by acting at sequence-specific targets within the messenger RNA 5’-untranslated region (5’UTR). We designed a set of short synthetic oligonucleotides (dGoligos), specifically targeting alternatively spliced 5’UTRs in transcripts expressed from the THRB and CDKN2A suppressor genes. The in vitro translation efficiency of reporter constructs containing alternative TRβ1 5’UTRs was increased by up to more than 55-fold following exposure to specific dGoligos. Moreover, we found that the most folded 5’UTR has higher translational regulatory potential when compared to the weakly folded TRβ1 variant. This suggests such a strategy may be especially applied to enhance translation from relatively inactive transcripts containing long 5’UTRs of complex structure. Significance This report represents the first method for gene-specific translation enhancement using selective trans-acting factors designed to target specific 5’UTR cis-acting elements. This simple strategy may be developed further to complement other available methods for gene expression regulation including gene silencing. The dGoligo-mediated translation-enhancing approach has the potential to be transferred to increase the translation efficiency of any suitable target gene

  7. ck2-Dependent Phosphorylation of Progesterone Receptors (PR) on Ser81 Regulates PR-B Isoform-Specific Target Gene Expression in Breast Cancer Cells ▿

    PubMed Central

    Hagan, Christy R.; Regan, Tarah M.; Dressing, Gwen E.; Lange, Carol A.

    2011-01-01

    Progesterone receptors (PR) are critical mediators of mammary gland development and contribute to breast cancer progression. Progestin-induced rapid activation of cytoplasmic protein kinases leads to selective regulation of growth-promoting genes by phospho-PR species. Herein, we show that phosphorylation of PR Ser81 is ck2 dependent and progestin regulated in intact cells but also occurs in the absence of PR ligands when cells enter the G1/S phase of the cell cycle. T47D breast cancer cells stably expressing a PR-B mutant receptor that cannot be phosphorylated at Ser79/81 (S79/81A) formed fewer soft agar colonies. Regulation of selected genes by PR-B, but not PR-A, also required Ser79/81 phosphorylation for basal and/or progestin-regulated (BIRC3, HSD11β2, and HbEGF) expression. Additionally, wild-type (wt) PR-B, but not S79/81A mutant PR, was robustly recruited to a progesterone response element (PRE)-containing transcriptional enhancer region of BIRC3; abundant ck2 also associated with this region in cells expressing wt but not S79/81A PR. We conclude that phospho-Ser81 PR provides a platform for ck2 recruitment and regulation of selected PR-B target genes. Understanding how ligand-independent PRs function in the context of high levels of kinase activities characteristic of breast cancer is critical to understanding the basis of tumor-specific changes in gene expression and will speed the development of highly selective treatments. PMID:21518957

  8. GENE EXPRESSION NETWORKS

    EPA Science Inventory

    "Gene expression network" is the term used to describe the interplay, simple or complex, between two or more gene products in performing a specific cellular function. Although the delineation of such networks is complicated by the existence of multiple and subtle types of intera...

  9. Gene expression analysis of six GC-rich Gram-negative phytopathogens.

    PubMed

    Fu, Qing-Shan; Li, Feng; Chen, Ling-Ling

    2005-07-01

    Predicted highly expressed (PHX) genes are comparatively analyzed for six GC-rich Gram-negative phytopathogens, i.e., Ralstonia solanacearum, Agrobacterium tumefaciens, Xanthomonas campestris pv. campestris (Xcc), Xanthomonas axonopodis pv. citri (Xac), Pseudomonas syringae pv. tomato, and Xylella fastidiosa. Enzymes involved in energy metabolism, such as ATP synthase, and genes involved in TCA cycle, are PHX in most bacteria except X. fastidiosa, which prefers an anaerobic environment. Most pathogenicity-related factors, including flagellar proteins and some outer membrane proteins, are PHX, except that flagellar proteins are missing in X. fastidiosa which is spread by insects and does not need to move during invasion. Although type III secretion system apparatus are homologous to flagellar proteins, none of them is PHX, which support the viewpoint that the two types of genes have evolved independently. Furthermore, it is revealed that some biosynthesis-related enzymes are highly expressed in certain bacteria. The PHX genes may provide potential drug targets for the design of new bactericide.

  10. Non-coding RNAs as regulators of gene expression and epigenetics

    PubMed Central

    Kaikkonen, Minna U.; Lam, Michael T.Y.; Glass, Christopher K.

    2011-01-01

    Genome-wide studies have revealed that mammalian genomes are pervasively transcribed. This has led to the identification and isolation of novel classes of non-coding RNAs (ncRNAs) that influence gene expression by a variety of mechanisms. Here we review the characteristics and functions of regulatory ncRNAs in chromatin remodelling and at multiple levels of transcriptional and post-transcriptional regulation. We also describe the potential roles of ncRNAs in vascular biology and in mediating epigenetic modifications that might play roles in cardiovascular disease susceptibility. The emerging recognition of the diverse functions of ncRNAs in regulation of gene expression suggests that they may represent new targets for therapeutic intervention. PMID:21558279

  11. Synergistic and Dose-Controlled Regulation of Cellulase Gene Expression in Penicillium oxalicum.

    PubMed

    Li, Zhonghai; Yao, Guangshan; Wu, Ruimei; Gao, Liwei; Kan, Qinbiao; Liu, Meng; Yang, Piao; Liu, Guodong; Qin, Yuqi; Song, Xin; Zhong, Yaohua; Fang, Xu; Qu, Yinbo

    2015-09-01

    Filamentous fungus Penicillium oxalicum produces diverse lignocellulolytic enzymes, which are regulated by the combinations of many transcription factors. Here, a single-gene disruptant library for 470 transcription factors was constructed and systematically screened for cellulase production. Twenty transcription factors (including ClrB, CreA, XlnR, Ace1, AmyR, and 15 unknown proteins) were identified to play putative roles in the activation or repression of cellulase synthesis. Most of these regulators have not been characterized in any fungi before. We identified the ClrB, CreA, XlnR, and AmyR transcription factors as critical dose-dependent regulators of cellulase expression, the core regulons of which were identified by analyzing several transcriptomes and/or secretomes. Synergistic and additive modes of combinatorial control of each cellulase gene by these regulatory factors were achieved, and cellulase expression was fine-tuned in a proper and controlled manner. With one of these targets, the expression of the major intracellular β-glucosidase Bgl2 was found to be dependent on ClrB. The Bgl2-deficient background resulted in a substantial gene activation by ClrB and proved to be closely correlated with the relief of repression mediated by CreA and AmyR during cellulase induction. Our results also signify that probing the synergistic and dose-controlled regulation mechanisms of cellulolytic regulators and using it for reconstruction of expression regulation network (RERN) may be a promising strategy for cellulolytic fungi to develop enzyme hyper-producers. Based on our data, ClrB was identified as focal point for the synergistic activation regulation of cellulase expression by integrating cellulolytic regulators and their target genes, which refined our understanding of transcriptional-regulatory network as a "seesaw model" in which the coordinated regulation of cellulolytic genes is established by counteracting activators and repressors.

  12. Synergistic and Dose-Controlled Regulation of Cellulase Gene Expression in Penicillium oxalicum

    PubMed Central

    Li, Zhonghai; Yao, Guangshan; Wu, Ruimei; Gao, Liwei; Kan, Qinbiao; Liu, Meng; Yang, Piao; Liu, Guodong; Qin, Yuqi; Song, Xin; Zhong, Yaohua; Fang, Xu; Qu, Yinbo

    2015-01-01

    Filamentous fungus Penicillium oxalicum produces diverse lignocellulolytic enzymes, which are regulated by the combinations of many transcription factors. Here, a single-gene disruptant library for 470 transcription factors was constructed and systematically screened for cellulase production. Twenty transcription factors (including ClrB, CreA, XlnR, Ace1, AmyR, and 15 unknown proteins) were identified to play putative roles in the activation or repression of cellulase synthesis. Most of these regulators have not been characterized in any fungi before. We identified the ClrB, CreA, XlnR, and AmyR transcription factors as critical dose-dependent regulators of cellulase expression, the core regulons of which were identified by analyzing several transcriptomes and/or secretomes. Synergistic and additive modes of combinatorial control of each cellulase gene by these regulatory factors were achieved, and cellulase expression was fine-tuned in a proper and controlled manner. With one of these targets, the expression of the major intracellular β-glucosidase Bgl2 was found to be dependent on ClrB. The Bgl2-deficient background resulted in a substantial gene activation by ClrB and proved to be closely correlated with the relief of repression mediated by CreA and AmyR during cellulase induction. Our results also signify that probing the synergistic and dose-controlled regulation mechanisms of cellulolytic regulators and using it for reconstruction of expression regulation network (RERN) may be a promising strategy for cellulolytic fungi to develop enzyme hyper-producers. Based on our data, ClrB was identified as focal point for the synergistic activation regulation of cellulase expression by integrating cellulolytic regulators and their target genes, which refined our understanding of transcriptional-regulatory network as a “seesaw model” in which the coordinated regulation of cellulolytic genes is established by counteracting activators and repressors. PMID:26360497

  13. Gene Expression Profiling of Liver Cancer Stem Cells by RNA-Sequencing

    PubMed Central

    Lam, Chi Tat; Ng, Michael N. P.; Yu, Wan Ching; Lau, Joyce; Wan, Timothy; Wang, Xiaoqi; Yan, Zhixiang; Liu, Hang; Fan, Sheung Tat

    2012-01-01

    Background Accumulating evidence supports that tumor growth and cancer relapse are driven by cancer stem cells. Our previous work has demonstrated the existence of CD90+ liver cancer stem cells (CSCs) in hepatocellular carcinoma (HCC). Nevertheless, the characteristics of these cells are still poorly understood. In this study, we employed a more sensitive RNA-sequencing (RNA-Seq) to compare the gene expression profiling of CD90+ cells sorted from tumor (CD90+CSCs) with parallel non-tumorous liver tissues (CD90+NTSCs) and elucidate the roles of putative target genes in hepatocarcinogenesis. Methodology/Principal Findings CD90+ cells were sorted respectively from tumor and adjacent non-tumorous human liver tissues using fluorescence-activated cell sorting. The amplified RNAs of CD90+ cells from 3 HCC patients were subjected to RNA-Seq analysis. A differential gene expression profile was established between CD90+CSCs and CD90+NTSCs, and validated by quantitative real-time PCR (qRT-PCR) on the same set of amplified RNAs, and further confirmed in an independent cohort of 12 HCC patients. Five hundred genes were differentially expressed (119 up-regulated and 381 down-regulated genes) between CD90+CSCs and CD90+NTSCs. Gene ontology analysis indicated that the over-expressed genes in CD90+CSCs were associated with inflammation, drug resistance and lipid metabolism. Among the differentially expressed genes, glypican-3 (GPC3), a member of glypican family, was markedly elevated in CD90+CSCs compared to CD90+NTSCs. Immunohistochemistry demonstrated that GPC3 was highly expressed in forty-two human liver tumor tissues but absent in adjacent non-tumorous liver tissues. Flow cytometry indicated that GPC3 was highly expressed in liver CD90+CSCs and mature cancer cells in liver cancer cell lines and human liver tumor tissues. Furthermore, GPC3 expression was positively correlated with the number of CD90+CSCs in liver tumor tissues. Conclusions/Significance The identified genes

  14. Programmable control of bacterial gene expression with the combined CRISPR and antisense RNA system

    PubMed Central

    Lee, Young Je; Hoynes-O'Connor, Allison; Leong, Matthew C.; Moon, Tae Seok

    2016-01-01

    A central goal of synthetic biology is to implement diverse cellular functions by predictably controlling gene expression. Though research has focused more on protein regulators than RNA regulators, recent advances in our understanding of RNA folding and functions have motivated the use of RNA regulators. RNA regulators provide an advantage because they are easier to design and engineer than protein regulators, potentially have a lower burden on the cell and are highly orthogonal. Here, we combine the CRISPR system from Streptococcus pyogenes and synthetic antisense RNAs (asRNAs) in Escherichia coli strains to repress or derepress a target gene in a programmable manner. Specifically, we demonstrate for the first time that the gene target repressed by the CRISPR system can be derepressed by expressing an asRNA that sequesters a small guide RNA (sgRNA). Furthermore, we demonstrate that tunable levels of derepression can be achieved (up to 95%) by designing asRNAs that target different regions of a sgRNA and by altering the hybridization free energy of the sgRNA–asRNA complex. This new system, which we call the combined CRISPR and asRNA system, can be used to reversibly repress or derepress multiple target genes simultaneously, allowing for rational reprogramming of cellular functions. PMID:26837577

  15. Evaluation of RNAi and CRISPR technologies by large-scale gene expression profiling in the Connectivity Map.

    PubMed

    Smith, Ian; Greenside, Peyton G; Natoli, Ted; Lahr, David L; Wadden, David; Tirosh, Itay; Narayan, Rajiv; Root, David E; Golub, Todd R; Subramanian, Aravind; Doench, John G

    2017-11-01

    The application of RNA interference (RNAi) to mammalian cells has provided the means to perform phenotypic screens to determine the functions of genes. Although RNAi has revolutionized loss-of-function genetic experiments, it has been difficult to systematically assess the prevalence and consequences of off-target effects. The Connectivity Map (CMAP) represents an unprecedented resource to study the gene expression consequences of expressing short hairpin RNAs (shRNAs). Analysis of signatures for over 13,000 shRNAs applied in 9 cell lines revealed that microRNA (miRNA)-like off-target effects of RNAi are far stronger and more pervasive than generally appreciated. We show that mitigating off-target effects is feasible in these datasets via computational methodologies to produce a consensus gene signature (CGS). In addition, we compared RNAi technology to clustered regularly interspaced short palindromic repeat (CRISPR)-based knockout by analysis of 373 single guide RNAs (sgRNAs) in 6 cells lines and show that the on-target efficacies are comparable, but CRISPR technology is far less susceptible to systematic off-target effects. These results will help guide the proper use and analysis of loss-of-function reagents for the determination of gene function.

  16. Comparison of CRISPR/Cas9 expression constructs for efficient targeted mutagenesis in rice.

    PubMed

    Mikami, Masafumi; Toki, Seiichi; Endo, Masaki

    2015-08-01

    The CRISPR/Cas9 system is an efficient tool used for genome editing in a variety of organisms. Despite several recent reports of successful targeted mutagenesis using the CRISPR/Cas9 system in plants, in each case the target gene of interest, the Cas9 expression system and guide-RNA (gRNA) used, and the tissues used for transformation and subsequent mutagenesis differed, hence the reported frequencies of targeted mutagenesis cannot be compared directly. Here, we evaluated mutation frequency in rice using different Cas9 and/or gRNA expression cassettes under standardized experimental conditions. We introduced Cas9 and gRNA expression cassettes separately or sequentially into rice calli, and assessed the frequency of mutagenesis at the same endogenous targeted sequences. Mutation frequencies differed significantly depending on the Cas9 expression cassette used. In addition, a gRNA driven by the OsU6 promoter was superior to one driven by the OsU3 promoter. Using an all-in-one expression vector harboring the best combined Cas9/gRNA expression cassette resulted in a much improved frequency of targeted mutagenesis in rice calli, and bi-allelic mutant plants were produced in the T0 generation. The approach presented here could be adapted to optimize the construction of Cas9/gRNA cassettes for genome editing in a variety of plants.

  17. PhotoMorphs™: A Novel Light-Activated Reagent for Controlling Gene Expression in Zebrafish

    PubMed Central

    Tomasini, Amber J.; Schuler, Aaron D.; Zebala, John A.; Mayer, Alan N.

    2009-01-01

    Manipulating gene expression in zebrafish is critical for exploiting the full potential of this vertebrate model organism. Morpholino oligos are the most commonly employed antisense technology for knocking down gene expression. However, morpholinos suffer from a lack of control over the timing and location of knockdown. In this report, we describe a novel light-activatable knockdown reagent called PhotoMorph™. PhotoMorphs can be generated from existing morpholinos by hybridization with a complementary caging strand containing a photocleavable linkage. The caging strand neutralizes the morpholino activity until irradiation of the PhotoMorph with UV light releases the morpholino. We generated PhotoMorphs to target genes encoding enhanced green fluorescent protein (EGFP), No tail, and E-cadherin to illustrate the utility of this approach. Temporal control of gene expression with PhotoMorphs permitted us to circumvent the early lethal phenotype of E-cadherin knockdown. A splice-blocking PhotoMorph directed to the rheb gene showed light-dependent gene knockdown up to 72 hpf. PhotoMorphs thus offer a new class of laboratory reagents suitable for the spatiotemporal control of gene expression in the zebrafish. PMID:19644983

  18. Aberrant expression of NKL homeobox gene HLX in Hodgkin lymphoma.

    PubMed

    Nagel, Stefan; Pommerenke, Claudia; Meyer, Corinna; Kaufmann, Maren; MacLeod, Roderick A F; Drexler, Hans G

    2018-03-06

    NKL homeobox genes are basic regulators of cell and tissue differentiation, many acting as oncogenes in T-cell leukemia. Recently, we described an hematopoietic NKL-code comprising six particular NKL homeobox genes expressed in hematopoietic stem cells and lymphoid progenitors, unmasking their physiological roles in the development of these cell types. Hodgkin lymphoma (HL) is a B-cell malignancy showing aberrant activity of several developmental genes resulting in disturbed B-cell differentiation. To examine potential concordances in abnormal lymphoid differentiation of T- and B-cell malignancies we analyzed the expression of the hematopoietic NKL-code associated genes in HL, comprising HHEX, HLX, MSX1, NKX2-3, NKX3-1 and NKX6-3. Our approach revealed aberrant HLX activity in 8 % of classical HL patients and additionally in HL cell line L-540. Accordingly, to identify upstream regulators and downstream target genes of HLX we used L-540 cells as a model and performed chromosome and genome analyses, comparative expression profiling and functional assays via knockdown and overexpression experiments therein. These investigations excluded chromosomal rearrangements of the HLX locus at 1q41 and demonstrated that STAT3 operated directly as transcriptional activator of the HLX gene. Moreover, subcellular analyses showed highly enriched STAT3 protein in the nucleus of L-540 cells which underwent cytoplasmic translocation by repressing deacetylation. Finally, HLX inhibited transcription of B-cell differentiation factors MSX1, BCL11A and SPIB and of pro-apoptotic factor BCL2L11/BIM, thereby suppressing Etoposide-induced cell death. Collectively, we propose that aberrantly expressed NKL homeobox gene HLX is part of a pathological gene network in HL, driving deregulated B-cell differentiation and survival.

  19. TITER AND PRODUCT AFFECTS THE DISTRIBUTION OF GENE EXPRESSION AFTER INTRAPUTAMINAL CONVECTION-ENHANCED DELIVERY

    PubMed Central

    Emborg, Marina E.; Hurley, Samuel A.; Joers, Valerie; Tromp, Do P.M.; Swanson, Christine R.; Ohshima-Hosoyama, Sachiko; Bondarenko, Viktorya; Cummisford, Kyle; Sonnemans, Marc; Hermening, Stephan; Blits, Bas; Alexander, Andrew L.

    2014-01-01

    Background Efficacy and safety of intracerebral gene therapy for brain disorders, like Parkinson’s disease, depends on appropriate distribution of gene expression. Objectives To assess if the distribution of gene expression is affected by vector titer and protein type. Methods Four adult macaque monkeys seronegative for adeno-associated virus 5 (AAV5) received in the right and left ventral postcommisural putamen 30μl inoculation of a high or low titer suspension of AAV5 encoding glial derived neurotrophic factor (GDNF) or green fluorescent protein (GFP). Inoculations were performed using convection enhanced delivery and intraoperative MRI (IMRI). Results IMRI confirmed targeting and infusion cloud irradiating from the catheter tip into surrounding area. Postmortem analysis six weeks after surgery revealed GFP and GDNF expression ipsilateral to the injection side that had a titer-dependent distribution. GFP and GDNF expression was also observed in fibers in the Substantia Nigra (SN) pars reticulata (pr), demonstrating anterograde transport. Few GFP-positive neurons were present in the SN pars compacta (pc), possibly by direct retrograde transport of the vector. GDNF was present in many SNpc and SNpr neurons. Conclusions After controlling for target and infusate volume, intracerebral distribution of gene product is affected by vector titer and product biology. PMID:24943657

  20. Differential Gene Expression in Primary Human Skin Keratinocytes and Fibroblasts in Response to Ionizing Radiation

    PubMed Central

    Warters, Raymond L.; Packard, Ann T.; Kramer, Gwen F.; Gaffney, David K.; Moos, Philip J.

    2009-01-01

    Although skin is usually exposed during human exposures to ionizing radiation, there have been no thorough examinations of the transcriptional response of skin fibroblasts and keratinocytes to radiation. The transcriptional response of quiescent primary fibroblasts and keratinocytes exposed to from 10 cGy to 5 Gy and collected 4 h after treatment was examined. RNA was isolated and examined by microarray analysis for changes in the levels of gene expression. Exposure to ionizing radiation altered the expression of 279 genes across both cell types. Changes in RNA expression could be arranged into three main categories: (1) changes in keratinocytes but not in fibroblasts, (2) changes in fibroblasts but not in keratinocytes, and (3) changes in both. All of these changes were primarily of p53 target genes. Similar radiation-induced changes were induced in immortalized fibroblasts or keratinocytes. In separate experiments, protein was collected and analyzed by Western blotting for expression of proteins observed in microarray experiments to be overexpressed at the mRNA level. Both Q-PCR and Western blot analysis experiments validated these transcription changes. Our results are consistent with changes in the expression of p53 target genes as indicating the magnitude of cell responses to ionizing radiation. PMID:19580510

  1. Two White Spot Syndrome Virus MicroRNAs Target the Dorsal Gene To Promote Virus Infection in Marsupenaeus japonicus Shrimp.

    PubMed

    Ren, Qian; Huang, Xin; Cui, Yalei; Sun, Jiejie; Wang, Wen; Zhang, Xiaobo

    2017-04-15

    In eukaryotes, microRNAs (miRNAs) serve as regulators of many biological processes, including virus infection. An miRNA can generally target diverse genes during virus-host interactions. However, the regulation of gene expression by multiple miRNAs has not yet been extensively explored during virus infection. This study found that the Spaztle (Spz)-Toll-Dorsal-antilipopolysaccharide factor (ALF) signaling pathway plays a very important role in antiviral immunity against invasion of white spot syndrome virus (WSSV) in shrimp ( Marsupenaeus japonicus ). Dorsal , the central gene in the Toll pathway, was targeted by two viral miRNAs (WSSV-miR-N13 and WSSV-miR-N23) during WSSV infection. The regulation of Dorsal expression by viral miRNAs suppressed the Spz-Toll-Dorsal-ALF signaling pathway in shrimp in vivo , leading to virus infection. Our study contributes novel insights into the viral miRNA-mediated Toll signaling pathway during the virus-host interaction. IMPORTANCE An miRNA can target diverse genes during virus-host interactions. However, the regulation of gene expression by multiple miRNAs during virus infection has not yet been extensively explored. The results of this study indicated that the shrimp Dorsal gene, the central gene in the Toll pathway, was targeted by two viral miRNAs during infection with white spot syndrome virus. Regulation of Dorsal expression by viral miRNAs suppressed the Spz-Toll-Dorsal-ALF signaling pathway in shrimp in vivo , leading to virus infection. Our study provides new insight into the viral miRNA-mediated Toll signaling pathway in virus-host interactions. Copyright © 2017 American Society for Microbiology.

  2. The effect of mutation on Rhodococcus equi virulence plasmid gene expression and mouse virulence.

    PubMed

    Ren, Jun; Prescott, John F

    2004-11-15

    An 81 kb virulence plasmid containing a pathogenicity island (PI) plays a crucial role in the pathogenesis of Rhodococcus equi pneumonia in foals but its specific function in virulence and regulation of plasmid-encoded virulence genes is unclear. Using a LacZ selection marker developed for R. equi in this study, in combination with an apramycin resistance gene, an efficient two-stage homologous recombination targeted gene mutation procedure was used to mutate three virulence plasmid genes, a LysR regulatory gene homologue (ORF4), a ResD-like two-component response regulator homologue (ORF8), and a gene (ORF10) of unknown function that is highly expressed by R. equi inside macrophages, as well as the chromosomal gene operon, phoPR. Virulence testing by liver clearance after intravenous injection in mice showed that the ORF4 and ORF8 mutants were fully attenuated, that the phoPR mutant was hypervirulent, and that virulence of the ORF10 mutant remained unchanged. A virulence plasmid DNA microarray was used to compare the plasmid gene expression profile of each of the four gene-targeted mutants against the parental R. equi strain. Changes were limited to PI genes and gene induction was observed for all mutants, suggesting that expression of virulence plasmid genes is dominated by a negative regulatory network. The finding of attenuation of ORF4 and ORF8 mutants despite enhanced transcription of vapA suggests that factors other than VapA are important for full expression of virulence. ORF1, a putative Lsr antigen gene, was strongly and similarly induced in all mutants, implying a common regulatory pathway affecting this gene for all four mutated genes. ORF8 is apparently the centre of this common pathway. Two distinct highly correlated gene induction patterns were observed, that of the ORF4 and ORF8 mutants, and that of the ORF10 and phoPR mutants. The gene induction pattern distinguishing these two groups paralleled their virulence in mice.

  3. Genome-wide DNA methylation drives human embryonic stem cell erythropoiesis by remodeling gene expression dynamics.

    PubMed

    Liu, Zhijing; Feng, Qiang; Sun, Pengpeng; Lu, Yan; Yang, Minlan; Zhang, Xiaowei; Jin, Xiangshu; Li, Yulin; Lu, Shi-Jiang; Quan, Chengshi

    2017-12-01

    To investigate the role of DNA methylation during erythrocyte production by human embryonic stem cells (hESCs). We employed an erythroid differentiation model from hESCs, and then tracked the genome-wide DNA methylation maps and gene expression patterns through an Infinium HumanMethylation450K BeadChip and an Ilumina Human HT-12 v4 Expression Beadchip, respectively. A negative correlation between DNA methylation and gene expression was substantially enriched during the later differentiation stage and was present in both the promoter and the gene body. Moreover, erythropoietic genes with differentially methylated CpG sites that were primarily enriched in nonisland regions were upregulated, and demethylation of their gene bodies was associated with the presence of enhancers and DNase I hypersensitive sites. Finally, the components of JAK-STAT-NF-κB signaling were DNA hypomethylated and upregulated, which targets the key genes for erythropoiesis. Erythroid lineage commitment by hESCs requires genome-wide DNA methylation modifications to remodel gene expression dynamics.

  4. Arabidopsis gene expression patterns during spaceflight

    NASA Astrophysics Data System (ADS)

    Paul, A.-L.; Ferl, R. J.

    The exposure of Arabidopsis thaliana (Arabidopsis) plants to spaceflight environments resulted in the differential expression of hundreds of genes. A 5 day mission on orbiter Columbia in 1999 (STS-93) carried transgenic Arabidopsis plants engineered with a transgene composed of the alcohol dehydrogenase (Adh) gene promoter linked to the β -Glucuronidase (GUS) reporter gene. The plants were used to evaluate the effects of spaceflight on two fronts. First, expression patterns visualized with the Adh/GUS transgene were used to address specifically the possibility that spaceflight induces a hypoxic stress response, and to assess whether any spaceflight response was similar to control terrestrial hypoxia-induced gene expression patterns. (Paul et al., Plant Physiol. 2001, 126:613). Second, genome-wide patterns of native gene expression were evaluated utilizing the Affymetrix ATH1 GeneChip? array of 8,000 Arabidopsis genes. As a control for the veracity of the array analyses, a selection of genes identified with the arrays was further characterized with quantitative Real-Time RT PCR (ABI - TaqmanTM). Comparison of the patterns of expression for arrays of hybridized with RNA isolated from plants exposed to spaceflight compared to the control arrays revealed hundreds of genes that were differentially expressed in response to spaceflight, yet most genes that are hallmarks of hypoxic stress were unaffected. These results will be discussed in light of current models for plant responses to the spaceflight environment, and with regard to potential future flight opportunities.

  5. Combined antitumor gene therapy with herpes simplex virus-thymidine kinase and short hairpin RNA specific for mammalian target of rapamycin.

    PubMed

    Woo, Ha-Na; Lee, Won Il; Kim, Ji Hyun; Ahn, Jeonghyun; Han, Jeong Hee; Lim, Sue Yeon; Lee, Won Woo; Lee, Heuiran

    2015-12-01

    A proof-of-concept study is presented using dual gene therapy that employed a small hairpin RNA (shRNA) specific for mammalian target of rapamycin (mTOR) and a herpes simplex virus-thymidine kinase (HSV-TK) gene to inhibit the growth of tumors. Recombinant adeno-associated virus (rAAV) vectors containing a mutant TK gene (sc39TK) were transduced into HeLa cells, and the prodrug ganciclovir (GCV) was administered to establish a suicide gene-therapy strategy. Additionally, rAAV vectors expressing an mTOR-targeted shRNA were employed to suppress mTOR-dependent tumor growth. GCV selectively induced death in tumor cells expressing TK, and the mTOR-targeted shRNA altered the cell cycle to impair tumor growth. Combining the TK-GCV system with mTOR inhibition suppressed tumor growth to a greater extent than that achieved with either treatment alone. Furthermore, HSV-TK expression and mTOR inhibition did not mutually interfere with each other. In conclusion, gene therapy that combines the TK-GCV system and mTOR inhibition shows promise as a novel strategy for cancer therapy.

  6. oPOSSUM: identification of over-represented transcription factor binding sites in co-expressed genes

    PubMed Central

    Ho Sui, Shannan J.; Mortimer, James R.; Arenillas, David J.; Brumm, Jochen; Walsh, Christopher J.; Kennedy, Brian P.; Wasserman, Wyeth W.

    2005-01-01

    Targeted transcript profiling studies can identify sets of co-expressed genes; however, identification of the underlying functional mechanism(s) is a significant challenge. Established methods for the analysis of gene annotations, particularly those based on the Gene Ontology, can identify functional linkages between genes. Similar methods for the identification of over-represented transcription factor binding sites (TFBSs) have been successful in yeast, but extension to human genomics has largely proved ineffective. Creation of a system for the efficient identification of common regulatory mechanisms in a subset of co-expressed human genes promises to break a roadblock in functional genomics research. We have developed an integrated system that searches for evidence of co-regulation by one or more transcription factors (TFs). oPOSSUM combines a pre-computed database of conserved TFBSs in human and mouse promoters with statistical methods for identification of sites over-represented in a set of co-expressed genes. The algorithm successfully identified mediating TFs in control sets of tissue-specific genes and in sets of co-expressed genes from three transcript profiling studies. Simulation studies indicate that oPOSSUM produces few false positives using empirically defined thresholds and can tolerate up to 50% noise in a set of co-expressed genes. PMID:15933209

  7. Pleiotropic and Epistatic Network-Based Discovery: Integrated Networks for Target Gene Discovery

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Weighill, Deborah; Jones, Piet; Shah, Manesh

    Biological organisms are complex systems that are composed of functional networks of interacting molecules and macro-molecules. Complex phenotypes are the result of orchestrated, hierarchical, heterogeneous collections of expressed genomic variants. However, the effects of these variants are the result of historic selective pressure and current environmental and epigenetic signals, and, as such, their co-occurrence can be seen as genome-wide correlations in a number of different manners. Biomass recalcitrance (i.e., the resistance of plants to degradation or deconstruction, which ultimately enables access to a plant's sugars) is a complex polygenic phenotype of high importance to biofuels initiatives. This study makes usemore » of data derived from the re-sequenced genomes from over 800 different Populus trichocarpa genotypes in combination with metabolomic and pyMBMS data across this population, as well as co-expression and co-methylation networks in order to better understand the molecular interactions involved in recalcitrance, and identify target genes involved in lignin biosynthesis/degradation. A Lines Of Evidence (LOE) scoring system is developed to integrate the information in the different layers and quantify the number of lines of evidence linking genes to target functions. This new scoring system was applied to quantify the lines of evidence linking genes to lignin-related genes and phenotypes across the network layers, and allowed for the generation of new hypotheses surrounding potential new candidate genes involved in lignin biosynthesis in P. trichocarpa, including various AGAMOUS-LIKE genes. Lastly, the resulting Genome Wide Association Study networks, integrated with Single Nucleotide Polymorphism (SNP) correlation, co-methylation, and co-expression networks through the LOE scores are proving to be a powerful approach to determine the pleiotropic and epistatic relationships underlying cellular functions and, as such, the molecular basis for

  8. Pleiotropic and Epistatic Network-Based Discovery: Integrated Networks for Target Gene Discovery

    DOE PAGES

    Weighill, Deborah; Jones, Piet; Shah, Manesh; ...

    2018-05-11

    Biological organisms are complex systems that are composed of functional networks of interacting molecules and macro-molecules. Complex phenotypes are the result of orchestrated, hierarchical, heterogeneous collections of expressed genomic variants. However, the effects of these variants are the result of historic selective pressure and current environmental and epigenetic signals, and, as such, their co-occurrence can be seen as genome-wide correlations in a number of different manners. Biomass recalcitrance (i.e., the resistance of plants to degradation or deconstruction, which ultimately enables access to a plant's sugars) is a complex polygenic phenotype of high importance to biofuels initiatives. This study makes usemore » of data derived from the re-sequenced genomes from over 800 different Populus trichocarpa genotypes in combination with metabolomic and pyMBMS data across this population, as well as co-expression and co-methylation networks in order to better understand the molecular interactions involved in recalcitrance, and identify target genes involved in lignin biosynthesis/degradation. A Lines Of Evidence (LOE) scoring system is developed to integrate the information in the different layers and quantify the number of lines of evidence linking genes to target functions. This new scoring system was applied to quantify the lines of evidence linking genes to lignin-related genes and phenotypes across the network layers, and allowed for the generation of new hypotheses surrounding potential new candidate genes involved in lignin biosynthesis in P. trichocarpa, including various AGAMOUS-LIKE genes. Lastly, the resulting Genome Wide Association Study networks, integrated with Single Nucleotide Polymorphism (SNP) correlation, co-methylation, and co-expression networks through the LOE scores are proving to be a powerful approach to determine the pleiotropic and epistatic relationships underlying cellular functions and, as such, the molecular basis for

  9. Hippocampal chromatin-modifying enzymes are pivotal for scopolamine-induced synaptic plasticity gene expression changes and memory impairment.

    PubMed

    Singh, Padmanabh; Konar, Arpita; Kumar, Ashish; Srivas, Sweta; Thakur, Mahendra K

    2015-08-01

    The amnesic potential of scopolamine is well manifested through synaptic plasticity gene expression changes and behavioral paradigms of memory impairment. However, the underlying mechanism remains obscure and consequently ideal therapeutic target is lacking. In this context, chromatin-modifying enzymes, which regulate memory gene expression changes, deserve major attention. Therefore, we analyzed the expression of chromatin-modifying enzymes and recovery potential of enzyme modulators in scopolamine-induced amnesia. Scopolamine administration drastically up-regulated DNA methyltransferases (DNMT1) and HDAC2 expression while CREB-binding protein (CBP), DNMT3a and DNMT3b remained unaffected. HDAC inhibitor sodium butyrate and DNMT inhibitor Aza-2'deoxycytidine recovered scopolamine-impaired hippocampal-dependent memory consolidation with concomitant increase in the expression of synaptic plasticity genes Brain-derived neurotrophic factor (BDNF) and Arc and level of histone H3K9 and H3K14 acetylation and decrease in DNA methylation level. Sodium butyrate showed more pronounced effect than Aza-2'deoxycytidine and their co-administration did not exhibit synergistic effect on gene expression. Taken together, we showed for the first time that scopolamine-induced up-regulation of chromatin-modifying enzymes, HDAC2 and DNMT1, leads to gene expression changes and consequent decline in memory consolidation. Our findings on the action of scopolamine as an epigenetic modulator can pave a path for ideal therapeutic targets. We propose the following putative pathway for scopolamine-mediated memory impairment; scopolamine up-regulates hippocampal DNMT1 and HDAC2 expression, induces methylation and deacetylation of BDNF and Arc promoter, represses gene expression and eventually impairs memory consolidation. On the other hand, Aza-2 and NaB inhibit DNMT1 and HDAC2 respectively, up-regulate BDNF and Arc expression and recover memory consolidation. We elucidate the action of

  10. Targeted Antiangiogenesis Gene Therapy Using Targeted Cationic Microbubbles Conjugated with CD105 Antibody Compared with Untargeted Cationic and Neutral Microbubbles

    PubMed Central

    Zhou, Yu; Gu, Haitao; Xu, Yan; Li, Fan; Kuang, Shaojing; Wang, Zhigang; Zhou, Xiyuan; Ma, Huafeng; Li, Pan; Zheng, Yuanyi; Ran, Haitao; Jian, Jia; Zhao, Yajing; Song, Weixiang; Wang, Qiushi; Wang, Dong

    2015-01-01

    Objective This study aimed to develop targeted cationic microbubbles conjugated with a CD105 antibody (CMB105) for use in targeted vascular endothelial cell gene therapy and ultrasound imaging. We compared the results with untargeted cationic microbubbles (CMB) and neutral microbubbles (NMB). Methods CMB105 were prepared and compared with untargeted CMB and NMB. First, the microbubbles were characterized in terms of size, zeta-potential, antibody binding ability and plasmid DNA loading capacity. A tumor model of subcutaneous breast cancer in nude mice was used for our experiments. The ability of different types of microbubbles to target HUVECs in vitro and tumor neovascularization in vivo was measured. The endostatin gene was selected for its outstanding antiangiogenesis effect. For in vitro experiments, the transfection efficiency and cell cycle were analyzed using flow cytometry, and the transcription and expression of endostatin were measured by qPCR and Western blotting, respectively. Vascular tube cavity formation and tumor cell invasion were used to evaluate the antiangiogenesis gene therapy efficiency in vitro. Tumors were exposed to ultrasound irradiation with different types of microbubbles, and the gene therapy effects were investigated by detecting apoptosis induction and changes in tumor volume. Results CMB105 and CMB differed significantly from NMB in terms of zeta-potential, and the DNA loading capacities were 16.76±1.75 μg, 18.21±1.22 μg, and 0.48±0.04 μg per 5×108 microbubbles, respectively. The charge coupling of plasmid DNA to CMB105 was not affected by the presence of the CD105 antibody. Both CMB105 and CMB could target to HUVECs in vitro, whereas only CMB105 could target to tumor neovascularization in vivo. In in vitro experiments, the transfection efficiency of CMB105 was 24.7-fold higher than the transfection efficiency of NMB and 1.47-fold higher than the transfection efficiency of CMB (P<0.05). With ultrasound-targeted microbubble

  11. Targeted antiangiogenesis gene therapy using targeted cationic microbubbles conjugated with CD105 antibody compared with untargeted cationic and neutral microbubbles.

    PubMed

    Zhou, Yu; Gu, Haitao; Xu, Yan; Li, Fan; Kuang, Shaojing; Wang, Zhigang; Zhou, Xiyuan; Ma, Huafeng; Li, Pan; Zheng, Yuanyi; Ran, Haitao; Jian, Jia; Zhao, Yajing; Song, Weixiang; Wang, Qiushi; Wang, Dong

    2015-01-01

    This study aimed to develop targeted cationic microbubbles conjugated with a CD105 antibody (CMB105) for use in targeted vascular endothelial cell gene therapy and ultrasound imaging. We compared the results with untargeted cationic microbubbles (CMB) and neutral microbubbles (NMB). CMB105 were prepared and compared with untargeted CMB and NMB. First, the microbubbles were characterized in terms of size, zeta-potential, antibody binding ability and plasmid DNA loading capacity. A tumor model of subcutaneous breast cancer in nude mice was used for our experiments. The ability of different types of microbubbles to target HUVECs in vitro and tumor neovascularization in vivo was measured. The endostatin gene was selected for its outstanding antiangiogenesis effect. For in vitro experiments, the transfection efficiency and cell cycle were analyzed using flow cytometry, and the transcription and expression of endostatin were measured by qPCR and Western blotting, respectively. Vascular tube cavity formation and tumor cell invasion were used to evaluate the antiangiogenesis gene therapy efficiency in vitro. Tumors were exposed to ultrasound irradiation with different types of microbubbles, and the gene therapy effects were investigated by detecting apoptosis induction and changes in tumor volume. CMB105 and CMB differed significantly from NMB in terms of zeta-potential, and the DNA loading capacities were 16.76±1.75 μg, 18.21±1.22 μg, and 0.48±0.04 μg per 5×10(8) microbubbles, respectively. The charge coupling of plasmid DNA to CMB105 was not affected by the presence of the CD105 antibody. Both CMB105 and CMB could target to HUVECs in vitro, whereas only CMB105 could target to tumor neovascularization in vivo. In in vitro experiments, the transfection efficiency of CMB105 was 24.7-fold higher than the transfection efficiency of NMB and 1.47-fold higher than the transfection efficiency of CMB (P<0.05). With ultrasound-targeted microbubble destruction (UTMD

  12. Identification of a set of genes showing regionally enriched expression in the mouse brain

    PubMed Central

    D'Souza, Cletus A; Chopra, Vikramjit; Varhol, Richard; Xie, Yuan-Yun; Bohacec, Slavita; Zhao, Yongjun; Lee, Lisa LC; Bilenky, Mikhail; Portales-Casamar, Elodie; He, An; Wasserman, Wyeth W; Goldowitz, Daniel; Marra, Marco A; Holt, Robert A; Simpson, Elizabeth M; Jones, Steven JM

    2008-01-01

    Background The Pleiades Promoter Project aims to improve gene therapy by designing human mini-promoters (< 4 kb) that drive gene expression in specific brain regions or cell-types of therapeutic interest. Our goal was to first identify genes displaying regionally enriched expression in the mouse brain so that promoters designed from orthologous human genes can then be tested to drive reporter expression in a similar pattern in the mouse brain. Results We have utilized LongSAGE to identify regionally enriched transcripts in the adult mouse brain. As supplemental strategies, we also performed a meta-analysis of published literature and inspected the Allen Brain Atlas in situ hybridization data. From a set of approximately 30,000 mouse genes, 237 were identified as showing specific or enriched expression in 30 target regions of the mouse brain. GO term over-representation among these genes revealed co-involvement in various aspects of central nervous system development and physiology. Conclusion Using a multi-faceted expression validation approach, we have identified mouse genes whose human orthologs are good candidates for design of mini-promoters. These mouse genes represent molecular markers in several discrete brain regions/cell-types, which could potentially provide a mechanistic explanation of unique functions performed by each region. This set of markers may also serve as a resource for further studies of gene regulatory elements influencing brain expression. PMID:18625066

  13. Identification of a set of genes showing regionally enriched expression in the mouse brain.

    PubMed

    D'Souza, Cletus A; Chopra, Vikramjit; Varhol, Richard; Xie, Yuan-Yun; Bohacec, Slavita; Zhao, Yongjun; Lee, Lisa L C; Bilenky, Mikhail; Portales-Casamar, Elodie; He, An; Wasserman, Wyeth W; Goldowitz, Daniel; Marra, Marco A; Holt, Robert A; Simpson, Elizabeth M; Jones, Steven J M

    2008-07-14

    The Pleiades Promoter Project aims to improve gene therapy by designing human mini-promoters (< 4 kb) that drive gene expression in specific brain regions or cell-types of therapeutic interest. Our goal was to first identify genes displaying regionally enriched expression in the mouse brain so that promoters designed from orthologous human genes can then be tested to drive reporter expression in a similar pattern in the mouse brain. We have utilized LongSAGE to identify regionally enriched transcripts in the adult mouse brain. As supplemental strategies, we also performed a meta-analysis of published literature and inspected the Allen Brain Atlas in situ hybridization data. From a set of approximately 30,000 mouse genes, 237 were identified as showing specific or enriched expression in 30 target regions of the mouse brain. GO term over-representation among these genes revealed co-involvement in various aspects of central nervous system development and physiology. Using a multi-faceted expression validation approach, we have identified mouse genes whose human orthologs are good candidates for design of mini-promoters. These mouse genes represent molecular markers in several discrete brain regions/cell-types, which could potentially provide a mechanistic explanation of unique functions performed by each region. This set of markers may also serve as a resource for further studies of gene regulatory elements influencing brain expression.

  14. [Study of testicular cancer gene expression in samples of oral leukoplakia and squamous cell carcinoma of the mouth].

    PubMed

    Skorodumova, L O; Muraev, A A; Zakharova, E S; Shepelev, M V; Korobko, I V; Zaderenko, I A; Ivanov, S Iu; Gnuchev, N V; Georgiev, G P; Larin, S S

    2012-01-01

    Cancer-testis (CT) antigens are normally expressed mostly in human germ cells, there is also an aberrant expression in some tumor cells. This expression profile makes them potential tumor growth biomarkers and a promising target for tumor immunotherapy. Specificity of CT genes expression in oral malignant and potentially malignant diseases, e.g. oral leukoplakia, is not yet studied. Data on CT genes expression profile in leukoplakia would allow developing new diagnostic methods with potential value for immunotherapy and prophylaxis of leukoplakia malignization. In our study we compared CT genes expression in normal oral mucosa, oral leukoplakia and oral squamous cell carcinoma. We are the first to describe CT genes expression in oral leukoplakia without dysplasia. This findings make impossible differential diagnosis of oral leukoplakia and squamous cell carcinoma on the basis of CT genes expression. The prognostic value of CT genes expression is still unclear, therefore the longitudinal studies are necessary.

  15. Genome-Wide Identification, Evolution and Expression Analysis of mTERF Gene Family in Maize

    PubMed Central

    Zhao, Yanxin; Cai, Manjun; Zhang, Xiaobo; Li, Yurong; Zhang, Jianhua; Zhao, Hailiang; Kong, Fei; Zheng, Yonglian; Qiu, Fazhan

    2014-01-01

    Plant mitochondrial transcription termination factor (mTERF) genes comprise a large family with important roles in regulating organelle gene expression. In this study, a comprehensive database search yielded 31 potential mTERF genes in maize (Zea mays L.) and most of them were targeted to mitochondria or chloroplasts. Maize mTERF were divided into nine main groups based on phylogenetic analysis, and group IX represented the mitochondria and species-specific clade that diverged from other groups. Tandem and segmental duplication both contributed to the expansion of the mTERF gene family in the maize genome. Comprehensive expression analysis of these genes, using microarray data and RNA-seq data, revealed that these genes exhibit a variety of expression patterns. Environmental stimulus experiments revealed differential up or down-regulation expression of maize mTERF genes in seedlings exposed to light/dark, salts and plant hormones, respectively, suggesting various important roles of maize mTERF genes in light acclimation and stress-related responses. These results will be useful for elucidating the roles of mTERF genes in the growth, development and stress response of maize. PMID:24718683

  16. Epidermal Growth Factor Receptor (EGFR) mutation analysis, gene expression profiling and EGFR protein expression in primary prostate cancer

    PubMed Central

    2011-01-01

    Background Activating mutations of the epidermal growth factor receptor (EGFR) confer sensitivity to the tyrosine kinase inhibitors (TKi), gefitinib and erlotinib. We analysed EGFR expression, EGFR mutation status and gene expression profiles of prostate cancer (PC) to supply a rationale for EGFR targeted therapies in this disease. Methods Mutational analysis of EGFR TK domain (exons from 18 to 21) and immunohistochemistry for EGFR were performed on tumour tissues derived from radical prostatectomy from 100 PC patients. Gene expression profiling using oligo-microarrays was also carried out in 51 of the PC samples. Results EGFR protein overexpression (EGFRhigh) was found in 36% of the tumour samples, and mutations were found in 13% of samples. Patients with EGFRhigh tumours experienced a significantly increased risk of biochemical relapse (hazard ratio-HR 2.52, p=0.02) compared with patients with tumours expressing low levels of EGFR (EGFRlow). Microarray analysis did not reveal any differences in gene expression between EGFRhigh and EGFRlow tumours. Conversely, in EGFRhigh tumours, we were able to identify a 79 gene signature distinguishing mutated from non-mutated tumours. Additionally, 29 genes were found to be differentially expressed between mutated/EGFRhigh (n=3) and mutated/EGFRlow tumours (n=5). Four of the down-regulated genes, U19/EAF2, ABCC4, KLK3 and ANXA3 and one of the up-regulated genes, FOXC1, are involved in PC progression. Conclusions Based on our findings, we hypothesize that accurate definition of the EGFR status could improve prognostic stratification and we suggest a possible role for EGFR-directed therapies in PC patients. Having been generated in a relatively small sample of patients, our results warrant confirmation in larger series. PMID:21266046

  17. Epidermal Growth Factor Receptor (EGFR) mutation analysis, gene expression profiling and EGFR protein expression in primary prostate cancer.

    PubMed

    Peraldo-Neia, Caterina; Migliardi, Giorgia; Mello-Grand, Maurizia; Montemurro, Filippo; Segir, Raffaella; Pignochino, Ymera; Cavalloni, Giuliana; Torchio, Bruno; Mosso, Luciano; Chiorino, Giovanna; Aglietta, Massimo

    2011-01-25

    Activating mutations of the epidermal growth factor receptor (EGFR) confer sensitivity to the tyrosine kinase inhibitors (TKi), gefitinib and erlotinib. We analysed EGFR expression, EGFR mutation status and gene expression profiles of prostate cancer (PC) to supply a rationale for EGFR targeted therapies in this disease. Mutational analysis of EGFR TK domain (exons from 18 to 21) and immunohistochemistry for EGFR were performed on tumour tissues derived from radical prostatectomy from 100 PC patients. Gene expression profiling using oligo-microarrays was also carried out in 51 of the PC samples. EGFR protein overexpression (EGFRhigh) was found in 36% of the tumour samples, and mutations were found in 13% of samples. Patients with EGFRhigh tumours experienced a significantly increased risk of biochemical relapse (hazard ratio-HR 2.52, p=0.02) compared with patients with tumours expressing low levels of EGFR (EGFRlow). Microarray analysis did not reveal any differences in gene expression between EGFRhigh and EGFRlow tumours. Conversely, in EGFRhigh tumours, we were able to identify a 79 gene signature distinguishing mutated from non-mutated tumours. Additionally, 29 genes were found to be differentially expressed between mutated/EGFRhigh (n=3) and mutated/EGFRlow tumours (n=5). Four of the down-regulated genes, U19/EAF2, ABCC4, KLK3 and ANXA3 and one of the up-regulated genes, FOXC1, are involved in PC progression. Based on our findings, we hypothesize that accurate definition of the EGFR status could improve prognostic stratification and we suggest a possible role for EGFR-directed therapies in PC patients. Having been generated in a relatively small sample of patients, our results warrant confirmation in larger series.

  18. Transcriptional dynamics of a conserved gene expression network associated with craniofacial divergence in Arctic charr.

    PubMed

    Ahi, Ehsan Pashay; Kapralova, Kalina Hristova; Pálsson, Arnar; Maier, Valerie Helene; Gudbrandsson, Jóhannes; Snorrason, Sigurdur S; Jónsson, Zophonías O; Franzdóttir, Sigrídur Rut

    2014-01-01

    targets and upstream regulators showed differential expression between the contrasting morphotypes. Interestingly, although selected network genes showed overlapping expression patterns in situ and no morph differences, Timp2 expression patterns differed between morphs. Our comparative study of transcriptional dynamics in divergent craniofacial morphologies of Arctic charr revealed a conserved network of coexpressed genes sharing functional roles in structural morphogenesis. We also implicate transcriptional regulators of the network as targets for future functional studies.

  19. Gene Expression Measurement Module (GEMM) - a fully automated, miniaturized instrument for measuring gene expression in space

    NASA Astrophysics Data System (ADS)

    Karouia, Fathi; Ricco, Antonio; Pohorille, Andrew; Peyvan, Kianoosh

    2012-07-01

    The capability to measure gene expression on board spacecrafts opens the doors to a large number of experiments on the influence of space environment on biological systems that will profoundly impact our ability to conduct safe and effective space travel, and might also shed light on terrestrial physiology or biological function and human disease and aging processes. Measurements of gene expression will help us to understand adaptation of terrestrial life to conditions beyond the planet of origin, identify deleterious effects of the space environment on a wide range of organisms from microbes to humans, develop effective countermeasures against these effects, determine metabolic basis of microbial pathogenicity and drug resistance, test our ability to sustain and grow in space organisms that can be used for life support and in situ resource utilization during long-duration space exploration, and monitor both the spacecraft environment and crew health. These and other applications hold significant potential for discoveries in space biology, biotechnology and medicine. Accordingly, supported by funding from the NASA Astrobiology Science and Technology Instrument Development Program, we are developing a fully automated, miniaturized, integrated fluidic system for small spacecraft capable of in-situ measuring microbial expression of thousands of genes from multiple samples. The instrument will be capable of (1) lysing bacterial cell walls, (2) extracting and purifying RNA released from cells, (3) hybridizing it on a microarray and (4) providing electrochemical readout, all in a microfluidics cartridge. The prototype under development is suitable for deployment on nanosatellite platforms developed by the NASA Small Spacecraft Office. The first target application is to cultivate and measure gene expression of the photosynthetic bacterium Synechococcus elongatus, i.e. a cyanobacterium known to exhibit remarkable metabolic diversity and resilience to adverse conditions

  20. Gene-Expression Biomarkers for Application to High-Throughput Radiation Biodosimetry

    DTIC Science & Technology

    2005-01-01

    nuclear disaster . Even with the delayed onset of symptoms, sometimes several days after exposure, gene-expression biomarkers can identify these exposed individuals very early after exposure, allowing for prompt medical intervention. This early assessment of a radiation dose after exposure would enhance the operational commander’s situational awareness of the radiation exposure status of deployed units and increase the prospect of reduced morbidity and mortality through early medical intervention. Candidate gene targets were selected from microarray studies of ex