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Sample records for aav-mediated rnai knockdown

  1. Morphological Profiles of RNAi-Induced Gene Knockdown Are Highly Reproducible but Dominated by Seed Effects

    PubMed Central

    Singh, Shantanu; Wu, Xiaoyun; Ljosa, Vebjorn; Bray, Mark-Anthony; Piccioni, Federica; Root, David E.; Doench, John G.; Boehm, Jesse S.; Carpenter, Anne E.

    2015-01-01

    RNA interference and morphological profiling—the measurement of thousands of phenotypes from individual cells by microscopy and image analysis—are a potentially powerful combination. We show that morphological profiles of RNAi-induced knockdown using the Cell Painting assay are in fact highly sensitive and reproducible. However, we find that the magnitude and prevalence of off-target effects via the RNAi seed-based mechanism make morphological profiles of RNAi reagents targeting the same gene look no more similar than reagents targeting different genes. Pairs of RNAi reagents that share the same seed sequence produce image-based profiles that are much more similar to each other than profiles from pairs designed to target the same gene, a phenomenon previously observed in small-scale gene-expression profiling experiments. Various strategies have been used to enrich on-target versus off-target effects in the context of RNAi screening where a narrow set of phenotypes are measured, mostly based on comparing multiple sequences targeting the same gene; however, new approaches will be needed to make RNAi morphological profiling (that is, comparing multi-dimensional phenotypes) viable. We have shared our raw data and computational pipelines to facilitate research. PMID:26197079

  2. RNAi-mediated knockdown of INHBB increases apoptosis and inhibits steroidogenesis in mouse granulosa cells

    PubMed Central

    M’BAYE, Mohamed; HUA, Guohua; KHAN, Hamid Ali; YANG, Liguo

    2015-01-01

    Inhibins are members of the TGFβ superfamily and act as suppressors of follicle stimulating hormone (FSH) secretion from pituitary glands via a negative feedback mechanism to regulate folliculogenesis. In this study, the INHBB gene was knocked down by three RNAi-Ready pSIREN-RetroQ-ZsGreen vector- mediated recombinant plasmids to explore the effects of INHBB silencing on granulosa cell (GC) cell cycle, apoptosis and steroid production in vitro. Quantitative real-time polymerase chain reaction, Western blot, flow cytometry and ELISA were performed to evaluate the role of INHBB in the mouse GC cell cycle, apoptosis and steroid production in vitro. The results showed that the relative mRNA and protein expression of INHBB in mouse GCs can be significantly reduced by RNAi with pshRNA-B1, pshRNA-B2 and pshRNA-B3 plasmids, with pshRNA-B3 having the best knockdown efficiency. Downregulation of the expression of INHBB significantly arrests cells in the G1 phase of the cell cycle and increases the apoptosis rate in GCs. This was further confirmed by downregulation of the protein expressions of Cyclin D1, Cyclin E and Bcl2, while the protein expression of Bax was upregulated. In addition, specific downregulation of INHBB markedly decreased the concentration of estradiol and progesterone, which was further validated by the decrease in the mRNA levels of CYP19A1and CYP11A1. These findings suggest that inhibin βB is important in the regulation of apoptosis and cell cycle progression in granulosa cells. Furthermore, the inhibin βB subunit has a role in the regulation of steroid hormone biosynthesis. Evidence is accumulating to support the concept that inhibin βB is physiologically essential for early folliculogenesis in the mouse. PMID:26063610

  3. Persistence of RNAi-Mediated Knockdown in Drosophila Complicates Mosaic Analysis Yet Enables Highly Sensitive Lineage Tracing.

    PubMed

    Bosch, Justin A; Sumabat, Taryn M; Hariharan, Iswar K

    2016-05-01

    RNA interference (RNAi) has emerged as a powerful way of reducing gene function in Drosophila melanogaster tissues. By expressing synthetic short hairpin RNAs (shRNAs) using the Gal4/UAS system, knockdown is efficiently achieved in specific tissues or in clones of marked cells. Here we show that knockdown by shRNAs is so potent and persistent that even transient exposure of cells to shRNAs can reduce gene function in their descendants. When using the FLP-out Gal4 method, in some instances we observed unmarked "shadow RNAi" clones adjacent to Gal4-expressing clones, which may have resulted from brief Gal4 expression following recombination but prior to cell division. Similarly, Gal4 driver lines with dynamic expression patterns can generate shadow RNAi cells after their activity has ceased in those cells. Importantly, these effects can lead to erroneous conclusions regarding the cell autonomy of knockdown phenotypes. We have investigated the basis of this phenomenon and suggested experimental designs for eliminating ambiguities in interpretation. We have also exploited the persistence of shRNA-mediated knockdown to design a sensitive lineage-tracing method, i-TRACE, which is capable of detecting even low levels of past reporter expression. Using i-TRACE, we demonstrate transient infidelities in the expression of some cell-identity markers near compartment boundaries in the wing imaginal disc. PMID:26984059

  4. Persistence of RNAi-Mediated Knockdown in Drosophila Complicates Mosaic Analysis Yet Enables Highly Sensitive Lineage Tracing

    PubMed Central

    Bosch, Justin A.; Sumabat, Taryn M.; Hariharan, Iswar K.

    2016-01-01

    RNA interference (RNAi) has emerged as a powerful way of reducing gene function in Drosophila melanogaster tissues. By expressing synthetic short hairpin RNAs (shRNAs) using the Gal4/UAS system, knockdown is efficiently achieved in specific tissues or in clones of marked cells. Here we show that knockdown by shRNAs is so potent and persistent that even transient exposure of cells to shRNAs can reduce gene function in their descendants. When using the FLP-out Gal4 method, in some instances we observed unmarked “shadow RNAi” clones adjacent to Gal4-expressing clones, which may have resulted from brief Gal4 expression following recombination but prior to cell division. Similarly, Gal4 driver lines with dynamic expression patterns can generate shadow RNAi cells after their activity has ceased in those cells. Importantly, these effects can lead to erroneous conclusions regarding the cell autonomy of knockdown phenotypes. We have investigated the basis of this phenomenon and suggested experimental designs for eliminating ambiguities in interpretation. We have also exploited the persistence of shRNA-mediated knockdown to design a sensitive lineage-tracing method, i-TRACE, which is capable of detecting even low levels of past reporter expression. Using i-TRACE, we demonstrate transient infidelities in the expression of some cell-identity markers near compartment boundaries in the wing imaginal disc. PMID:26984059

  5. Simultaneous knockdown of six non-family genes using a single synthetic RNAi fragment in Arabidopsis thaliana

    DOE PAGESBeta

    Czarnecki, Olaf; Bryan, Anthony C.; Jawdy, Sara S.; Yang, Xiaohan; Cheng, Zong-Ming; Chen, Jin-Gui; Tuskan, Gerald A.

    2016-02-17

    Genetic engineering of plants that results in successful establishment of new biochemical or regulatory pathways requires stable introduction of one or more genes into the plant genome. It might also be necessary to down-regulate or turn off expression of endogenous genes in order to reduce activity of competing pathways. An established way to knockdown gene expression in plants is expressing a hairpin-RNAi construct, eventually leading to degradation of a specifically targeted mRNA. Knockdown of multiple genes that do not share homologous sequences is still challenging and involves either sophisticated cloning strategies to create vectors with different serial expression constructs ormore » multiple transformation events that is often restricted by a lack of available transformation markers. Synthetic RNAi fragments were assembled in yeast carrying homologous sequences to six or seven non-family genes and introduced into pAGRIKOLA. Transformation of Arabidopsis thaliana and subsequent expression analysis of targeted genes proved efficient knockdown of all target genes. In conclusion, we present a simple and cost-effective method to create constructs to simultaneously knockdown multiple non-family genes or genes that do not share sequence homology. The presented method can be applied in plant and animal synthetic biology as well as traditional plant and animal genetic engineering.« less

  6. Using RNAi in C. "elegans" to Demonstrate Gene Knockdown Phenotypes in the Undergraduate Biology Lab Setting

    ERIC Educational Resources Information Center

    Roy, Nicole M.

    2013-01-01

    RNA interference (RNAi) is a powerful technology used to knock down genes in basic research and medicine. In 2006 RNAi technology using "Caenorhabditis elegans" ("C. elegans") was awarded the Nobel Prize in medicine and thus students graduating in the biological sciences should have experience with this technology. However,…

  7. Therapeutic impact of systemic AAV-mediated RNA interference in a mouse model of myotonic dystrophy.

    PubMed

    Bisset, Darren R; Stepniak-Konieczna, Ewa A; Zavaljevski, Maja; Wei, Jessica; Carter, Gregory T; Weiss, Michael D; Chamberlain, Joel R

    2015-09-01

    RNA interference (RNAi) offers a promising therapeutic approach for dominant genetic disorders that involve gain-of-function mechanisms. One candidate disease for RNAi therapy application is myotonic dystrophy type 1 (DM1), which results from toxicity of a mutant mRNA. DM1 is caused by expansion of a CTG repeat in the 3' UTR of the DMPK gene. The expression of DMPK mRNA containing an expanded CUG repeat (CUG(exp)) leads to defects in RNA biogenesis and turnover. We designed miRNA-based RNAi hairpins to target the CUG(exp) mRNA in the human α-skeletal muscle actin long-repeat (HSA(LR)) mouse model of DM1. RNAi expression cassettes were delivered to HSA(LR) mice using recombinant adeno-associated viral (rAAV) vectors injected intravenously as a route to systemic gene therapy. Vector delivery significantly reduced disease pathology in muscles of the HSA(LR) mice, including a reduction in the CUG(exp) mRNA, a reduction in myotonic discharges, a shift toward adult pre-mRNA splicing patterns, reduced myofiber hypertrophy and a decrease in myonuclear foci containing the CUG(exp) mRNA. Significant reversal of hallmarks of DM1 in the rAAV RNAi-treated HSA(LR) mice indicate that defects characteristic of DM1 can be mitigated with a systemic RNAi approach targeting the nuclei of terminally differentiated myofibers. Efficient rAAV-mediated delivery of RNAi has the potential to provide a long-term therapy for DM1 and other dominant muscular dystrophies. PMID:26082468

  8. RNAi-mediated knockdown of the voltage gated sodium ion channel TcNav causes mortality in Tribolium castaneum.

    PubMed

    Abd El Halim, Hesham M; Alshukri, Baida M H; Ahmad, Munawar S; Nakasu, Erich Y T; Awwad, Mohammed H; Salama, Elham M; Gatehouse, Angharad M R; Edwards, Martin G

    2016-01-01

    The voltage-gated sodium ion channel (VGSC) belongs to the largest superfamily of ion channels. Since VGSCs play key roles in physiological processes they are major targets for effective insecticides. RNA interference (RNAi) is widely used to analyse gene function, but recently, it has shown potential to contribute to novel strategies for selectively controlling agricultural insect pests. The current study evaluates the delivery of dsRNA targeted to the sodium ion channel paralytic A (TcNav) gene in Tribolium castaneum as a viable means of controlling this insect pest. Delivery of TcNav dsRNA caused severe developmental arrest with larval mortalities up to 73% post injection of dsRNA. Injected larvae showed significant (p < 0.05) knockdown in gene expression between 30-60%. Expression was also significantly (p < 0.05) reduced in pupae following injection causing 30% and 42% knockdown for early and late pupal stages, respectively. Oral delivery of dsRNA caused dose-dependant mortalities of between 19 and 51.34%; this was accompanied by significant (p < 0.05) knockdown in gene expression following 3 days of continuous feeding. The majority of larvae injected with, or fed, dsRNA died during the final larval stage prior to pupation. This work provides evidence of a viable RNAi-based strategy for insect control. PMID:27411529

  9. RNAi-mediated knockdown of the voltage gated sodium ion channel TcNav causes mortality in Tribolium castaneum

    PubMed Central

    Abd El Halim, Hesham M.; Alshukri, Baida M. H.; Ahmad, Munawar S.; Nakasu, Erich Y. T.; Awwad, Mohammed H.; Salama, Elham M.; Gatehouse, Angharad M. R.; Edwards, Martin G.

    2016-01-01

    The voltage-gated sodium ion channel (VGSC) belongs to the largest superfamily of ion channels. Since VGSCs play key roles in physiological processes they are major targets for effective insecticides. RNA interference (RNAi) is widely used to analyse gene function, but recently, it has shown potential to contribute to novel strategies for selectively controlling agricultural insect pests. The current study evaluates the delivery of dsRNA targeted to the sodium ion channel paralytic A (TcNav) gene in Tribolium castaneum as a viable means of controlling this insect pest. Delivery of TcNav dsRNA caused severe developmental arrest with larval mortalities up to 73% post injection of dsRNA. Injected larvae showed significant (p < 0.05) knockdown in gene expression between 30–60%. Expression was also significantly (p < 0.05) reduced in pupae following injection causing 30% and 42% knockdown for early and late pupal stages, respectively. Oral delivery of dsRNA caused dose-dependant mortalities of between 19 and 51.34%; this was accompanied by significant (p < 0.05) knockdown in gene expression following 3 days of continuous feeding. The majority of larvae injected with, or fed, dsRNA died during the final larval stage prior to pupation. This work provides evidence of a viable RNAi-based strategy for insect control. PMID:27411529

  10. Core RNAi machinery and gene knockdown in the emerald ash borer (Agrilus planipennis).

    PubMed

    Zhao, Chaoyang; Alvarez Gonzales, Miguel A; Poland, Therese M; Mittapalli, Omprakash

    2015-01-01

    The RNA interference (RNAi) technology has been widely used in insect functional genomics research and provides an alternative approach for insect pest management. To understand whether the emerald ash borer (Agrilus planipennis), an invasive and destructive coleopteran insect pest of ash tree (Fraxinus spp.), possesses a strong RNAi machinery that is capable of degrading target mRNA as a response to exogenous double-stranded RNA (dsRNA) induction, we identified three RNAi pathway core component genes, Dicer-2, Argonaute-2 and R2D2, from the A. planipennis genome sequence. Characterization of these core components revealed that they contain conserved domains essential for the proteins to function in the RNAi pathway. Phylogenetic analyses showed that they are closely related to homologs derived from other coleopteran species. We also delivered the dsRNA fragment of AplaScrB-2, a β-fructofuranosidase-encoding gene horizontally acquired by A. planipennis as we reported previously, into A. planipennis adults through microinjection. Quantitative real-time PCR analysis on the dsRNA-treated beetles demonstrated a significantly decreased gene expression level of AplaScrB-2 appearing on day 2 and lasting until at least day 6. This study is the first record of RNAi applied in A. planipennis. PMID:25541004

  11. RNAi-mediated knockdown of IKK1 in transgenic mice using a transgenic construct containing the human H1 promoter.

    PubMed

    Moreno-Maldonado, Rodolfo; Murillas, Rodolfo; Navarro, Manuel; Page, Angustias; Suarez-Cabrera, Cristian; Alameda, Josefa P; Bravo, Ana; Casanova, M Llanos; Ramirez, Angel

    2014-01-01

    Inhibition of gene expression through siRNAs is a tool increasingly used for the study of gene function in model systems, including transgenic mice. To achieve perdurable effects, the stable expression of siRNAs by an integrated transgenic construct is necessary. For transgenic siRNA expression, promoters transcribed by either RNApol II or III (such as U6 or H1 promoters) can be used. Relatively large amounts of small RNAs synthesis are achieved when using RNApol III promoters, which can be advantageous in knockdown experiments. To study the feasibility of H1 promoter-driven RNAi-expressing constructs for protein knockdown in transgenic mice, we chose IKK1 as the target gene. Our results indicate that constructs containing the H1 promoter are sensitive to the presence of prokaryotic sequences and to transgene position effects, similar to RNApol II promoters-driven constructs. We observed variable expression levels of transgenic siRNA among different tissues and animals and a reduction of up to 80% in IKK1 expression. Furthermore, IKK1 knockdown led to hair follicle alterations. In summary, we show that constructs directed by the H1 promoter can be used for knockdown of genes of interest in different organs and for the generation of animal models complementary to knockout and overexpression models. PMID:24523631

  12. Ac45 silencing mediated by AAV-sh-Ac45-RNAi prevents both bone loss and inflammation caused by periodontitis

    PubMed Central

    Zhu, Zheng; Chen, Wei; Hao, Liang; Zhu, Guochun; Lu, Yun; Li, Sheng; Wang, Lin; Li, Yi-Ping

    2015-01-01

    Aim Periodontitis induced by oral pathogens leads to severe periodontal tissue damage and osteoclast-mediated bone resorption caused by inflammation. Based on the importance of Ac45 in osteoclast formation and function, we performed this study to evaluate the therapeutic potential of periodontitis by local adeno-associated virus (AAV)-mediated Ac45 gene knockdown. Material and Methods We used AAV-mediated short hairpin RNAi knockdown of Ac45 gene expression (AAV-sh-Ac45) to inhibit bone erosion and gingival inflammation simultaneously in a well-established periodontitis mouse model induced by Porphyromonas gingivalis W50. Histological studies were performed to evaluate the bone protection of AAV-sh-Ac45. Immunochemistry, ELISA and qRT-PCR were performed to reveal the role of Ac45 knockdown on inflammation, immune response and expression of cytokine. Results We found that Ac45 knockdown impaired osteoclast-mediated extracellular acidification and bone resorption in vitro and in vivo. Furthermore, local administration of AAV-sh-Ac45 protected mice from bone erosion by >85% and attenuated inflammation and decreased infiltration of T-cells, dendritic cells and macrophages in the periodontal lesion. Notably, the expression of pro-inflammatory cytokines was also reduced. Conclusions Local AAV-sh-Ac45 gene therapy efficiently protects against periodontal tissue damage and bone erosion through both inhibition of osteoclast function and attenuating inflammation, and may represent a powerful new treatment strategy for periodontitis. PMID:25952706

  13. Transcriptome analysis of the synganglion from the honey bee mite, Varroa destructor and RNAi knockdown of neural peptide targets.

    PubMed

    Campbell, Ewan M; Budge, Giles E; Watkins, Max; Bowman, Alan S

    2016-03-01

    Varroa mites (Varroa destructor) and the viruses that they transmit are one of the major contributing factors to the global honey bee crisis. Gene products within the nervous system are the targets of all the insecticides currently used to control Varroa but there is a paucity of transcriptomic data available for Varroa neural tissues. A cDNA library from the synganglia ("brains") of adult female Varroa was constructed and 600 ESTs sequenced and analysed revealing several current and potential druggable targets. Contigs coding for the deformed wing virus (DWV) variants V. destructor virus-1 (VDV-1) and the recombinant (VDV-1DVD) were present in the synganglion library. Negative-sense RNA-specific PCR indicated that VDV-1 replicates in the Varroa synganglion and all other tissues tested, but we could not detect DWV replicating in any Varroa tissue. Two neuropeptides were identified in the synganlion EST library: a B-type allatostatin and a member of the crustacean hyperglycaemic hormone (CHH) superfamily. Knockdown of the allatostatin or the CHH-like gene by double-stranded RNA-interference (dsRNAi) resulted in 85% and 55% mortality, respectively, of Varroa. Here, we present the first transcriptomic survey in Varroa and demonstrate that neural genes can be targeted by dsRNAi either for genetic validation of putative targets during drug discovery programmes or as a potential control measure in itself. PMID:26721201

  14. Knockdown of asparagine synthetase by RNAi suppresses cell growth in human melanoma cells and epidermoid carcinoma cells.

    PubMed

    Li, Hui; Zhou, Fusheng; Du, Wenhui; Dou, Jinfa; Xu, Yu; Gao, Wanwan; Chen, Gang; Zuo, Xianbo; Sun, Liangdan; Zhang, Xuejun; Yang, Sen

    2016-05-01

    Melanoma, the most aggressive form of skin cancer, causes more than 40,000 deaths each year worldwide. And epidermoid carcinoma is another major form of skin cancer, which could be studied together with melanoma in several aspects. Asparagine synthetase (ASNS) gene encodes an enzyme that catalyzes the glutamine- and ATP-dependent conversion of aspartic acid to asparagine, and its expression is associated with the chemotherapy resistance and prognosis in several human cancers. The present study aims to explore the potential role of ASNS in melanoma cells A375 and human epidermoid carcinoma cell line A431. We applied a lentivirus-mediated RNA interference (RNAi) system to study its function in cell growth of both cells. The results revealed that inhibition of ASNS expression by RNAi significantly suppressed the growth of melanoma cells and epidermoid carcinoma cells, and induced a G0/G1 cell cycle arrest in melanoma cells. Knockdown of ASNS in A375 cells remarkably downregulated the expression levels of CDK4, CDK6, and Cyclin D1, and upregulated the expression of p21. Therefore, our study provides evidence that ASNS may represent a potential therapeutic target for the treatment of melanoma. PMID:25858017

  15. RNAi-mediated knockdown of the Halloween gene spookiest (CYP307B1) impedes adult eclosion in the western tarnished plant bug, Lygus hesperus

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Ecdysteroids play a critical role in coordinating insect growth, development, and reproduction. A suite of cytochrome P450 monooxygenases coded by what are collectively termed Halloween genes mediate ecdysteroid biosynthesis. In this study, we describe cloning and RNAi-mediated knockdown of the CYP3...

  16. Knockdown of myostatin expression by RNAi enhances muscle growth in transgenic sheep.

    PubMed

    Hu, Shengwei; Ni, Wei; Sai, Wujiafu; Zi, Ha; Qiao, Jun; Wang, Pengyang; Sheng, Jinliang; Chen, Chuangfu

    2013-01-01

    Myostatin (MSTN) has been shown to be a negative regulator of skeletal muscle development and growth. MSTN dysfunction therefore offers a strategy for promoting animal growth performance in livestock production. In this study, we investigated the possibility of using RNAi-based technology to generate transgenic sheep with a double-muscle phenotype. A shRNA expression cassette targeting sheep MSTN was used to generate stable shRNA-expressing fibroblast clones. Transgenic sheep were further produced by somatic cell nuclear transfer (SCNT) technology. Five lambs developed to term and three live lambs were obtained. Integration of shRNA expression cassette in three live lambs was confirmed by PCR. RNase protection assay showed that the shRNAs targeting MSTN were expressed in muscle tissues of three transgenic sheep. MSTN expression was significantly inhibited in muscle tissues of transgenic sheep when compared with control sheep. Moreover, transgenic sheep showed a tendency to faster increase in body weight than control sheep. Histological analysis showed that myofiber diameter of transgenic sheep M17 were bigger than that of control sheep. Our findings demonstrate a promising approach to promoting muscle growth in livestock production. PMID:23526994

  17. RNAi knockdown of Nopp140 induces Minute-like phenotypes in Drosophila.

    PubMed

    Cui, Zhengfang; DiMario, Patrick J

    2007-06-01

    Nopp140 associates with small nucleolar RNPs to chaperone pre-rRNA processing and ribosome assembly. Alternative splicing yields two isoforms in Drosophila: Nopp140-True is homologous to vertebrate Nopp140 particularly in its carboxy terminus, whereas Nopp140-RGG contains a glycine and arginine-rich (RGG) carboxy terminus typically found in vertebrate nucleolin. Loss of ribosome function or production at critical points in development leads to Minute phenotypes in Drosophila or the Treacher Collins syndrome (TCS) in humans. To ascertain the functional significance of Nopp140 in Drosophila development, we expressed interfering RNA using the GAL4/UAS system. Reverse transcription-PCR showed variable losses of Nopp140 mRNA in larvae from separate RNAi-expressing transgenic lines, whereas immunofluorescence microscopy with isoform-specific antibodies showed losses of Nopp140 in imaginal and polyploid tissues. Phenotypic expression correlated with the percent loss of Nopp140 transcripts: a >or=50% loss correlated with larval and pupal lethality, disrupted nuclear structures, and in some cases melanotic tumors, whereas a 30% loss correlated with adult wing, leg, and tergite deformities. We consider these adult phenotypes to be Minute-like and reminiscent of human craniofacial malformations associated with TCS. Similarly, overexpression of either isoform caused embryonic and larval lethality, thus indicating proper expression of Nopp140 is critical for normal development. PMID:17392509

  18. Lentivirus-mediated RNAi knockdown of NUPR1 inhibits human nonsmall cell lung cancer growth in vitro and in vivo.

    PubMed

    Guo, Xiaotong; Wang, Wei; Hu, Jing; Feng, Kejian; Pan, Yanming; Zhang, Linyou; Feng, Yukuan

    2012-12-01

    NUPR1 (nuclear protein 1) was found to play a key role in the development of several malignancies including pancreas, breast, and prostate cancers. However, the functional role of NUPR1 in nonsmall cell lung cancer (NSCLC) progression and development is little known. Here, lentivirus-mediated small interfering RNA (siRNA) was employed to downregulate endogenous NUPR1 expression to study the function of NUPR1 in growth of nonsmall cell lung cancer. A lentivirus-mediated RNAi technology was used to specifically knock down the expression of NUPR1 in H1299 cells. Quantitative real-time reverse transcriptase polymerase chain reaction, flow cytometry, western blot and cell count assays were studied to characterize NUPR1 expression in vitro. Furthermore, nonsmall cell lung cancer xenograft models in nude mice were established to investigate whether knockdown of NUPR1 reduces the tumor growth in vivo. We found that downregulation of NUPR1 expression significantly inhibited nonsmall cell lung cancer H1299 cells proliferation and colony formation in vitro. Moreover, the specific downregulation of NUPR1 arrested cells in G0 phase of cell cycle and increased apoptosis rate. Silencing of NUPR1 also suppressed tumor growth by tail vein injection of lentivirus encoded shRNA against NUPR1 in vivo. Our findings revealed that the NUPR1 gene represents a promising target for gene silencing therapy in nonsmall cell lung cancer. PMID:22961798

  19. Nodes-and-connections RNAi knockdown screening: identification of a signaling molecule network involved in fulvestrant action and breast cancer prognosis

    PubMed Central

    Miyoshi, N; Wittner, B S; Shioda, K; Hitora, T; Ito, T; Ramaswamy, S; Isselbacher, K J; Sgroi, D C; Shioda, T

    2015-01-01

    Although RNA interference (RNAi) knockdown screening of cancer cell cultures is an effective approach to predict drug targets or therapeutic/prognostic biomarkers, interactions among identified targets often remain obscure. Here, we introduce the nodes-and-connections RNAi knockdown screening that generates a map of target interactions through systematic iterations of in silico prediction of targets and their experimental validation. An initial RNAi knockdown screening of MCF-7 human breast cancer cells targeting 6560 proteins identified four signaling molecules required for their fulvestrant-induced apoptosis. Signaling molecules physically or functionally interacting with these four primary node targets were computationally predicted and experimentally validated, resulting in identification of four second-generation nodes. Three rounds of further iterations of the prediction–validation cycle generated third, fourth and fifth generation of nodes, completing a 19-node interaction map that contained three predicted nodes but without experimental validation because of technical limitations. The interaction map involved all three members of the death-associated protein kinases (DAPKs) as well as their upstream and downstream signaling molecules (calmodulins and myosin light chain kinases), suggesting that DAPKs play critical roles in the cytocidal action of fulvestrant. The in silico Kaplan–Meier analysis of previously reported human breast cancer cohorts demonstrated significant prognostic predictive power for five of the experimentally validated nodes and for three of the prediction-only nodes. Immunohistochemical studies on the expression of 10 nodal proteins in human breast cancer tissues not only supported their prognostic prediction power but also provided statistically significant evidence of their synchronized expression, implying functional interactions among these nodal proteins. Thus, the Nodes-and-Connections approach to RNAi knockdown screening yields

  20. RNAi-mediated knockdown of pituitary tumor-transforming gene-1 (PTTG1) suppresses the proliferation and invasive potential of PC3 human prostate cancer cells

    PubMed Central

    Huang, S.Q.; Liao, Q.J.; Wang, X.W.; Xin, D.Q.; Chen, S.X.; Wu, Q.J.; Ye, G.

    2012-01-01

    Pituitary tumor-transforming gene-1 (PTTG1) is a proto-oncogene that promotes tumorigenesis and metastasis in numerous cell types and is overexpressed in a variety of human tumors. We have demonstrated that PTTG1 expression was up-regulated in both human prostate cancer specimens and prostate cancer cell lines. For a more direct assessment of the function of PTTG1 in prostate tumorigenesis, RNAi-mediated knockdown was used to selectively decrease PTTG1 expression in PC3 human prostate tumor cells. After three weeks of selection, colonies stably transfected with PTTG1-targeted RNAi (the knockdown PC3 cell line) or empty vector (the control PC3 cell line) were selected and expanded to investigate the role of PTTG1 expression in PC3 cell growth and invasion. Cell proliferation rate was significantly slower (28%) in the PTTG1 knockdown line after 6 days of growth as indicated by an MTT cell viability assay (P < 0.05). Similarly, a soft agar colony formation assay revealed significantly fewer (66.7%) PTTG1 knockdown PC3 cell colonies than control colonies after three weeks of growth. In addition, PTTG1 knockdown resulted in cell cycle arrest at G1 as indicated by fluorescence-activated cell sorting. The PTTG1 knockdown PC3 cell line also exhibited significantly reduced migration through Matrigel in a transwell assay of invasive potential, and down-regulation of PTTG1 could lead to increased sensitivity of these prostate cancer cells to a commonly used anticancer drug, taxol. Thus, PTTG1 expression is crucial for PC3 cell proliferation and invasion, and could be a promising new target for prostate cancer therapy. PMID:22872288

  1. RNAi-mediated knock-down of arylamine N-acetyltransferase-1 expression induces E-cadherin up-regulation and cell-cell contact growth inhibition.

    PubMed

    Tiang, Jacky M; Butcher, Neville J; Cullinane, Carleen; Humbert, Patrick O; Minchin, Rodney F

    2011-01-01

    Arylamine N-acetyltransferase-1 (NAT1) is an enzyme that catalyzes the biotransformation of arylamine and hydrazine substrates. It also has a role in the catabolism of the folate metabolite p-aminobenzoyl glutamate. Recent bioinformatics studies have correlated NAT1 expression with various cancer subtypes. However, a direct role for NAT1 in cell biology has not been established. In this study, we have knocked down NAT1 in the colon adenocarcinoma cell-line HT-29 and found a marked change in cell morphology that was accompanied by an increase in cell-cell contact growth inhibition and a loss of cell viability at confluence. NAT1 knock-down also led to attenuation in anchorage independent growth in soft agar. Loss of NAT1 led to the up-regulation of E-cadherin mRNA and protein levels. This change in E-cadherin was not attributed to RNAi off-target effects and was also observed in the prostate cancer cell-line 22Rv1. In vivo, NAT1 knock-down cells grew with a longer doubling time compared to cells stably transfected with a scrambled RNAi or to parental HT-29 cells. This study has shown that NAT1 affects cell growth and morphology. In addition, it suggests that NAT1 may be a novel drug target for cancer therapeutics. PMID:21347396

  2. Knockdown of CEBPβ by RNAi in porcine granulosa cells resulted in S phase cell cycle arrest and decreased progesterone and estradiol synthesis.

    PubMed

    Zhen, Yan-Hong; Wang, Li; Riaz, Hasan; Wu, Jia-Bin; Yuan, Yi-Feng; Han, Li; Wang, Yan-Ling; Zhao, Yi; Dan, Yi; Huo, Li-Jun

    2014-09-01

    Cultured ovarian granulosa cells (GCs) are essential models to study molecular mechanisms of gene regulation during folliculogenesis. CCAAT enhancer binding proteins β (CEBPβ) has been identified in the ovary and is critical for follicular growth, ovulation and luteinization in mice. In the present study, hormonal treatment indicated that luteinizing hormone (LH) and exogenous human chorionic gonadotropins (hCG) significantly increased the expression of CEBPβ in porcine GCs. By RNAi-Ready pSIREN-RetroQ-ZsGreen Vector mediated recombinant pshRNA vectors, CEBPβ gene was successfully knocked down in porcine GCs, confirmed by mRNA and protein level analyzed by real time PCR and western blot, respectively. We further found that knockdown of CEBPβ significantly increased the expression of p-ERK1/2. Furthermore, CEBPβ knockdown arrested the GCs at S phase of cell cycle, but had no effects on cell apoptosis. More importantly, it markedly down regulated the concentration of estradiol (E2) and progesterone (P4) in the culture medium. To uncover the regulatory mechanism of CEBPβ knockdown on cell cycle and steroids synthesis, we found that the mRNA expression of bcl-2 (anti-apoptosis), StAR and Runx2 (steroid hormone synthesis) was up-regulated, while genes related to apoptosis (Caspase-3 and p53), hormonal synthesis (CYP11A1) and cell cycle (cyclinA1, cyclinB1, cyclinD1) were down-regulated, suggesting that knockdown of CEBPβ may inhibit apoptosis, regulate cell cycle and hormone secretions at the transcriptional level in porcine GCs. Furthermore, knockdown of CEBPβ significantly increased the expression of PTGS2 and decreased the expression of IGFBP4, Has2 and PTGFR which are important for folliculogenesis in porcine GCs. In conclusion, this study reveals that CEBPβ is a key regulator of porcine GCs through modulation of cell cycle, apoptosis, steroid synthesis, and other regulators of folliculogenesis. PMID:24607812

  3. RNAi-mediated knockdown of inhibin α subunit increased apoptosis in granulosa cells and decreased fertility in mice.

    PubMed

    Kadariya, Ishwari; Wang, Jiaxing; ur Rehman, Zia; Ali, Hamid; Riaz, Hasan; He, JiuYa; Bhattarai, Dinesh; Liu, Jia Jia; Zhang, Shu Jun

    2015-08-01

    Inhibin α (INHα), a member of TGFβ superfamily, is an important modulator of reproductive function that plays a vital role in follicular changes, cell differentiation, oocyte development, and ultimately in mammalian reproduction. However, the role of inhibin α in female fertility and ovarian function remains largely unknown. To define its role in reproduction, transgenic mice of RNAi-INHα that knock down the INHα expression by shRNAi were used. Inhibin α subunit gene was knocked down successfully at both transcriptional and translational levels by RNAi PiggyBac transposon (Pbi) mediated recombinant pshRNA vectors and purified DNA fragments were microinjected into mouse zygotes. Results showed that transgenic female mice were sub-fertile and exhibited 35.28% reduction in litter size in F1 generation relative to wild type. The decreased litter size associated with the reduction in the number of oocytes ovulated after puberty. Serum INHα level was significantly decreased in both 3 and 6 weeks; whereas, FSH was significantly increased in 3 weeks but not in 6 weeks. Furthermore, suppression of INHα expression significantly promoted apoptosis by up-regulating Caspase-3, bcl2, INHβB and GDF9 and down regulated Kitl and TGFβRIII genes both at transcriptional and translational levels. Moreover, it also dramatically reduced the progression of G1 phase of cell cycle and the number of cells in S phase as determined by flow cytometer. These results indicate that suppression of INHα expression in RNAi-transgenic mice leads to disruption of normal ovarian regulatory mechanism and causes reproductive deficiencies by promoting cellular apoptosis, arresting cellular progression and altering hormonal signaling. PMID:25998417

  4. RNAi KNOCKDOWN OF BmRab3 LED TO LARVA AND PUPA LETHALITY IN SILKWORM Bombyx mori L.

    PubMed

    Singh, Chabungbam Orville; Xin, Hu-hu; Chen, Rui-ting; Wang, Mei-xian; Liang, Shuang; Lu, Yan; Cai, Zi-zheng; Zhang, Deng-pan; Miao, Yun-gen

    2015-06-01

    Rab3 GTPases are known to play key a role in vesicular trafficking, and express highest in brain and endocrine tissues. In mammals, Rab3 GTPases are paralogs unlike in insect. In this study, we cloned Rab3 from the silk gland tissue of silkworm Bombyx mori, and identified it as BmRab3. Our in silico analysis indicated that BmRab3 is an isoform with a theoretical isoelectric point and molecular weight of 5.52 and 24.3 kDa, respectively. Further, BmRab3 showed the C-terminal hypervariability for GGT2 site but having two other putative guanine nucleotide exchange factor/GDP dissociation inhibitor interaction sites. Multiple alignment sequence indicated high similarities of BmRab3 with Rab3 isoforms of other species. The phylogeny tree showed BmRab3 clustered between the species of Tribolium castaneum and Aedes aegypti. Meanwhile, the expression analysis of BmRab3 showed the highest expression in middle silk glands (MSGs) than all other tissues in the third day of fifth-instar larva. Simultaneously, we showed the differential expression of BmRab3 in the early instar larva development, followed by higher expression in male than female pupae. In vivo dsRNA interference of BmRab3 reduced the expression of BmRab3 by 75% compared to the control in the MSGs in the first day. But as the worm grew to the third day, the difference of BmRab3 between knockdown and control was only about 10%. The knockdown later witnessed underdevelopment of the larvae and pharate pupae lethality in the overall development of silkworm B. mori L. PMID:25735242

  5. Lentiviral-Mediated RNAi Knockdown of Cbfa1 Gene Inhibits Endochondral Ossification of Antler Stem Cells in Micromass Culture

    PubMed Central

    Sun, Hongmei; Yang, Fuhe; Chu, Wenhui; Zhao, Haiping; McMahon, Chris; Li, Chunyi

    2012-01-01

    Articular cartilage (AC) lacks ability to repair defects due to its avascular nature as healing process relies on cells being brought in by blood vessels. Multiple approaches have been taken to facilitate cartilage repair in clinics, to date there is no effective treatment available that can restores the AC lesion to a normally functioning level over extended periods. In this regard, antler cartilage is unique in being richly vascularised and hence can effectively repair and regenerate. Interestingly, antler stem cells, from which the vascularised cartilage is derived, can form avascular cartilage when taken away from their original niche, suggesting that the vascular or avascular state of antler cartilage is controlled by extrinsic factors. Understanding the mechanisms underlying this phenotype switch may help us to devise a way to trigger the effective intrinsic repair of AC. However, adoption of antler cartilage model for AC repair requires the demonstration that the cartilage specific signalling pathways also prevail in antler chondrogenesis. To achieve this, in the present study we silenced expression of Cbfa1, a key factor regulatingendochondral ossification, using RNAi, and showed that expression of the downstream genes type I collagen and osteocalcin were suppressed which, in turn, inhibited endochondral ossification process taking place in the antler stem cell-formed nodules. Therefore, we provided further evidence at molecular level that antler could be developed as novel model for the study of AC repair. The eventual identification of the extrinsic factors dictating the phenotype switch between the vascular and avascular state of antler cartilage will open up a new avenue for the cure of osteoarthritis. PMID:23056636

  6. Computational and molecular tools for scalable rAAV-mediated genome editing.

    PubMed

    Stoimenov, Ivaylo; Ali, Muhammad Akhtar; Pandzic, Tatjana; Sjöblom, Tobias

    2015-03-11

    The rapid discovery of potential driver mutations through large-scale mutational analyses of human cancers generates a need to characterize their cellular phenotypes. Among the techniques for genome editing, recombinant adeno-associated virus (rAAV)-mediated gene targeting is suited for knock-in of single nucleotide substitutions and to a lesser degree for gene knock-outs. However, the generation of gene targeting constructs and the targeting process is time-consuming and labor-intense. To facilitate rAAV-mediated gene targeting, we developed the first software and complementary automation-friendly vector tools to generate optimized targeting constructs for editing human protein encoding genes. By computational approaches, rAAV constructs for editing ~71% of bases in protein-coding exons were designed. Similarly, ~81% of genes were predicted to be targetable by rAAV-mediated knock-out. A Gateway-based cloning system for facile generation of rAAV constructs suitable for robotic automation was developed and used in successful generation of targeting constructs. Together, these tools enable automated rAAV targeting construct design, generation as well as enrichment and expansion of targeted cells with desired integrations. PMID:25488813

  7. RNAi knockdown of oxytocin receptor in the nucleus accumbens inhibits social attachment and parental care in monogamous female prairie voles

    PubMed Central

    Keebaugh, Alaine C.; Barrett, Catherine E.; LaPrairie, Jamie L.; Jenkins, Jasmine J.; Young, Larry J.

    2015-01-01

    Oxytocin modulates many aspects of social cognition and behaviors, including maternal nurturing, social recognition and bonding. Natural variation in oxytocin receptor (OXTR) density in the nucleus accumbens (NAcc) is associated with variation in alloparental behavior, and artificially enhancing OXTR expression in the NAcc enhances alloparental behavior and pair bonding in socially monogamous prairie voles. Furthermore, infusion of an OXTR antagonist into the nucleus accumbens (NAcc) inhibits alloparental behavior and partner preference formation. However, antagonists can promiscuously interact with other neuropeptide receptors. To directly examine the role of OXTR signaling in social bonding, we used RNA interference to selectively knockdown, but not eliminate, OXTR in the NAcc of female prairie voles and examined the impact on social behaviors. Using an adeno-associated viral vector expressing a short hairpin RNA (shRNA) targeting Oxtr mRNA, we reduced accumbal OXTR density in female prairie voles from juvenile age through adulthood. Females receiving the shRNA vector displayed a significant reduction in alloparental behavior and disrupted partner preference formation. These are the first direct demonstrations that OXTR plays a critical role in alloparental behavior and adult social attachment, and suggest that natural variation in OXTR expression in this region alone can create variation in social behavior. PMID:25874849

  8. RNAi Knock-Down of LHCBM1, 2 and 3 Increases Photosynthetic H2 Production Efficiency of the Green Alga Chlamydomonas reinhardtii

    PubMed Central

    Oey, Melanie; Ross, Ian L.; Stephens, Evan; Steinbeck, Janina; Wolf, Juliane; Radzun, Khairul Adzfa; Kügler, Johannes; Ringsmuth, Andrew K.; Kruse, Olaf; Hankamer, Ben

    2013-01-01

    Single cell green algae (microalgae) are rapidly emerging as a platform for the production of sustainable fuels. Solar-driven H2 production from H2O theoretically provides the highest-efficiency route to fuel production in microalgae. This is because the H2-producing hydrogenase (HYDA) is directly coupled to the photosynthetic electron transport chain, thereby eliminating downstream energetic losses associated with the synthesis of carbohydrate and oils (feedstocks for methane, ethanol and oil-based fuels). Here we report the simultaneous knock-down of three light-harvesting complex proteins (LHCMB1, 2 and 3) in the high H2-producing Chlamydomonas reinhardtii mutant Stm6Glc4 using an RNAi triple knock-down strategy. The resultant Stm6Glc4L01 mutant exhibited a light green phenotype, reduced expression of LHCBM1 (20.6% ±0.27%), LHCBM2 (81.2% ±0.037%) and LHCBM3 (41.4% ±0.05%) compared to 100% control levels, and improved light to H2 (180%) and biomass (165%) conversion efficiencies. The improved H2 production efficiency was achieved at increased solar flux densities (450 instead of ∼100 µE m−2 s−1) and high cell densities which are best suited for microalgae production as light is ideally the limiting factor. Our data suggests that the overall improved photon-to-H2 conversion efficiency is due to: 1) reduced loss of absorbed energy by non-photochemical quenching (fluorescence and heat losses) near the photobioreactor surface; 2) improved light distribution in the reactor; 3) reduced photoinhibition; 4) early onset of HYDA expression and 5) reduction of O2-induced inhibition of HYDA. The Stm6Glc4L01 phenotype therefore provides important insights for the development of high-efficiency photobiological H2 production systems. PMID:23613840

  9. RNAi knock-down of LHCBM1, 2 and 3 increases photosynthetic H2 production efficiency of the green alga Chlamydomonas reinhardtii.

    PubMed

    Oey, Melanie; Ross, Ian L; Stephens, Evan; Steinbeck, Janina; Wolf, Juliane; Radzun, Khairul Adzfa; Kügler, Johannes; Ringsmuth, Andrew K; Kruse, Olaf; Hankamer, Ben

    2013-01-01

    Single cell green algae (microalgae) are rapidly emerging as a platform for the production of sustainable fuels. Solar-driven H2 production from H2O theoretically provides the highest-efficiency route to fuel production in microalgae. This is because the H2-producing hydrogenase (HYDA) is directly coupled to the photosynthetic electron transport chain, thereby eliminating downstream energetic losses associated with the synthesis of carbohydrate and oils (feedstocks for methane, ethanol and oil-based fuels). Here we report the simultaneous knock-down of three light-harvesting complex proteins (LHCMB1, 2 and 3) in the high H2-producing Chlamydomonas reinhardtii mutant Stm6Glc4 using an RNAi triple knock-down strategy. The resultant Stm6Glc4L01 mutant exhibited a light green phenotype, reduced expression of LHCBM1 (20.6% ±0.27%), LHCBM2 (81.2% ±0.037%) and LHCBM3 (41.4% ±0.05%) compared to 100% control levels, and improved light to H2 (180%) and biomass (165%) conversion efficiencies. The improved H2 production efficiency was achieved at increased solar flux densities (450 instead of ∼100 µE m(-2) s(-1)) and high cell densities which are best suited for microalgae production as light is ideally the limiting factor. Our data suggests that the overall improved photon-to-H2 conversion efficiency is due to: 1) reduced loss of absorbed energy by non-photochemical quenching (fluorescence and heat losses) near the photobioreactor surface; 2) improved light distribution in the reactor; 3) reduced photoinhibition; 4) early onset of HYDA expression and 5) reduction of O2-induced inhibition of HYDA. The Stm6Glc4L01 phenotype therefore provides important insights for the development of high-efficiency photobiological H2 production systems. PMID:23613840

  10. Homologous recombination is required for AAV-mediated gene targeting

    PubMed Central

    Vasileva, Ana; Linden, R. Michael; Jessberger, Rolf

    2006-01-01

    High frequencies of gene targeting can be achieved by infection of mammalian cells with recombinant adeno-associated virus (rAAV) vectors [D. W. Russell and R. K. Hirata (1998) Nature Genet., 18, 325–330; D. W. Russell and R. K. Hirata (2000) J. Virol., 74, 4612–4620; R. Hirata et al. (2002) Nat. Biotechnol., 20, 735–738], but the mechanism of targeting is unclear and random integration often occurs in parallel. We assessed the role of specific DNA repair and recombination pathways in rAAV gene targeting by measuring correction of a mutated enhanced green fluorescent protein (EGFP) gene in cells where homologous recombination (HR) or non-homologous end-joining (NHEJ) had been suppressed by RNAi. EGFP-negative cells were transduced with rAAV vectors carrying a different inactivating deletion in the EGFP, and in parallel with rAAV vectors carrying red fluorescent protein (RFP). Expression of RFP accounted for viral transduction efficiency and long-term random integration. Approximately 0.02% of the infected GFP-negative cells were stably converted to GFP positive cells. Silencing of the essential NHEJ component DNA-PK had no significant effect on the frequency of targeting at any time point examined. Silencing of the SNF2/SWI2 family members RAD54L or RAD54B, which are important for HR, reduced the rate of stable rAAV gene targeting ∼5-fold. Further, partial silencing of the Rad51 paralogue XRCC3 completely abolished stable long-term EGFP expression. These results show that rAAV gene targeting requires the Rad51/Rad54 pathway of HR. PMID:16822856

  11. RNAi mediated IL-6 in vitro knockdown in psoriasis skin model with topical siRNA delivery system based on liquid crystalline phase.

    PubMed

    Depieri, Lívia Vieira; Borgheti-Cardoso, Lívia Neves; Campos, Patrícia Mazureki; Otaguiri, Katia Kaori; Vicentini, Fabiana Testa Moura de Carvalho; Lopes, Luciana Biagini; Fonseca, Maria José Vieira; Bentley, M Vitória Lopes Badra

    2016-08-01

    Gene therapy by RNA interference (RNAi) is a post-transcriptional silencing process that can suppress the expression of a particular gene and it is a promising therapeutic approach for the treatment of many severe diseases, including cutaneous disorders. However, difficulties related to administration and body distribution limit the clinical use of small interfering RNA (siRNA) molecules. In this study, we proposed to use nanocarriers to enable siRNA application in the topical treatment of skin disorders. A siRNA nanodispersion based on liquid crystalline phase and composed of monoolein (MO), oleic acid (OA) and polyethylenimine (PEI) was developed and its physicochemical properties, efficiency of complexation and carrier/siRNA stability were assessed. Subsequently, cell viability, cellular uptake, in vitro skin irritation test using reconstructed human epidermis (RHE) and in vitro IL-6 knockdown in psoriasis skin model were evaluated. The results showed that the liquid crystalline nanodispersion is a promising topical delivery system for administration of siRNA, being able to overcome the limitations of the route of administration, as well those resulting from the characteristics of siRNA molecules. The formulation was effective at complexing the siRNA, presented high rate of cell uptake (∼90%), increased the skin penetration of siRNA in vitro, and did not cause skin irritation compared with Triton-X (a moderate irritant), resulting in a 4-fold higher viability of reconstructed human epidermis and a 15.6-fold lower release of IL-1α. A single treatment with the liquid crystalline nanodispersion carrying IL-6 siRNA for 6h was able to reduce the extracellular IL-6 levels by 3.3-fold compared with control treatment in psoriasis skin model. Therefore, liquid crystalline nanodispersion is a suitable nanocarrier for siRNA with therapeutic potential to suppress skin disease-specific genes. This study also highlights the applicability of reconstructed skin models in

  12. Recombinant Human Myelin-Associated Glycoprotein Promoter Drives Selective AAV-Mediated Transgene Expression in Oligodendrocytes

    PubMed Central

    von Jonquieres, Georg; Fröhlich, Dominik; Klugmann, Claudia B.; Wen, Xin; Harasta, Anne E.; Ramkumar, Roshini; Spencer, Ziggy H. T.; Housley, Gary D.; Klugmann, Matthias

    2016-01-01

    Leukodystrophies are hereditary central white matter disorders caused by oligodendrocyte dysfunction. Recent clinical trials for some of these devastating neurological conditions have employed an ex vivo gene therapy approach that showed improved endpoints because cross-correction of affected myelin-forming cells occurred following secretion of therapeutic proteins by transduced autologous grafts. However, direct gene transfer to oligodendrocytes is required for the majority of leukodystrophies with underlying mutations in genes encoding non-secreted oligodendroglial proteins. Recombinant adeno-associated viral (AAV) vectors are versatile tools for gene transfer to the central nervous system (CNS) and proof-of-concept studies in rodents have shown that the use of cellular promoters is sufficient to target AAV-mediated transgene expression to glia. The potential of this strategy has not been exploited. The major caveat of the AAV system is its limited packaging capacity of ~5 kb, providing the rationale for identifying small yet selective recombinant promoters. Here, we characterize the human myelin associated glycoprotein (MAG) promoter for reliable targeting of AAV-mediated transgene expression to oligodendrocytes in vivo. A homology screen revealed highly conserved genomic regions among mammalian species upstream of the transcription start site. Recombinant AAV expression cassettes carrying the cDNA encoding enhanced green fluorescent protein (GFP) driven by truncated versions of the recombinant MAG promoter (2.2, 1.5 and 0.3 kb in size) were packaged as cy5 vectors and delivered into the dorsal striatum of mice. At 3 weeks post-injection, oligodendrocytes, neurons and astrocytes expressing the reporter were quantified by immunohistochemical staining. Our results revealed that both 2.2 and 1.5 kb MAG promoters targeted more than 95% of transgene expression to oligodendrocytes. Even the short 0.3 kb fragment conveyed high oligodendroglial specific transgene

  13. Recombinant Human Myelin-Associated Glycoprotein Promoter Drives Selective AAV-Mediated Transgene Expression in Oligodendrocytes.

    PubMed

    von Jonquieres, Georg; Fröhlich, Dominik; Klugmann, Claudia B; Wen, Xin; Harasta, Anne E; Ramkumar, Roshini; Spencer, Ziggy H T; Housley, Gary D; Klugmann, Matthias

    2016-01-01

    Leukodystrophies are hereditary central white matter disorders caused by oligodendrocyte dysfunction. Recent clinical trials for some of these devastating neurological conditions have employed an ex vivo gene therapy approach that showed improved endpoints because cross-correction of affected myelin-forming cells occurred following secretion of therapeutic proteins by transduced autologous grafts. However, direct gene transfer to oligodendrocytes is required for the majority of leukodystrophies with underlying mutations in genes encoding non-secreted oligodendroglial proteins. Recombinant adeno-associated viral (AAV) vectors are versatile tools for gene transfer to the central nervous system (CNS) and proof-of-concept studies in rodents have shown that the use of cellular promoters is sufficient to target AAV-mediated transgene expression to glia. The potential of this strategy has not been exploited. The major caveat of the AAV system is its limited packaging capacity of ~5 kb, providing the rationale for identifying small yet selective recombinant promoters. Here, we characterize the human myelin associated glycoprotein (MAG) promoter for reliable targeting of AAV-mediated transgene expression to oligodendrocytes in vivo. A homology screen revealed highly conserved genomic regions among mammalian species upstream of the transcription start site. Recombinant AAV expression cassettes carrying the cDNA encoding enhanced green fluorescent protein (GFP) driven by truncated versions of the recombinant MAG promoter (2.2, 1.5 and 0.3 kb in size) were packaged as cy5 vectors and delivered into the dorsal striatum of mice. At 3 weeks post-injection, oligodendrocytes, neurons and astrocytes expressing the reporter were quantified by immunohistochemical staining. Our results revealed that both 2.2 and 1.5 kb MAG promoters targeted more than 95% of transgene expression to oligodendrocytes. Even the short 0.3 kb fragment conveyed high oligodendroglial specific transgene

  14. Current Challenges and Future Directions in Recombinant AAV-Mediated Gene Therapy of Duchenne Muscular Dystrophy.

    PubMed

    Okada, Takashi; Takeda, Shin'ichi

    2013-01-01

    Various characteristics of adeno-associated virus (AAV)-based vectors with long-term safe expression have made it an exciting transduction tool for clinical gene therapy of Duchenne muscular dystrophy (DMD). Although host immune reactions against the vector as well as transgene products were detected in some instances of the clinical studies, there have been promising observations. Methods of producing AAV vectors for considerable in vivo experimentation and clinical investigations have been developed and a number of studies with AAV vector-mediated muscle transduction were attempted. Notably, an intravenous limb perfusion transduction technique enables extensive transgene expression in the skeletal muscles without noticeable adverse events. Furthermore, cardiac transduction by the rAAV9-microdystrophin would be promising to prevent development of cardiac dysfunction. Recent achievements in transduction technology suggest that long-term transgene expression with therapeutic benefits in DMD treatment would be achieved by the rAAV-mediated transduction strategy with an adequate regimen to regulate host immune response. PMID:24276316

  15. RNAi-Mediated Knock-Down of transformer and transformer 2 to Generate Male-Only Progeny in the Oriental Fruit Fly, Bactrocera dorsalis (Hendel)

    PubMed Central

    Li, Jianwei; Zhang, Guifen; Wan, Fanghao

    2015-01-01

    The transformer (tra) gene appears to act as the genetic switch that promotes female development by interaction with the transformer2 (tra-2) gene in several dipteran species including the Medfly, housefly and Drosophila melanogaster. In this study, we describe the isolation, expression and function of tra and tra-2 in the economically important agricultural pest, the oriental fruit fly, Bactrocera dorsalis (Hendel). Bdtra and Bdtra-2 are similar to their homologs from other tephritid species. Bdtra demonstrated sex-specific transcripts: one transcript in females and two transcripts in males. In contrast, Bdtra-2 only had one transcript that was common to males and females, which was transcribed continuously in different adult tissues and developmental stages. Bdtra-2 and the female form of Bdtra were maternally inherited in eggs, whereas the male form of Bdtra was not detectable until embryos of 1 and 2 h after egg laying. Function analyses of Bdtra and Bdtra-2 indicated that both were indispensable for female development, as nearly 100% males were obtained with embryonic RNAi against either Bdtra or Bdtra-2. The fertility of these RNAi-generated males was subsequently tested. More than 80% of RNAi-generated males could mate and the mated females could lay eggs, but only 40-48.6% males gave rise to progeny. In XX-reversed males and intersex individuals, no clear female gonadal morphology was observed after dissection. These results shed light on the development of a genetic sexing system with male-only release for this agricultural pest. PMID:26057559

  16. Knockdown of neuropeptide Y in the dorsomedial hypothalamus reverses high-fat diet-induced obesity and impaired glucose tolerance in rats.

    PubMed

    Kim, Yonwook J; Bi, Sheng

    2016-01-15

    Neuropeptide Y (NPY) in the dorsomedial hypothalamus (DMH) plays an important role in the regulation of energy balance. While DMH NPY overexpression causes hyperphagia and obesity in rats, knockdown of NPY in the DMH via adeno-associated virus (AAV)-mediated RNAi (AAVshNPY) ameliorates these alterations. Whether this knockdown has a therapeutic effect on obesity and glycemic disorder has yet to be determined. The present study sought to test this potential using a rat model of high-fat diet (HFD)-induced obesity and insulin resistance, mimicking human obesity with impaired glucose homeostasis. Rats had ad libitum access to rodent regular chow (RC) or HFD. Six weeks later, an oral glucose tolerance test (OGTT) was performed for verifying HFD-induced glucose intolerance. After verification, obese rats received bilateral DMH injections of AAVshNPY or the control vector AAVshCTL, and OGTT and insulin tolerance test (ITT) were performed at 16 and 18 wk after viral injection (23 and 25 wk on HFD), respectively. Rats were killed at 26 wk on HFD. We found that AAVshCTL rats on HFD remained hyperphagic, obese, glucose intolerant, and insulin resistant relative to lean control RC-fed rats receiving DMH injection of AAVshCTL, whereas these alterations were reversed in NPY knockdown rats fed a HFD. NPY knockdown rats exhibited normal food intake, body weight, glucose tolerance, and insulin sensitivity, as seen in lean control rats. Together, these results demonstrate a therapeutic action of DMH NPY knockdown against obesity and impaired glucose homeostasis in rats, providing a potential target for the treatment of obesity and diabetes. PMID:26561644

  17. RNAi-mediated knockdown of the CLN3 gene inhibits proliferation and promotes apoptosis in drug-resistant ovarian cancer cells

    PubMed Central

    MAO, DONGWEI; CHE, JIANHUA; HAN, SHIYU; ZHAO, HONGHUI; ZHU, YUMEI; ZHU, HONG

    2015-01-01

    CLN3 is a recently identified anti-apoptotic gene, which has been demonstrated to be highly expressed in a diverse range of cancer cell lines, including ovarian cancer. In the present study, RNA interference, mediated by a lentivirus expressing CLN3 short hairpin RNA (shRNA) was utilized to knockdown the expression of CLN3 in the A2780 human ovarian cancer cell line, and its cisplatin-resistant and carboplatin-resistant sublines, A2780/DDP and A2780/CBP cells. It was revealed that the mRNA and protein expression levels of CLN3 were significantly reduced in the CLN3-specific shRNA-transduced cells, compared with the untransduced and control shRNA-transduced cells. In addition, specific knockdown of CLN3 in these cells inhibited cell proliferation and led to cell cycle arrest at the G0/G1 phase, with eventual apoptosis. CLN3 knockdown caused increases in the levels of Bax, FAX, cleaved-caspase 3, cleaved-caspase 8 and cleaved-RARP, but decreased the level of Bcl-2. Finally, it was observed that CLN3 depletion markedly reduced the half maximum inhibitory concentration in the A2780/DDP and A2780/CBP cells. Taken together, these data suggested that CLN3 is involved in tumorigenesis and drug resistance in ovarian cancer, and may serve as a promising therapeutic target for its treatment. PMID:26299671

  18. Sustained correction of FVII deficiency in dogs using AAV-mediated expression of zymogen FVII

    PubMed Central

    Marcos-Contreras, Oscar A.; Smith, Shannon M.; Bellinger, Dwight A.; Raymer, Robin A.; Merricks, Elizabeth; Faella, Armida; Pavani, Giulia; Zhou, Shangzhen; Nichols, Timothy C.; High, Katherine A.

    2016-01-01

    Factor VII (FVII) deficiency is a rare autosomal recessive bleeding disorder treated by infusion of fresh-frozen plasma, plasma-derived FVII concentrates and low-dose recombinant activated FVII. Clinical data suggest that a mild elevation of plasma FVII levels (>10% normal) results in improved hemostasis. Research dogs with a G96E missense FVII mutation (FVII-G96E) have <1% FVII activity. By western blot, we show that they have undetectable plasmatic antigen, thus representing the most prevalent type of human FVII deficiency (low antigen/activity). In these dogs, we determine the feasibility of a gene therapy approach using liver-directed, adeno-associated viral (AAV) serotype 8 vector delivery of a canine FVII (cFVII) zymogen transgene. FVII-G96E dogs received escalating AAV doses (2E11 to 4.95E13 vector genomes [vg] per kg). Clinically therapeutic expression (15% normal) was attained with as low as 6E11 vg/kg of AAV and has been stable for >1 year (ongoing) without antibody formation to the cFVII transgene. Sustained and supraphysiological expression of 770% normal was observed using 4.95E13 vg/kg of AAV (2.6 years, ongoing). No evidence of pathological activation of coagulation or detrimental animal physiology was observed as platelet counts, d-dimer, fibrinogen levels, and serum chemistries remained normal in all dogs (cumulative 6.4 years). We observed a transient and noninhibitory immunoglobulin G class 2 response against cFVII only in the dog receiving the highest AAV dose. In conclusion, in the only large-animal model representing the majority of FVII mutation types, our data are first to demonstrate the feasibility, safety, and long-term duration of AAV-mediated correction of FVII deficiency. PMID:26702064

  19. AAV-mediated delivery of zinc finger nucleases targeting hepatitis B virus inhibits active replication.

    PubMed

    Weber, Nicholas D; Stone, Daniel; Sedlak, Ruth Hall; De Silva Feelixge, Harshana S; Roychoudhury, Pavitra; Schiffer, Joshua T; Aubert, Martine; Jerome, Keith R

    2014-01-01

    Despite an existing effective vaccine, hepatitis B virus (HBV) remains a major public health concern. There are effective suppressive therapies for HBV, but they remain expensive and inaccessible to many, and not all patients respond well. Furthermore, HBV can persist as genomic covalently closed circular DNA (cccDNA) that remains in hepatocytes even during otherwise effective therapy and facilitates rebound in patients after treatment has stopped. Therefore, the need for an effective treatment that targets active and persistent HBV infections remains. As a novel approach to treat HBV, we have targeted the HBV genome for disruption to prevent viral reactivation and replication. We generated 3 zinc finger nucleases (ZFNs) that target sequences within the HBV polymerase, core and X genes. Upon the formation of ZFN-induced DNA double strand breaks (DSB), imprecise repair by non-homologous end joining leads to mutations that inactivate HBV genes. We delivered HBV-specific ZFNs using self-complementary adeno-associated virus (scAAV) vectors and tested their anti-HBV activity in HepAD38 cells. HBV-ZFNs efficiently disrupted HBV target sites by inducing site-specific mutations. Cytotoxicity was seen with one of the ZFNs. scAAV-mediated delivery of a ZFN targeting HBV polymerase resulted in complete inhibition of HBV DNA replication and production of infectious HBV virions in HepAD38 cells. This effect was sustained for at least 2 weeks following only a single treatment. Furthermore, high specificity was observed for all ZFNs, as negligible off-target cleavage was seen via high-throughput sequencing of 7 closely matched potential off-target sites. These results show that HBV-targeted ZFNs can efficiently inhibit active HBV replication and suppress the cellular template for HBV persistence, making them promising candidates for eradication therapy. PMID:24827459

  20. AAV-Mediated Gene Editing via Double-Strand Break Repair

    PubMed Central

    Hirsch, Matthew L.; Samulski, R. Jude

    2016-01-01

    Traditionally, the ability to edit the mammalian genome was inhibited by the inherent low efficiency of homologous recombination (HR; approximately <1 in a million events) and the inability to deliver DNA efficiently to dividing and non-dividing cells/tissue. Despite these limitations, creative selections designed over 20 years ago, clearly demonstrated the powerful implications of gene knock-in and knockout technology for the genetic engineering of mice (Doetschman et al. Nat 330(6148): 576–578, 1987; Thomas and Capecchi. Cell 51(3): 503–512, 1987). The development and application of recombinant vectors based on adeno-associated virus (rAAV) have helped to overcome both of the initial limitations regarding DNA delivery and the frequency of HR. Considering DNA delivery, rAAV infects non-dividing and dividing cultured cells as well as most tissues in mouse and larger animal models (including humans). At the DNA editing level, rAAV genomes have been reported to increase the frequency of HR several orders of magnitude by serving as the repair substrate (Russell and Hirata. Nat Genet 18(4): 325–330, 1998). However, reports on the ability of rAAV genomes to stimulate HR, compared to plasmid DNA and oligonucleotides, are variable, and many labs have found it necessary to augment the frequency of rAAV-induced HR using site-specific endonucleases (Ellis et al. Gene Ther, 2012; Hirsch et al. Gene Ther 17(9): 1175–1180, 2010; Porteus et al. Mol Cell Biol 23(10): 3558–3565, 2003; Radecke et al. Mol Ther 14(6): 798–808, 2006). In this protocol, we describe a method to perform rAAV-mediated double-strand break (DSB) repair for precise genetic engineering in human cells. PMID:24557911

  1. AAV-Mediated Delivery of Zinc Finger Nucleases Targeting Hepatitis B Virus Inhibits Active Replication

    PubMed Central

    Weber, Nicholas D.; Stone, Daniel; Sedlak, Ruth Hall; De Silva Feelixge, Harshana S.; Roychoudhury, Pavitra; Schiffer, Joshua T.; Aubert, Martine; Jerome, Keith R.

    2014-01-01

    Despite an existing effective vaccine, hepatitis B virus (HBV) remains a major public health concern. There are effective suppressive therapies for HBV, but they remain expensive and inaccessible to many, and not all patients respond well. Furthermore, HBV can persist as genomic covalently closed circular DNA (cccDNA) that remains in hepatocytes even during otherwise effective therapy and facilitates rebound in patients after treatment has stopped. Therefore, the need for an effective treatment that targets active and persistent HBV infections remains. As a novel approach to treat HBV, we have targeted the HBV genome for disruption to prevent viral reactivation and replication. We generated 3 zinc finger nucleases (ZFNs) that target sequences within the HBV polymerase, core and X genes. Upon the formation of ZFN-induced DNA double strand breaks (DSB), imprecise repair by non-homologous end joining leads to mutations that inactivate HBV genes. We delivered HBV-specific ZFNs using self-complementary adeno-associated virus (scAAV) vectors and tested their anti-HBV activity in HepAD38 cells. HBV-ZFNs efficiently disrupted HBV target sites by inducing site-specific mutations. Cytotoxicity was seen with one of the ZFNs. scAAV-mediated delivery of a ZFN targeting HBV polymerase resulted in complete inhibition of HBV DNA replication and production of infectious HBV virions in HepAD38 cells. This effect was sustained for at least 2 weeks following only a single treatment. Furthermore, high specificity was observed for all ZFNs, as negligible off-target cleavage was seen via high-throughput sequencing of 7 closely matched potential off-target sites. These results show that HBV-targeted ZFNs can efficiently inhibit active HBV replication and suppress the cellular template for HBV persistence, making them promising candidates for eradication therapy. PMID:24827459

  2. RNAi knockdown of acetyl-CoA carboxylase gene eliminates jinggangmycin-enhanced reproduction and population growth in the brown planthopper, Nilaparvata lugens

    PubMed Central

    Zhang, Yi-Xin; Ge, Lin-Quan; Jiang, Yi-Ping; Lu, Xiu-Li; Li, Xin; Stanley, David; Song, Qi-Sheng; Wu, Jin-Cai

    2015-01-01

    A major challenge in ecology lies in understanding the coexistence of intraguild species, well documented at the organismal level, but not at the molecular level. This study focused on the effects of the antibiotic, jinggangmycin (JGM), a fungicide widely used in Asian rice agroecosystems, on reproduction of insects within the planthopper guild, including the brown planthopper (BPH) Nilaparvata lugens and the white-backed planthopper (WBPH) Sogatella furcifera, both serious resurgence rice pests. JGM exposure significantly increased BPH fecundity and population growth, but suppressed both parameters in laboratory and field WBPH populations. We used digital gene expression and transcriptomic analyses to identify a panel of differentially expressed genes, including a set of up-regulated genes in JGM-treated BPH, which were down-regulated in JGM-treated WBPH. RNAi silencing of Acetyl Co-A carboxylase (ACC), highly expressed in JGM-treated BPH, reduced ACC expression (by > 60%) and eliminated JGM-induced fecundity increases in BPH. These findings support our hypothesis that differences in ACC expression separates intraguild species at the molecular level. PMID:26482193

  3. A comprehensive transcriptomic view on the role of SMAD4 gene by RNAi-mediated knockdown in porcine follicular granulosa cells.

    PubMed

    Zhang, Lifan; Du, Xing; Wei, Shengjuan; Li, Dongfeng; Li, Qifa

    2016-07-01

    As a key mediator of the transforming growth factor-beta (TGF-β) signaling pathway, which plays a pivotal role in regulating mammalian reproductive performance, Sma- and Mad-related protein 4 (SMAD4) is closely associated with the development of ovarian follicular. However, current knowledge of the genome-wide view on the role of SMAD4 gene in mammalian follicular granulosa cells (GCs) is still largely unknown. In the present study, RNA-Seq was performed to investigate the effects of SMAD4 knockdown by RNA interference (SMAD4-siRNA) in porcine follicular GCs. A total of 1025 differentially expressed genes (DEGs), including 530 upregulated genes and 495 downregulated genes, were identified in SMAD4-siRNA treated GCs compared with that treated with NC-siRNA. Furthermore, functional enrichment analysis indicated that upregulated DEGs in SMAD4-siRNA treated cells were mainly enriched in cell-cycle related processes, interferon signaling pathway, and immune system process, while downregulated DEGs in SMAD4-siRNA treated cells were mainly involved in extracellular matrix organization/disassembly, pathogenesis, and cell adhesion. In particular, cell cycle and TGF-β signaling pathway were discovered as the canonical pathways changed under SMAD4-silencing. Taken together, our data reveals SMAD4 knockdown alters the expression of numerous genes involved in key biological processes of the development of follicular GCs and provides a novel global clue of the role of SMAD4 gene in porcine follicular GCs, thus improving our understanding of regulatory mechanisms of SMAD4 gene in follicular development. PMID:27107035

  4. The development of a high-yield recombinant protein bioreactor through RNAi induced knockdown of ATP/ADP transporter in Solanum tuberosum.

    PubMed

    Tremblay, Reynald; Diao, Hong; Hüner, Norm; Jevnikar, Anthony M; Ma, Shengwu

    2011-10-20

    There is an increased need for high-yield protein production platforms to meet growing demand. Tuber-based production in Solanum tuberosum offers several advantages, including high biomass yield, although protein concentration is typically low. In this work, we investigated the question whether minor interruption of starch biosynthesis can have a positive effect on tuber protein content and/or tuber biomass, as previous work suggested that partial obstruction of starch synthesis had variable effects on tuber yield. To this end, we used a RNAi approach to knock down ATP/ADP transporter and obtained a large number of transgenic lines for screening of lines with improved tuber protein content and/or tuber biomass. The initial screening was based on tuber biomass because of its relative simplicity. We identified a line, riAATP1-10, with minor (less than 15%) reduction in starch, that had a nearly 30% increase in biomass compared to wild-type, producing both more and larger tubers with altered morphological features compared to wild-type. riAATP1-10 tubers have a higher concentration of soluble protein compared to wild-type tubers, with nearly 50% more soluble protein. We assessed the suitability of this line as a new bioreactor by expressing a human scFv, reaching over 0.5% of total soluble protein, a 2-fold increase over the highest accumulating line in a wild-type background. Together with increased biomass and increased levels in total protein content, foreign protein expression in riAATP1-10 line would translate into a nearly 4-fold increase in recombinant protein yield per plant. Our results indicate that riAATP1-10 line provides an improved expression system for production of foreign proteins. PMID:21864587

  5. RNAi-mediated knockdown of catalase causes cell cycle arrest in SL-1 cells and results in low survival rate of Spodoptera litura (Fabricius).

    PubMed

    Zhao, Haiming; Yi, Xin; Hu, Zhen; Hu, Meiying; Chen, Shaohua; Muhammad, Rizwan-ul-Haq; Dong, Xiaolin; Gong, Liang

    2013-01-01

    Deregulated reactive oxygen species (ROS) production can lead to the disruption of structural and functional integrity of cells as a consequence of reactive interaction between ROS and various biological components. Catalase (CAT) is a common enzyme existing in nearly all organisms exposed to oxygen, which decomposes harmful hydrogen peroxide, into water and oxygen. In this study, the full length sequence that encodes CAT-like protein from Spodoptera litura named siltCAT (GenBank accession number: JQ_663444) was cloned and characterized. Amino acid sequence alignment showed siltCAT shared relatively high conservation with other insect, especially the conserved residues which defined heme and NADPH orientation. Expression pattern analysis showed that siltCAT mRNA was mainly expressed in the fat body, midgut, cuticle and malpighian tube, and as well as over last instar larvae, pupa and adult stages. RNA interference was used to silence CAT gene in SL-1 cells and the fourth-instar stage of S. litura larvae respectively. Our results provided evidence that CAT knockdown induced ROS generation, cell cycle arrest and apoptosis in SL-1 cells. It also confirmed the decrease in survival rate because of increased ROS production in experimental groups injected with double-stranded RNA of CAT (dsCAT). This study implied that ROS scavenging by CAT is important for S. litura survival. PMID:23555693

  6. RNA interference (RNAi) mediated stable knockdown of protein casein kinase 2-alpha (CK2α) inhibits migration and invasion and enhances cisplatin-induced apoptosis in HEp-2 laryngeal carcinoma cells.

    PubMed

    Zhang, Fang; Yang, Bo; Shi, Shengli; Jiang, Xuejun

    2014-07-01

    Laryngeal carcinoma is a common malignant neoplasm occurring in the head and neck, threatening human health. Protein casein kinase 2-alpha (CK2α) has been indicated to participate in the pathogenesis of this cancer; however, the underlying mechanisms still need to be elucidated. In this study, short hairpin RNA (shRNA)-mediated RNA interference (RNAi) technology was utilized to inhibit the CK2α expression in HEp-2 laryngeal carcinoma cells. Results showed that both mRNA and protein expression levels of endogenous CK2α were markedly decreased in HEp-2 cells transfected with CK2α specific shRNA. Transwell assays revealed that stable knockdown of CK2α significantly inhibited the migration and invasion of HEp-2 cells. As compared with cells treated with negative control shRNA, epithelial cadherin (E-cadherin) expression was increased, but snail, slug and vimentin were decreased in cells transfected with CK2α shRNA, indicating that inhibition of CK2α expression may suppress the epithelial-mesenchymal transition (EMT) process of laryngeal carcinoma in vitro. Moreover, suppression of CK2α was found to enhance the apoptosis induced by cisplatin in laryngeal carcinoma cells, probably through inhibition of permeability glycoprotein (P-glycoprotein) and multidrug-resistance protein (MRP1). In conclusion, our study may provide a promising therapeutic strategy for human laryngeal carcinoma by targeting CK2α. PMID:24831064

  7. Adeno-Associated Virus (AAV) Mediated Dystrophin Gene Transfer Studies and Exon Skipping Strategies for Duchenne Muscular Dystrophy (DMD).

    PubMed

    Kawecka, Klaudia; Theodoulides, Michael; Hasoglu, Yalin; Jarmin, Susan; Kymalainen, Hanna; Le-Heron, Anita; Popplewell, Linda; Malerba, Alberto; Dickson, George; Athanasopoulos, Takis

    2015-01-01

    Duchenne muscular dystrophy (DMD), an X-linked inherited musclewasting disease primarily affecting young boys with prevalence of between1:3,500- 1:5,000, is a rare genetic disease caused by defects in the gene for dystrophin. Dystrophin protein is critical to the stability of myofibers in skeletal and cardiac muscle. There is currently no cure available to ameliorate DMD and/or its patho-physiology. A number of therapeutic strategies including molecular-based therapeutics that replace or correct the missing or nonfunctional dystrophin protein have been devised to correct the patho-physiological consequences induced by dystrophin absence. We will review the current in vivo experimentation status (including preclinical models and clinical trials) for two of these approaches, namely: 1) Adeno-associated virus (AAV) mediated (micro) dystrophin gene augmentation/ supplementation and 2) Antisense oligonucleotide (AON)-mediated exon skipping strategies. PMID:26159373

  8. Novel rat Alzheimer's disease models based on AAV-mediated gene transfer to selectively increase hippocampal Aβ levels

    PubMed Central

    Lawlor, Patricia A; Bland, Ross J; Das, Pritam; Price, Robert W; Holloway, Vallie; Smithson, Lisa; Dicker, Bridget L; During, Matthew J; Young, Deborah; Golde, Todd E

    2007-01-01

    Background Alzheimer's disease (AD) is characterized by a decline in cognitive function and accumulation of amyloid-β peptide (Aβ) in extracellular plaques. Mutations in amyloid precursor protein (APP) and presenilins alter APP metabolism resulting in accumulation of Aβ42, a peptide essential for the formation of amyloid deposits and proposed to initiate the cascade leading to AD. However, the role of Aβ40, the more prevalent Aβ peptide secreted by cells and a major component of cerebral Aβ deposits, is less clear. In this study, virally-mediated gene transfer was used to selectively increase hippocampal levels of human Aβ42 and Aβ40 in adult Wistar rats, allowing examination of the contribution of each to the cognitive deficits and pathology seen in AD. Results Adeno-associated viral (AAV) vectors encoding BRI-Aβ cDNAs were generated resulting in high-level hippocampal expression and secretion of the specific encoded Aβ peptide. As a comparison the effect of AAV-mediated overexpression of APPsw was also examined. Animals were tested for development of learning and memory deficits (open field, Morris water maze, passive avoidance, novel object recognition) three months after infusion of AAV. A range of impairments was found, with the most pronounced deficits observed in animals co-injected with both AAV-BRI-Aβ40 and AAV-BRI-Aβ42. Brain tissue was analyzed by ELISA and immunohistochemistry to quantify levels of detergent soluble and insoluble Aβ peptides. BRI-Aβ42 and the combination of BRI-Aβ40+42 overexpression resulted in elevated levels of detergent-insoluble Aβ. No significant increase in detergent-insoluble Aβ was seen in the rats expressing APPsw or BRI-Aβ40. No pathological features were noted in any rats, except the AAV-BRI-Aβ42 rats which showed focal, amorphous, Thioflavin-negative Aβ42 deposits. Conclusion The results show that AAV-mediated gene transfer is a valuable tool to model aspects of AD pathology in vivo, and demonstrate that

  9. A Preclinical Animal Model to Assess the Effect of Pre-existing Immunity on AAV-mediated Gene Transfer

    PubMed Central

    Li, Hua; Lin, Shih-Wen; Giles-Davis, Wynetta; Li, Yan; Zhou, Dongming; Xiang, Zhi Quan; High, Katherine A; Ertl, Hildegund CJ

    2009-01-01

    Hepatic adeno-associated virus (AAV)-serotype 2–mediated gene transfer results in sustained transgene expression in experimental animals but not in human subjects. We hypothesized that loss of transgene expression in humans might be caused by immune memory mechanisms that become reactivated upon AAV vector transfer. Here, we tested the effect of immunological memory to AAV capsid on AAV-mediated gene transfer in a mouse model. Upon hepatic transfer of an AAV2 vector expressing human factor IX (hF.IX), mice immunized with adenovirus (Ad) vectors expressing AAV8 capsid before AAV2 transfer developed less circulating hF.IX and showed a gradual loss of hF.IX gene copies in liver cells as compared to control animals. This was not observed in mice immunized with an Ad vectors expressing AAV2 capsid before transfer of rAAV8-hF.IX vectors. The lower hF.IX expression was primarily linked to AAV-binding antibodies that lacked AAV-neutralizing activity in vitro rather than to AAV capsid–specific CD8+ T cells. PMID:19367258

  10. scAAV-Mediated Gene Transfer of Interleukin 1-Receptor Antagonist to Synovium and Articular Cartilage in Large Mammalian Joints

    PubMed Central

    Watson, Rachael S.; Broome, Ted A.; Levings, Padraic P.; Rice, Bret L.; Kay, Jesse D.; Smith, Andrew D.; Gouze, Elvire; Gouze, Jean-Noel; Dacanay, E. Anthony; Hauswirth, William W.; Nickerson, David M.; Dark, Michael J.; Colahan, Patrick T.; Ghivizzani, Steven C.

    2012-01-01

    With the long-term goal of developing a gene-based treatment for osteoarthritis (OA), we performed studies to evaluate the equine joint as a model for AAV-mediated gene transfer to large, weight-bearing human joints. A self-complementary AAV2 vector containing the coding regions for human interleukin-1 receptor antagonist (hIL-1Ra) or green fluorescent protein (GFP) was packaged in AAV capsid serotypes 1, 2, 5, 8 and 9. Following infection of human and equine synovial fibroblasts in culture, we found that both were only receptive to transduction with AAV1, 2 and 5. For these serotypes, however, transgene expression from the equine cells was consistently at least 10-fold higher. Analyses of AAV surface receptor molecules and intracellular trafficking of vector genomes implicate enhanced viral uptake by the equine cells. Following delivery of 1 × 1011 vector genomes of serotypes 2, 5 and 8 into the forelimb joints of the horse, all three enabled hIL-1Ra expression at biologically relevant levels and effectively transduced the same cell types, primarily synovial fibroblasts and, to a lesser degree, chondrocytes in articular cartilage. These results provide optimism that AAV vectors can be effectively adapted for gene delivery to large human joints affected by OA. PMID:23151520

  11. Improved methods of AAV-mediated gene targeting for human cell lines using ribosome-skipping 2A peptide

    PubMed Central

    Karnan, Sivasundaram; Ota, Akinobu; Konishi, Yuko; Wahiduzzaman, Md; Hosokawa, Yoshitaka; Konishi, Hiroyuki

    2016-01-01

    The adeno-associated virus (AAV)-based targeting vector has been one of the tools commonly used for genome modification in human cell lines. It allows for relatively efficient gene targeting associated with 1–4-log higher ratios of homologous-to-random integration of targeting vectors (H/R ratios) than plasmid-based targeting vectors, without actively introducing DNA double-strand breaks. In this study, we sought to improve the efficiency of AAV-mediated gene targeting by introducing a 2A-based promoter-trap system into targeting constructs. We generated three distinct AAV-based targeting vectors carrying 2A for promoter trapping, each targeting a GFP-based reporter module incorporated into the genome, PIGA exon 6 or PIGA intron 5. The absolute gene targeting efficiencies and H/R ratios attained using these vectors were assessed in multiple human cell lines and compared with those attained using targeting vectors carrying internal ribosome entry site (IRES) for promoter trapping. We found that the use of 2A for promoter trapping increased absolute gene targeting efficiencies by 3.4–28-fold and H/R ratios by 2–5-fold compared to values obtained with IRES. In CRISPR-Cas9-assisted gene targeting using plasmid-based targeting vectors, the use of 2A did not enhance the H/R ratios but did upregulate the absolute gene targeting efficiencies compared to the use of IRES. PMID:26657635

  12. Development of genetically engineered human intestinal cells for regulated insulin secretion using rAAV-mediated gene transfer.

    PubMed

    Tang, Shiue-Cheng; Sambanis, Athanassios

    2003-04-01

    Cell-based therapies for treating insulin-dependent diabetes (IDD) can provide a more physiologic regulation of blood glucose levels in a less invasive fashion than daily insulin injections. Promising cells include intestinal enteroendocrine cells genetically engineered to secrete insulin in response to physiologic stimuli; responsiveness occurs at the exocytosis level to regulate the acute release of recombinant insulin. In this work, we established a human cellular model to demonstrate that meat hydrolysate can simultaneously stimulate glucagon-like peptide-1 (GLP-1, an enteroendocrine cell-derived incretin hormone) and recombinant insulin secretion from the engineered human NCI-H716 intestinal cell line. Cells were genetically modified using the recombinant adeno-associated virus (rAAV)-mediated insulin gene transfer. Recombinant cells were then differentiated to display endocrine features, in particular the formation of granule-like compartments. A fusion protein of insulin and enhanced green fluorescence protein (EGFP) was designed to reveal the compartments of localization of the fusion protein and assess its co-localization with endogenous GLP-1. Our work provides a unique human cellular model for regulated insulin release through genetic engineering of GLP-1-secreting intestinal cells, which is expected to be useful for cell-based therapies of IDD. PMID:12659868

  13. Depressive-like phenotype induced by AAV-mediated overexpression of human α-synuclein in midbrain dopaminergic neurons.

    PubMed

    Caudal, D; Alvarsson, A; Björklund, A; Svenningsson, P

    2015-11-01

    Parkinson's disease (PD) is a neurodegenerative disorder characterized by a progressive loss of nigral dopaminergic neurons and by the presence of aggregates containing α-synuclein called Lewy bodies. Viral vector-induced overexpression of α-synuclein in dopaminergic neurons represents a model of PD which recapitulates disease progression better than commonly used neurotoxin models. Previous studies using this model have reported motor and cognitive impairments, whereas depression, mood and anxiety phenotypes are less described. To investigate these psychiatric phenotypes, Sprague-Dawley rats received bilateral injections of a recombinant adeno-associated virus (AAV) vector expressing human α-synuclein or GFP into the substantia nigra pars compacta. Behavior was assessed at two timepoints: 3 and 8 weeks post-injection. We report that nigral α-synuclein overexpression led to a pronounced nigral dopaminergic cell loss accompanied by a smaller cell loss in the ventral tegmental area, and to a decreased striatal density of dopaminergic fibers. The AAV-α-synuclein group exhibited modest, but significant motor impairments 8 weeks after vector administration. The AAV-α-synuclein group displayed depressive-like behavior in the forced swim test after 3 weeks, and reduced sucrose preference at week 8. At both timepoints, overexpression of α-synuclein was linked to a hyperactive hypothalamic-pituitary-adrenal (HPA) axis regulation of corticosterone. The depressive-like phenotype was also correlated with decreased nigral brain-derived neurotrophic factor and spinophilin levels, and with decreased striatal levels of the activity-regulated cytoskeleton-associated protein. This study demonstrates that AAV-mediated α-synuclein overexpression in dopamine neurons is not only useful to model motor impairments of PD, but also depression. This study also provides evidence that depression in experimental Parkinsonism is correlated to dysregulation of the HPA axis and to

  14. RNAi-Assisted Genome Evolution (RAGE) in Saccharomyces cerevisiae.

    PubMed

    Si, Tong; Zhao, Huimin

    2016-01-01

    RNA interference (RNAi)-assisted genome evolution (RAGE) applies directed evolution principles to engineer Saccharomyces cerevisiae genomes. Here, we use acetic acid tolerance as a target trait to describe the key steps of RAGE. Briefly, iterative cycles of RNAi screening are performed to accumulate multiplex knockdown modifications, enabling directed evolution of the yeast genome and continuous improvement of a target phenotype. Detailed protocols are provided on the reconstitution of RNAi machinery, creation of genome-wide RNAi libraries, identification and integration of beneficial knockdown cassettes, and repeated RAGE cycles. PMID:27581294

  15. AAV-Mediated Gene Therapy in the Guanylate Cyclase (RetGC1/RetGC2) Double Knockout Mouse Model of Leber Congenital Amaurosis

    PubMed Central

    Boye, Sanford L.; Peshenko, Igor V.; Huang, Wei Chieh; Min, Seok Hong; McDoom, Issam; Kay, Christine N.; Liu, Xuan; Dyka, Frank M.; Foster, Thomas C.; Umino, Yumiko; Karan, Sukanya; Jacobson, Samuel G.; Baehr, Wolfgang; Dizhoor, Alexander; Hauswirth, William W.

    2013-01-01

    Mutations in GUCY2D are associated with recessive Leber congenital amaurosis-1 (LCA1). GUCY2D encodes photoreceptor-specific, retinal guanylate cyclase-1 (RetGC1). Reports of retinal degeneration in LCA1 are conflicting; some describe no obvious degeneration and others report loss of both rods and cones. Proof of concept studies in models representing the spectrum of phenotypes is warranted. We have previously demonstrated adeno-associated virus (AAV)-mediated RetGC1 is therapeutic in GC1ko mice, a model exhibiting loss of cones only. The purpose of this study was to characterize AAV-mediated gene therapy in the RetGC1/RetGC2 double knockout (GCdko) mouse, a model lacking rod and cone function and exhibiting progressive loss of both photoreceptor subclasses. Use of this model also allowed for the evaluation of the functional efficiency of transgenic RetGC1 isozyme. Subretinal delivery of AAV8(Y733F) vector containing the human rhodopsin kinase (hGRK1) promoter driving murine Gucy2e was performed in GCdko mice at various postnatal time points. Treatment resulted in restoration of rod and cone function at all treatment ages and preservation of retinal structure in GCdko mice treated as late as 7 weeks of age. Functional gains and structural preservation were stable for at least 1 year. Treatment also conferred cortical- and subcortical-based visually-guided behavior. Functional efficiency of transgenic RetGC1 was indistinguishable from that of endogenous isozyme in congenic wild-type (WT) mice. This study clearly demonstrates AAV-mediated RetGC1 expression restores function to and preserves structure of rod and cone photoreceptors in a degenerative model of retinal guanylate cyclase deficiency, further supporting development of an AAV-based vector for treatment of LCA1. PMID:23210611

  16. AAV-mediated transduction and targeting of retinal bipolar cells with improved mGluR6 promoters in rodents and primates.

    PubMed

    Lu, Q; Ganjawala, T H; Ivanova, E; Cheng, J G; Troilo, D; Pan, Z-H

    2016-08-01

    Adeno-associated virus (AAV) vectors have been a powerful gene delivery vehicle to the retina for basic research and gene therapy. For many of these applications, achieving cell type-specific targeting and high transduction efficiency is desired. Recently, there has been increasing interest in AAV-mediated gene targeting to specific retinal bipolar cell types. A 200-bp enhancer in combination with a basal SV40 promoter has been commonly used to target transgenes into ON-type bipolar cells. In the current study, we searched for additional cis-regulatory elements in the mGluR6 gene for improving AAV-mediated transduction efficiency into retinal bipolar cells. Our results showed that the combination of the endogenous mGluR6 promoter with additional enhancers in the introns of the mGluR6 gene markedly enhanced AAV transduction efficiency as well as made the targeting more selective for rod bipolar cells in mice. Furthermore, the AAV vectors with the improved promoter could target to ON bipolar cells with robust transduction efficiency in the parafovea and the far peripheral retina of marmoset monkeys. The improved mGluR6 promoter constructs could provide a valuable tool for genetic manipulation in rod bipolar cells in mice and facilitate clinical applications for ON bipolar cell-based gene therapies. PMID:27115727

  17. AAV-Mediated Gene Transfer of the Obesity-Associated Gene Etv5 in Rat Midbrain Does Not Affect Energy Balance or Motivated Behavior

    PubMed Central

    Boender, Arjen J.; Koning, Nivard A.; van den Heuvel, José K.; Luijendijk, Mieneke C. M.; van Rozen, Andrea J.; la Fleur, Susanne E.; Adan, Roger A. H.

    2014-01-01

    Several genome-wide association studies have implicated the transcription factor E-twenty- six version 5 (Etv5) in the regulation of body mass index. Further substantiating the role of Etv5 in feeding behavior are the findings that targeted disruption of Etv5 in mice leads to decreased body weight gain and that expression of Etv5 is decreased in the ventral tegmental area and substantia nigra pars compacta (VTA/SNpc) after food restriction. As Etv5 has been suggested to influence dopaminergic neurotransmission by driving the expression of genes that are responsible for the synthesis and release of dopamine, we investigated if expression levels of Etv5 are dependent on nutritional state and subsequently influence the expression levels of tyrosine hydroxylase. While it was shown that Etv5 expression in the VTA/SNpc increases after central administration of leptin and that Etv5 was able to drive expression of tyrosine hydroxylase in vitro, AAV-mediated gene transfer of Etv5 into the VTA/SNpc of rats did not alter expression of tyrosine hydroxylase in vivo. Moreover, AAV-mediated gene transfer of Etv5 in the VTA/SNpc did not affect measures of energy balance or performances in a progressive ratio schedule. Thus, these data do not support a role for increased expression of Etv5 in the VTA/SNpc in the regulation of feeding behavior. PMID:24710089

  18. RNAi induced knockdown of a cadherin-like protein (EF531715) does not affect toxicity of Cry34/35Ab1 or Cry3Aa to Diabrotica virgifera virgifera larvae (Coleoptera: Chrysomelidae).

    PubMed

    Tan, Sek Yee; Rangasamy, Murugesan; Wang, Haichuan; Vélez, Ana María; Hasler, James; McCaskill, David; Xu, Tao; Chen, Hong; Jurzenski, Jessica; Kelker, Matthew; Xu, Xiaoping; Narva, Kenneth; Siegfried, Blair D

    2016-08-01

    The western corn rootworm (WCR), Diabrotica virgifera virgifera LeConte, is an important maize pest throughout most of the U.S. Corn Belt. Bacillus thuringiensis (Bt) insecticidal proteins including modified Cry3Aa and Cry34/35Ab1 have been expressed in transgenic maize to protect against WCR feeding damage. To date, there is limited information regarding the WCR midgut target sites for these proteins. In this study, we examined whether a cadherin-like gene from Diabrotica virgifera virgifera (DvvCad; GenBank accession # EF531715) associated with WCR larval midgut tissue is necessary for Cry3Aa or Cry34/35Ab1 toxicity. Experiments were designed to examine the sensitivity of WCR to trypsin activated Cry3Aa and Cry34/35Ab1 after oral feeding of the DvvCad dsRNA to knockdown gene expression. Quantitative real-time PCR confirmed that DvvCad mRNA transcript levels were reduced in larvae treated with cadherin dsRNA. Relative cadherin expression by immunoblot analysis and nano-liquid chromatography - mass spectrometry (nanoLC-MS) of WCR neonate brush border membrane vesicle (BBMV) preparations exposed to DvvCad dsRNA confirmed reduced cadherin expression when compared to BBMV from untreated larvae. However, the larval mortality and growth inhibition of WCR neonates exposed to cadherin dsRNA for two days followed by feeding exposure to either Cry3Aa or Cry34/35Ab1 for four days was not significantly different to that observed in insects exposed to either Cry3Aa or Cry34/35Ab1 alone. In combination, these results suggest that cadherin is unlikely to be involved in the toxicity of Cry3Aa or Cry34/35Ab1 to WCR. PMID:27334721

  19. RNAi2015 - Ten years of RNAi Oxford.

    PubMed

    Bewicke-Copley, Findlay; Samuel, Priya; Carter, David Rf

    2015-01-01

    The tenth RNAi conference was held at St. Hilda's College Oxford on the 24-26 March 2015. The conference offered researchers from all over the world the chance to present, discuss and discover work pertaining to the field of RNAi. RNAi has become an essential technique in genomic research for functional validation as well as an exciting avenue to explore in therapeutic medicine. Emerging techniques such as CRISPR as well as improvements in efficiency of existing techniques and expansions in libraries have cemented the importance of RNAi at the cutting edge of research. Featured presentations and posters showcased recent research in the field ranging from RNA detection in bio fluids through to potential oligonucleotide therapies. PMID:26557153

  20. AAV-mediated Liver-specific MPV17 Expression Restores mtDNA Levels and Prevents Diet-induced Liver Failure

    PubMed Central

    Bottani, Emanuela; Giordano, Carla; Civiletto, Gabriele; Di Meo, Ivano; Auricchio, Alberto; Ciusani, Emilio; Marchet, Silvia; Lamperti, Costanza; d'Amati, Giulia; Viscomi, Carlo; Zeviani, Massimo

    2014-01-01

    Mutations in human MPV17 cause a hepatocerebral form of mitochondrial DNA depletion syndrome (MDS) hallmarked by early-onset liver failure, leading to premature death. Liver transplantation and frequent feeding using slow-release carbohydrates are the only available therapies, although surviving patients eventually develop slowly progressive peripheral and central neuropathy. The physiological role of Mpv17, including its functional link to mitochondrial DNA (mtDNA) maintenance, is still unclear. We show here that Mpv17 is part of a high molecular weight complex of unknown composition, which is essential for mtDNA maintenance in critical tissues, i.e. liver, of a Mpv17 knockout mouse model. On a standard diet, Mpv17−/− mouse shows hardly any symptom of liver dysfunction, but a ketogenic diet (KD) leads these animals to liver cirrhosis and failure. However, when expression of human MPV17 is carried out by adeno-associated virus (AAV)-mediated gene replacement, the Mpv17 knockout mice are able to reconstitute the Mpv17-containing supramolecular complex, restore liver mtDNA copy number and oxidative phosphorylation (OXPHOS) proficiency, and prevent liver failure induced by the KD. These results open new therapeutic perspectives for the treatment of MPV17-related liver-specific MDS. PMID:24247928

  1. RNAi Trigger Delivery into Anopheles gambiae Pupae

    PubMed Central

    Regna, Kimberly; Harrison, Rachel M.; Heyse, Shannon A.; Chiles, Thomas C.; Michel, Kristin; Muskavitch, Marc A. T.

    2016-01-01

    RNA interference (RNAi), a naturally occurring phenomenon in eukaryotic organisms, is an extremely valuable tool that can be utilized in the laboratory for functional genomic studies. The ability to knockdown individual genes selectively via this reverse genetic technique has allowed many researchers to rapidly uncover the biological roles of numerous genes within many organisms, by evaluation of loss-of-function phenotypes. In the major human malaria vector Anopheles gambiae, the predominant method used to reduce the function of targeted genes involves injection of double-stranded (dsRNA) into the hemocoel of the adult mosquito. While this method has been successful, gene knockdown in adults excludes the functional assessment of genes that are expressed and potentially play roles during pre-adult stages, as well as genes that are expressed in limited numbers of cells in adult mosquitoes. We describe a method for the injection of Serine Protease Inhibitor 2 (SRPN2) dsRNA during the early pupal stage and validate SRPN2 protein knockdown by observing decreased target protein levels and the formation of melanotic pseudo-tumors in SRPN2 knockdown adult mosquitoes. This evident phenotype has been described previously for adult stage knockdown of SRPN2 function, and we have recapitulated this adult phenotype by SRPN2 knockdown initiated during pupal development. When used in conjunction with a dye-labeled dsRNA solution, this technique enables easy visualization by simple light microscopy of injection quality and distribution of dsRNA in the hemocoel. PMID:27023367

  2. Cytochromr b expression and RNAi knockdown in Aedes aegypti.

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Cytochrome b, coded by mitochondrial DNA, is one of the cytochromes involved in the electron transport in the respiratory chain of mitochondria. Cytochrome b is a critical intermediate in mitoptosis, i.e. a mitochondrial death pathway. To reveal whether cytochrome b of the mosquito Aedes aegypti (Ae...

  3. RNAi mediated Tiam1 gene knockdown inhibits invasion of retinoblastoma.

    PubMed

    Subramanian, Nithya; Navaneethakrishnan, Saranya; Biswas, Jyotirmay; Kanwar, Rupinder K; Kanwar, Jagat R; Krishnakumar, Subramanian

    2013-01-01

    T lymphoma invasion and metastasis protein (Tiam1) is up-regulated in variety of cancers and its expression level is related to metastatic potential of the type of cancer. Earlier, Tiam1 was shown to be overexpressed in retinoblastoma (RB) and we hypothesized that it was involved in invasiveness of RB. This was tested by silencing Tiam1 in RB cell lines (Y79 and Weri-Rb1) using siRNA pool, targeting different regions of Tiam1 mRNA. The cDNA microarray of Tiam1 silenced cells showed gene regulations altered by Tiam1 were predominantly on the actin cytoskeleton interacting proteins, apoptotic initiators and tumorogenic potential targets. The silenced phenotype resulted in decreased growth and increased apoptosis with non-invasive characteristics. Transfection of full length and N-terminal truncated construct (C1199) clearly revealed membrane localization of Tiam1 and not in the case of C580 construct. F-actin staining showed the interaction of Tiam1 with actin in the membrane edges that leads to ruffling, and also imparts varying invasive potential to the cell. The results obtained from our study show for the first time that Tiam1 modulates the cell invasion, mediated by actin cytoskeleton remodeling in RB. PMID:23950931

  4. Modulation of gene expression by RNAi.

    PubMed

    Wójcik, Cezary; Fabunmi, Rosalind; DeMartino, George N

    2005-01-01

    RNA interference (RNAi) is a form of posttranscriptional gene silencing in which the presence within the cell of double-stranded RNA (dsRNA) leads to the specific degradation of mRNA with a complimentary sequence. RNAi is a natural phenomenon that can be exploited as a powerful tool to study gene function by generating gene "knockdowns" in various cell types. RNAi is mediated by short interfering RNAs (siRNAs), which are generated within cells from long dsRNAs. To avoid generalized toxic effects, mammalian cells are transfected directly with 21-23-bp-long siRNAs generated either by chemical synthesis or obtained by a series of enzymatic reactions. The present chapter deals with siRNA design, synthesis, transfection, and readout of efficiency in a mammalian cell culture system. The general principle is illustrated by the functional knockdown of p97/VCP (valosin-containing protein) in HeLa cells using five different siRNA sequences. PMID:16028696

  5. Core RNAi Machinery and Sid1, a Component for Systemic RNAi, in the Hemipteran Insect, Aphis glycines

    PubMed Central

    Bansal, Raman; Michel, Andy P.

    2013-01-01

    RNA interference (RNAi) offers a novel tool to manage hemipteran pests. For the success of RNAi based pest control in the field, a robust and systemic RNAi response is a prerequisite. We identified and characterized major genes of the RNAi machinery, Dicer2 (Dcr2), Argonaute2 (Ago2), and R2d2 in Aphis glycines, a serious pest of soybean. The A. glycines genome encodes for at least one copy of Dcr2, R2d2 and Ago2. Comparative and molecular evolution analyses (dN/dS) showed that domain regions of encoded proteins are highly conserved, whereas linker (non-domain) regions are diversified. Sequence homology and phylogenetic analyses suggested that the RNAi machinery of A. glycines is more similar to that of Tribolium casteneum as compared to that of Drosophila melanogaster. We also characterized Sid1, a major gene implicated in the systemic response for RNAi-mediated gene knockdown. Through qPCR, Dcr2, R2d2, Ago2, and Sid1 were found to be expressed at similar levels in various tissues, but higher expression of Dcr2, R2d2, and Ago2 was seen in first and second instars. Characterization of RNAi pathway and Sid1 in A. glycines will provide the foundation of future work for controlling one of the most important insect pests of soybean in North America. PMID:23396108

  6. RNAi microarray analysis in cultured mammalian cells.

    PubMed

    Mousses, Spyro; Caplen, Natasha J; Cornelison, Robert; Weaver, Don; Basik, Mark; Hautaniemi, Sampsa; Elkahloun, Abdel G; Lotufo, Roberto A; Choudary, Ashish; Dougherty, Edward R; Suh, Ed; Kallioniemi, Olli

    2003-10-01

    RNA interference (RNAi) mediated by small interfering RNAs (siRNAs) is a powerful new tool for analyzing gene knockdown phenotypes in living mammalian cells. To facilitate large-scale, high-throughput functional genomics studies using RNAi, we have developed a microarray-based technology for highly parallel analysis. Specifically, siRNAs in a transfection matrix were first arrayed on glass slides, overlaid with a monolayer of adherent cells, incubated to allow reverse transfection, and assessed for the effects of gene silencing by digital image analysis at a single cell level. Validation experiments with HeLa cells stably expressing GFP showed spatially confined, sequence-specific, time- and dose-dependent inhibition of green fluorescence for those cells growing directly on microspots containing siRNA targeting the GFP sequence. Microarray-based siRNA transfections analyzed with a custom-made quantitative image analysis system produced results that were identical to those from traditional well-based transfection, quantified by flow cytometry. Finally, to integrate experimental details, image analysis, data display, and data archiving, we developed a prototype information management system for high-throughput cell-based analyses. In summary, this RNAi microarray platform, together with ongoing efforts to develop large-scale human siRNA libraries, should facilitate genomic-scale cell-based analyses of gene function. PMID:14525932

  7. Current issues of RNAi therapeutics delivery and development.

    PubMed

    Haussecker, D

    2014-12-10

    12 years following the discovery of the RNAi mechanism in Man, a number of RNAi therapeutics development candidates have emerged with profiles suggesting that they could become drugs of significant medical importance for diseases like TTR amyloidosis, HBV, solid cancers, and hemophilia. Despite this robust progress, the perception of RNAi therapeutics has been on a roller-coaster ride driven not only by science, but also regulatory trends, the stock markets, and Big Pharma business development decisions [1]. This presentation provides an update on the current state of RNAi therapeutics development with a particular focus on what RNAi delivery can achieve today and key challenges to be overcome to expand therapeutic opportunities. The delivery of RNAi triggers to disease-relevant cell types clearly represents the rate-limiting factor in broadly expanding the applicability of RNAi therapeutics. Today, with at least 3 delivery options (lipid nanoparticles/LNPs, GalNAc-siRNA conjugates, Dynamic PolyConjugates/DPCs) for which profound gene knockdowns have been demonstrated in non-human primates and in the clinic, RNAi therapeutics should in principle be able to address most diseases related to gene expression in the liver. Given the central importance of the liver in systemic physiology, this already represents a significant therapeutic and commercial opportunity rivaling that of e.g. monoclonal antibodies. Beyond the liver, there is a reason to believe that current RNAi therapeutics technologies can address a number of solid tumors (e.g. LNPs), diseases of the eye (e.g. self-delivering RNAi triggers) as well as diseases involving the respiratory epithelium (e.g. aerosolized LNPs), certain phagocytic cells (LNPs), hematopoietic stem cells and their progeny (lentiviral DNA-directed RNAi), vascular endothelial cells (cationic lipoplexes), and certain cell types in the kidney (self-delivering RNAi triggers, DPCs; Table 1). Despite this success, there has been a sense that

  8. Plant-mediated RNAi of a gap gene-enhanced tobacco tolerance against the Myzus persicae.

    PubMed

    Mao, Jianjun; Zeng, Fanrong

    2014-02-01

    Plant-mediated RNAi has been developed as a powerful weapon in the fight against agricultural insect pests. The gap gene hunchback (hb) is of crucial importance in insect axial patterning and knockdown of hb is deforming and lethal to the next generation. The peach potato aphid, Myzus persicae (Sulzer), has many host plants and can be found throughout the world. To investigate the effect of plant-mediated RNAi on control of this insect, the hb gene in M. persicae was cloned, plant RNAi vector was constructed, and transgenic tobacco expressing Mphb dsRNA was developed. Transgenic tobacco had a different integration pattern of the transgene. Bioassays were performed by applying neonate aphids to homozygous transgenic plants in the T2 generation. Results revealed that continuous feeding of transgenic diet reduced Mphb mRNA level in the fed aphids and inhibited insect reproduction, indicating successful knockdown of the target gene in M. persicae by plant-mediated RNAi. PMID:23949691

  9. RNAi phenotypes are influenced by the genetic background of the injected strain

    PubMed Central

    2013-01-01

    Background RNA interference (RNAi) is a powerful tool to study gene function in organisms that are not amenable to classical forward genetics. Hence, together with the ease of comprehensively identifying genes by new generation sequencing, RNAi is expanding the scope of animal species and questions that can be addressed in terms of gene function. In the case of genetic mutants, the genetic background of the strains used is known to influence the phenotype while this has not been described for RNAi experiments. Results Here we show in the red flour beetle Tribolium castaneum that RNAi against Tc-importin α1 leads to different phenotypes depending on the injected strain. We rule out off target effects and show that sequence divergence does not account for this difference. By quantitatively comparing phenotypes elicited by RNAi knockdown of four different genes we show that there is no general difference in RNAi sensitivity between these strains. Finally, we show that in case of Tc-importin α1 the difference depends on the maternal genotype. Conclusions These results show that in RNAi experiments strain specific differences have to be considered and that a proper documentation of the injected strain is required. This is especially important for the increasing number of emerging model organisms that are being functionally investigated using RNAi. In addition, our work shows that RNAi is suitable to systematically identify the differences in the gene regulatory networks present in populations of the same species, which will allow novel insights into the evolution of animal diversity. PMID:23324472

  10. High-Throughput, Liquid-Based Genome-Wide RNAi Screening in C. elegans.

    PubMed

    O'Reilly, Linda P; Knoerdel, Ryan R; Silverman, Gary A; Pak, Stephen C

    2016-01-01

    RNA interference (RNAi) is a process in which double-stranded RNA (dsRNA) molecules mediate the inhibition of gene expression. RNAi in C. elegans can be achieved by simply feeding animals with bacteria expressing dsRNA against the gene of interest. This "feeding" method has made it possible to conduct genome-wide RNAi experiments for the systematic knockdown and subsequent investigation of almost every single gene in the genome. Historically, these genome-scale RNAi screens have been labor and time intensive. However, recent advances in automated, high-throughput methodologies have allowed the development of more rapid and efficient screening protocols. In this report, we describe a fast and efficient, liquid-based method for genome-wide RNAi screening. PMID:27581291

  11. RNAi pathway participates in chromosome segregation in mammalian cells

    PubMed Central

    Huang, Chuan; Wang, Xiaolin; Liu, Xu; Cao, Shuhuan; Shan, Ge

    2015-01-01

    The RNAi machinery is a mighty regulator in a myriad of life events. Despite lines of evidence that small RNAs and components of the RNAi pathway may be associated with structure and behavior of mitotic chromosomes in diverse organisms, a direct role of the RNAi pathway in mammalian mitotic chromosome segregation remains elusive. Here we report that Dicer and AGO2, two central components of the mammalian RNAi pathway, participate in the chromosome segregation. Knockdown of Dicer or AGO2 results in a higher incidence of chromosome lagging, and this effect is independent from microRNAs as examined with DGCR8 knockout cells. Further investigation has revealed that α-satellite RNA, a noncoding RNA derived from centromeric repeat region, is managed by AGO2 under the guidance of endogenous small interference RNAs (ASAT siRNAs) generated by Dicer. Furthermore, the slicer activity of AGO2 is essential for the chromosome segregation. Level and distribution of chromosome-associated α-satellite RNA have crucial regulatory effect on the localization of centromeric proteins such as centromere protein C1 (CENPC1). With these results, we also provide a paradigm in which the RNAi pathway participates in vital cellular events through the maintenance of level and distribution of noncoding RNAs in cells.

  12. RNAi Screening in Spodoptera frugiperda.

    PubMed

    Ghosh, Subhanita; Singh, Gatikrushna; Sachdev, Bindiya; Kumar, Ajit; Malhotra, Pawan; Mukherjee, Sunil K; Bhatnagar, Raj K

    2016-01-01

    RNA interference is a potent and precise reverse genetic approach to carryout large-scale functional genomic studies in a given organism. During the past decade, RNAi has also emerged as an important investigative tool to understand the process of viral pathogenesis. Our laboratory has successfully generated transgenic reporter and RNAi sensor line of Spodoptera frugiperda (Sf21) cells and developed a reversal of silencing assay via siRNA or shRNA guided screening to investigate RNAi factors or viral pathogenic factors with extraordinary fidelity. Here we describe empirical approaches and conceptual understanding to execute successful RNAi screening in Spodoptera frugiperda 21-cell line. PMID:27581295

  13. Identification of Neural Outgrowth Genes using Genome-Wide RNAi

    PubMed Central

    Sepp, Katharine J.; Hong, Pengyu; Lizarraga, Sofia B.; Liu, Judy S.; Mejia, Luis A.; Walsh, Christopher A.; Perrimon, Norbert

    2008-01-01

    While genetic screens have identified many genes essential for neurite outgrowth, they have been limited in their ability to identify neural genes that also have earlier critical roles in the gastrula, or neural genes for which maternally contributed RNA compensates for gene mutations in the zygote. To address this, we developed methods to screen the Drosophila genome using RNA-interference (RNAi) on primary neural cells and present the results of the first full-genome RNAi screen in neurons. We used live-cell imaging and quantitative image analysis to characterize the morphological phenotypes of fluorescently labelled primary neurons and glia in response to RNAi-mediated gene knockdown. From the full genome screen, we focused our analysis on 104 evolutionarily conserved genes that when downregulated by RNAi, have morphological defects such as reduced axon extension, excessive branching, loss of fasciculation, and blebbing. To assist in the phenotypic analysis of the large data sets, we generated image analysis algorithms that could assess the statistical significance of the mutant phenotypes. The algorithms were essential for the analysis of the thousands of images generated by the screening process and will become a valuable tool for future genome-wide screens in primary neurons. Our analysis revealed unexpected, essential roles in neurite outgrowth for genes representing a wide range of functional categories including signalling molecules, enzymes, channels, receptors, and cytoskeletal proteins. We also found that genes known to be involved in protein and vesicle trafficking showed similar RNAi phenotypes. We confirmed phenotypes of the protein trafficking genes Sec61alpha and Ran GTPase using Drosophila embryo and mouse embryonic cerebral cortical neurons, respectively. Collectively, our results showed that RNAi phenotypes in primary neural culture can parallel in vivo phenotypes, and the screening technique can be used to identify many new genes that have

  14. Environmental RNAi in herbivorous insects

    PubMed Central

    Ivashuta, Sergey; Zhang, Yuanji; Wiggins, B. Elizabeth; Ramaseshadri, Partha; Segers, Gerrit C.; Johnson, Steven; Meyer, Steve E.; Kerstetter, Randy A.; McNulty, Brian C.; Bolognesi, Renata; Heck, Gregory R.

    2015-01-01

    Environmental RNAi (eRNAi) is a sequence-specific regulation of endogenous gene expression in a receptive organism by exogenous double-stranded RNA (dsRNA). Although demonstrated under artificial dietary conditions and via transgenic plant presentations in several herbivorous insects, the magnitude and consequence of exogenous dsRNA uptake and the role of eRNAi remains unknown under natural insect living conditions. Our analysis of coleopteran insects sensitive to eRNAi fed on wild-type plants revealed uptake of plant endogenous long dsRNAs, but not small RNAs. Subsequently, the dsRNAs were processed into 21 nt siRNAs by insects and accumulated in high quantities in insect cells. No accumulation of host plant-derived siRNAs was observed in lepidopteran larvae that are recalcitrant to eRNAi. Stability of ingested dsRNA in coleopteran larval gut followed by uptake and transport from the gut to distal tissues appeared to be enabling factors for eRNAi. Although a relatively large number of distinct coleopteran insect-processed plant-derived siRNAs had sequence complementarity to insect transcripts, the vast majority of the siRNAs were present in relatively low abundance, and RNA-seq analysis did not detect a significant effect of plant-derived siRNAs on insect transcriptome. In summary, we observed a broad genome-wide uptake of plant endogenous dsRNA and subsequent processing of ingested dsRNA into 21 nt siRNAs in eRNAi-sensitive insects under natural feeding conditions. In addition to dsRNA stability in gut lumen and uptake, dosage of siRNAs targeting a given insect transcript is likely an important factor in order to achieve measurable eRNAi-based regulation in eRNAi-competent insects that lack an apparent silencing amplification mechanism. PMID:25802407

  15. Preclinical evaluation of RNAi as a treatment for transthyretin-mediated amyloidosis.

    PubMed

    Butler, James S; Chan, Amy; Costelha, Susete; Fishman, Shannon; Willoughby, Jennifer L S; Borland, Todd D; Milstein, Stuart; Foster, Donald J; Gonçalves, Paula; Chen, Qingmin; Qin, June; Bettencourt, Brian R; Sah, Dinah W; Alvarez, Rene; Rajeev, Kallanthottathil G; Manoharan, Muthiah; Fitzgerald, Kevin; Meyers, Rachel E; Nochur, Saraswathy V; Saraiva, Maria J; Zimmermann, Tracy S

    2016-06-01

    ATTR amyloidosis is a systemic, debilitating and fatal disease caused by transthyretin (TTR) amyloid accumulation. RNA interference (RNAi) is a clinically validated technology that may be a promising approach to the treatment of ATTR amyloidosis. The vast majority of TTR, the soluble precursor of TTR amyloid, is expressed and synthesized in the liver. RNAi technology enables robust hepatic gene silencing, the goal of which would be to reduce systemic levels of TTR and mitigate many of the clinical manifestations of ATTR that arise from hepatic TTR expression. To test this hypothesis, TTR-targeting siRNAs were evaluated in a murine model of hereditary ATTR amyloidosis. RNAi-mediated silencing of hepatic TTR expression inhibited TTR deposition and facilitated regression of existing TTR deposits in pathologically relevant tissues. Further, the extent of deposit regression correlated with the level of RNAi-mediated knockdown. In comparison to the TTR stabilizer, tafamidis, RNAi-mediated TTR knockdown led to greater regression of TTR deposits across a broader range of affected tissues. Together, the data presented herein support the therapeutic hypothesis behind TTR lowering and highlight the potential of RNAi in the treatment of patients afflicted with ATTR amyloidosis. PMID:27033334

  16. Preclinical evaluation of RNAi as a treatment for transthyretin-mediated amyloidosis

    PubMed Central

    Butler, James S.; Chan, Amy; Costelha, Susete; Fishman, Shannon; Willoughby, Jennifer L. S.; Borland, Todd D.; Milstein, Stuart; Foster, Donald J.; Gonçalves, Paula; Chen, Qingmin; Qin, June; Bettencourt, Brian R.; Sah, Dinah W.; Alvarez, Rene; Rajeev, Kallanthottathil G.; Manoharan, Muthiah; Fitzgerald, Kevin; Meyers, Rachel E.; Nochur, Saraswathy V.; Saraiva, Maria J.; Zimmermann, Tracy S.

    2016-01-01

    Abstract ATTR amyloidosis is a systemic, debilitating and fatal disease caused by transthyretin (TTR) amyloid accumulation. RNA interference (RNAi) is a clinically validated technology that may be a promising approach to the treatment of ATTR amyloidosis. The vast majority of TTR, the soluble precursor of TTR amyloid, is expressed and synthesized in the liver. RNAi technology enables robust hepatic gene silencing, the goal of which would be to reduce systemic levels of TTR and mitigate many of the clinical manifestations of ATTR that arise from hepatic TTR expression. To test this hypothesis, TTR-targeting siRNAs were evaluated in a murine model of hereditary ATTR amyloidosis. RNAi-mediated silencing of hepatic TTR expression inhibited TTR deposition and facilitated regression of existing TTR deposits in pathologically relevant tissues. Further, the extent of deposit regression correlated with the level of RNAi-mediated knockdown. In comparison to the TTR stabilizer, tafamidis, RNAi-mediated TTR knockdown led to greater regression of TTR deposits across a broader range of affected tissues. Together, the data presented herein support the therapeutic hypothesis behind TTR lowering and highlight the potential of RNAi in the treatment of patients afflicted with ATTR amyloidosis. PMID:27033334

  17. Application of RNAi to Genomic Drug Target Validation in Schistosomes

    PubMed Central

    Guidi, Alessandra; Mansour, Nuha R.; Paveley, Ross A.; Carruthers, Ian M.; Besnard, Jérémy; Hopkins, Andrew L.; Gilbert, Ian H.; Bickle, Quentin D.

    2015-01-01

    Concerns over the possibility of resistance developing to praziquantel (PZQ), has stimulated efforts to develop new drugs for schistosomiasis. In addition to the development of improved whole organism screens, the success of RNA interference (RNAi) in schistosomes offers great promise for the identification of potential drug targets to initiate drug discovery. In this study we set out to contribute to RNAi based validation of putative drug targets. Initially a list of 24 target candidates was compiled based on the identification of putative essential genes in schistosomes orthologous of C. elegans essential genes. Knockdown of Calmodulin (Smp_026560.2) (Sm-Calm), that topped this list, produced a phenotype characterised by waves of contraction in adult worms but no phenotype in schistosomula. Knockdown of the atypical Protein Kinase C (Smp_096310) (Sm-aPKC) resulted in loss of viability in both schistosomula and adults and led us to focus our attention on other kinase genes that were identified in the above list and through whole organism screening of known kinase inhibitor sets followed by chemogenomic evaluation. RNAi knockdown of these kinase genes failed to affect adult worm viability but, like Sm-aPKC, knockdown of Polo-like kinase 1, Sm-PLK1 (Smp_009600) and p38-MAPK, Sm-MAPK p38 (Smp_133020) resulted in an increased mortality of schistosomula after 2-3 weeks, an effect more marked in the presence of human red blood cells (hRBC). For Sm-PLK-1 the same effects were seen with the specific inhibitor, BI2536, which also affected viable egg production in adult worms. For Sm-PLK-1 and Sm-aPKC the in vitro effects were reflected in lower recoveries in vivo. We conclude that the use of RNAi combined with culture with hRBC is a reliable method for evaluating genes important for larval development. However, in view of the slow manifestation of the effects of Sm-aPKC knockdown in adults and the lack of effects of Sm-PLK-1 and Sm-MAPK p38 on adult viability, these

  18. Application of RNAi to Genomic Drug Target Validation in Schistosomes.

    PubMed

    Guidi, Alessandra; Mansour, Nuha R; Paveley, Ross A; Carruthers, Ian M; Besnard, Jérémy; Hopkins, Andrew L; Gilbert, Ian H; Bickle, Quentin D

    2015-05-01

    Concerns over the possibility of resistance developing to praziquantel (PZQ), has stimulated efforts to develop new drugs for schistosomiasis. In addition to the development of improved whole organism screens, the success of RNA interference (RNAi) in schistosomes offers great promise for the identification of potential drug targets to initiate drug discovery. In this study we set out to contribute to RNAi based validation of putative drug targets. Initially a list of 24 target candidates was compiled based on the identification of putative essential genes in schistosomes orthologous of C. elegans essential genes. Knockdown of Calmodulin (Smp_026560.2) (Sm-Calm), that topped this list, produced a phenotype characterised by waves of contraction in adult worms but no phenotype in schistosomula. Knockdown of the atypical Protein Kinase C (Smp_096310) (Sm-aPKC) resulted in loss of viability in both schistosomula and adults and led us to focus our attention on other kinase genes that were identified in the above list and through whole organism screening of known kinase inhibitor sets followed by chemogenomic evaluation. RNAi knockdown of these kinase genes failed to affect adult worm viability but, like Sm-aPKC, knockdown of Polo-like kinase 1, Sm-PLK1 (Smp_009600) and p38-MAPK, Sm-MAPK p38 (Smp_133020) resulted in an increased mortality of schistosomula after 2-3 weeks, an effect more marked in the presence of human red blood cells (hRBC). For Sm-PLK-1 the same effects were seen with the specific inhibitor, BI2536, which also affected viable egg production in adult worms. For Sm-PLK-1 and Sm-aPKC the in vitro effects were reflected in lower recoveries in vivo. We conclude that the use of RNAi combined with culture with hRBC is a reliable method for evaluating genes important for larval development. However, in view of the slow manifestation of the effects of Sm-aPKC knockdown in adults and the lack of effects of Sm-PLK-1 and Sm-MAPK p38 on adult viability, these

  19. Benefits of Recombinant Adeno-Associated Virus (rAAV)-Mediated Insulinlike Growth Factor I (IGF-I) Overexpression for the Long-Term Reconstruction of Human Osteoarthritic Cartilage by Modulation of the IGF-I Axis

    PubMed Central

    Weimer, Anja; Madry, Henning; Venkatesan, Jagadeesh K; Schmitt, Gertrud; Frisch, Janina; Wezel, Anna; Jung, Jochen; Kohn, Dieter; Terwilliger, Ernest F; Trippel, Stephen B; Cucchiarini, Magali

    2012-01-01

    Administration of therapeutic genes to human osteoarthritic (OA) cartilage is a potential approach to generate effective, durable treatments against this slow, progressive disorder. Here, we tested the ability of recombinant adeno-associated virus (rAAV)-mediated overexpression of human insulinlike growth factor (hIGF)-I to reproduce an original surface in human OA cartilage in light of the pleiotropic activities of the factor. We examined the proliferative, survival and anabolic effects of the rAAV-hIGF-I treatment in primary human normal and OA chondrocytes in vitro and in explant cultures in situ compared with control (reporter) vector delivery. Efficient, prolonged IGF-I secretion via rAAV stimulated the biological activities of OA chondrocytes in all the systems evaluated over extended periods of time, especially in situ, where it allowed for the long-term reconstruction of OA cartilage (at least for 90 d). Remarkably, production of high, stable amounts of IGF-I in OA cartilage using rAAV advantageously modulated the expression of central effectors of the IGF-I axis by downregulating IGF-I inhibitors (IGF binding protein [IGFBP]-3 and IGFBP4) while up-regulating key potentiators (IGFBP5, the IGF-I receptor and downstream mitogen-activated protein kinase/extracellular signal–regulated kinase 1/2 [MAPK/ERK-1/2] and phosphatidylinisitol-3/Akt [PI3K/Akt] signal transduction pathways), probably explaining the enhanced responsiveness of OA cartilage to IGF-I treatment. These findings show the benefits of directly providing an IGF-I sequence to articular cartilage via rAAV for the future treatment of human osteoarthritis. PMID:22160392

  20. FlyRNAi: the Drosophila RNAi screening center database

    PubMed Central

    Flockhart, Ian; Booker, Matthew; Kiger, Amy; Boutros, Michael; Armknecht, Susan; Ramadan, Nadire; Richardson, Kris; Xu, Andrew; Perrimon, Norbert; Mathey-Prevot, Bernard

    2006-01-01

    RNA interference (RNAi) has become a powerful tool for genetic screening in Drosophila. At the Drosophila RNAi Screening Center (DRSC), we are using a library of over 21 000 double-stranded RNAs targeting known and predicted genes in Drosophila. This library is available for the use of visiting scientists wishing to perform full-genome RNAi screens. The data generated from these screens are collected in the DRSC database () in a flexible format for the convenience of the scientist and for archiving data. The long-term goal of this database is to provide annotations for as many of the uncharacterized genes in Drosophila as possible. Data from published screens are available to the public through a highly configurable interface that allows detailed examination of the data and provides access to a number of other databases and bioinformatics tools. PMID:16381918

  1. RNAI: Future in insect management

    Technology Transfer Automated Retrieval System (TEKTRAN)

    RNA interference is a post-transcriptional, gene regulation mechanism found in virtually all plants and animals including insects. The demonstration of RNAi in insects and its successful use as a tool in the study of functional genomics opened the door to the development of a variety of novel, envir...

  2. Gene transfer as a strategy to achieve permanent cardioprotection II: rAAV-mediated gene therapy with heme oxygenase-1 limits infarct size 1 year later without adverse functional consequences

    PubMed Central

    Li, Qianhong; Guo, Yiru; Ou, Qinghui; Wu, Wen-Jian; Chen, Ning; Zhu, Xiaoping; Tan, Wei; Yuan, Fangping; Dawn, Buddhadeb; Luo, Li; Hunt, Gregory N.

    2013-01-01

    Extensive evidence indicates that heme oxygenase-1 (HO-1) exerts potent cytoprotective effects in response to stress. Previous studies have shown that gene therapy with HO-1 protects against myocardial ischemia/reperfusion injury for up to 8 weeks after gene transfer. However, the long-term effects of HO-1 gene therapy on myocardial ischemic injury and function are unknown. To address this issue, we created a recombinant adeno-associated viral vector carrying the HO-1 gene (rAAV/HO-1) that enables long-lasting transgene expression. Mice received injections in the anterior LV wall of rAAV/LacZ (LacZ group) or rAAV/HO-1 (HO-1 group); 1 year later, they were subjected to a 30-min coronary occlusion (O) and 4 h of reperfusion (R). Cardiac HO-1 gene expression was confirmed at 1 month and 1 year after gene transfer by immunoblotting and immunohistochemistry analyses. In the HO-1 group, infarct size (% of risk region) was dramatically reduced at 1 year after gene transfer (11.2 ± 2.1%, n = 12, vs. 44.7 ± 3.6%, n = 8, in the LacZ group; P < 0.05). The infarct-sparing effects of HO-1 gene therapy at 1 year were as powerful as those observed 24 h after ischemic PC (six 4-min O/4-min R cycles) (15.0 ± 1.7%, n = 10). There were no appreciable changes in LV fractional shortening, LV ejection fraction, or LV end-diastolic or end-systolic diameter at 1 year after HO-1 gene transfer as compared to the age-matched controls or with the LacZ group. Histology showed no inflammation in the myocardium 1 year after rAAV/HO-1-mediated gene transfer. These results demonstrate, for the first time, that rAAV-mediated HO-1 gene transfer confers long-term (1 year), possibly permanent, cardioprotection without adverse functional consequences, providing proof of principle for the concept of achieving prophylactic cardioprotection (i.e., “immunization against infarction”). PMID:21785893

  3. Gene transfer as a strategy to achieve permanent cardioprotection I: rAAV-mediated gene therapy with inducible nitric oxide synthase limits infarct size 1 year later without adverse functional consequences

    PubMed Central

    Li, Qianhong; Guo, Yiru; Wu, Wen-Jian; Ou, Qinghui; Zhu, Xiaoping; Tan, Wei; Yuan, Fangping; Chen, Ning; Dawn, Buddhadeb; Luo, Li; O’Brien, Erin

    2013-01-01

    The ultimate goal of prophylactic gene therapy is to confer permanent protection against ischemia. Although gene therapy with inducible nitric oxide synthase (iNOS) is known to protect against myocardial infarction at 3 days and up to 2 months, the long-term effects on myocardial ischemic injury and function are unknown. To address this issue, we created a recombinant adeno-associated viral vector carrying the iNOS gene (rAAV/iNOS), which enables long-lasting transgene expression. The ability of rAAV/iNOS to direct the expression of functional iNOS protein was confirmed in COS-7 cells before in vivo gene transfer. Mice received injections in the anterior LV wall of rAAV/LacZ or rAAV/iNOS; 1 year later, they underwent a 30-min coronary occlusion (O) and 4 h of reperfusion (R). iNOS gene transfer resulted in elevated iNOS protein expression (+3-fold vs. the LacZ group, n = 6; P < 0.05) and iNOS activity (+4.4-fold vs. the LacZ group, n = 6; P < 0.05) 1 year later. Infarct size (% of risk region) was dramatically reduced at 1 year after iNOS gene transfer (13.5 ± 2.2%, n = 12, vs. 41.7 ± 2.9%, n = 10, in the LacZ group; P < 0.05). The infarct-sparing effect of iNOS gene therapy at 1 year was as powerful as that observed 24 h after ischemic preconditioning (six 4-min O/4-min R cycles) (19.3 ± 2.3%, n = 11; P < 0.05). Importantly, compared with the LacZ group (n = 11), iNOS gene transfer (n = 10) had no effect on LV dimensions or function for up to 1 year (at 1 year: FS 34.5 ± 2.0 vs. 34.6 ± 2.6%, EF 57.0 ± 2.0 vs. 59.7 ± 2.9%, LVEDD 4.3 ± 0.1 vs. 4.2 ± 0.2 mm, LVESD 2.8 ± 0.1 vs. 2.9 ± 0.2 mm) (echocardiography). These data demonstrate, for the first time, that rAAV-mediated iNOS gene transfer affords long-term, probably permanent (1 year), cardioprotection without adverse functional consequences, providing a strong rationale for further preclinical testing of prophylactic gene therapy. PMID:21779912

  4. Cellular localization of long non-coding RNAs affects silencing by RNAi more than by antisense oligonucleotides.

    PubMed

    Lennox, Kim A; Behlke, Mark A

    2016-01-29

    Thousands of long non-coding RNAs (lncRNAs) have been identified in mammalian cells. Some have important functions and their dysregulation can contribute to a variety of disease states. However, most lncRNAs have not been functionally characterized. Complicating their study, lncRNAs have widely varying subcellular distributions: some reside predominantly in the nucleus, the cytoplasm or in both compartments. One method to query function is to suppress expression and examine the resulting phenotype. Methods to suppress expression of mRNAs include antisense oligonucleotides (ASOs) and RNA interference (RNAi). Antisense and RNAi-based gene-knockdown methods vary in efficacy between different cellular compartments. It is not known if this affects their ability to suppress lncRNAs. To address whether localization of the lncRNA influences susceptibility to degradation by either ASOs or RNAi, nuclear lncRNAs (MALAT1 and NEAT1), cytoplasmic lncRNAs (DANCR and OIP5-AS1) and dual-localized lncRNAs (TUG1, CasC7 and HOTAIR) were compared for knockdown efficiency. We found that nuclear lncRNAs were more effectively suppressed using ASOs, cytoplasmic lncRNAs were more effectively suppressed using RNAi and dual-localized lncRNAs were suppressed using both methods. A mixed-modality approach combining ASOs and RNAi reagents improved knockdown efficacy, particularly for those lncRNAs that localize to both nuclear and cytoplasmic compartments. PMID:26578588

  5. Catalytic in vivo protein knockdown by small-molecule PROTACs.

    PubMed

    Bondeson, Daniel P; Mares, Alina; Smith, Ian E D; Ko, Eunhwa; Campos, Sebastien; Miah, Afjal H; Mulholland, Katie E; Routly, Natasha; Buckley, Dennis L; Gustafson, Jeffrey L; Zinn, Nico; Grandi, Paola; Shimamura, Satoko; Bergamini, Giovanna; Faelth-Savitski, Maria; Bantscheff, Marcus; Cox, Carly; Gordon, Deborah A; Willard, Ryan R; Flanagan, John J; Casillas, Linda N; Votta, Bartholomew J; den Besten, Willem; Famm, Kristoffer; Kruidenier, Laurens; Carter, Paul S; Harling, John D; Churcher, Ian; Crews, Craig M

    2015-08-01

    The current predominant therapeutic paradigm is based on maximizing drug-receptor occupancy to achieve clinical benefit. This strategy, however, generally requires excessive drug concentrations to ensure sufficient occupancy, often leading to adverse side effects. Here, we describe major improvements to the proteolysis targeting chimeras (PROTACs) method, a chemical knockdown strategy in which a heterobifunctional molecule recruits a specific protein target to an E3 ubiquitin ligase, resulting in the target's ubiquitination and degradation. These compounds behave catalytically in their ability to induce the ubiquitination of super-stoichiometric quantities of proteins, providing efficacy that is not limited by equilibrium occupancy. We present two PROTACs that are capable of specifically reducing protein levels by >90% at nanomolar concentrations. In addition, mouse studies indicate that they provide broad tissue distribution and knockdown of the targeted protein in tumor xenografts. Together, these data demonstrate a protein knockdown system combining many of the favorable properties of small-molecule agents with the potent protein knockdown of RNAi and CRISPR. PMID:26075522

  6. Catalytic in vivo protein knockdown by small-molecule PROTACs

    PubMed Central

    Bondeson, Daniel P; Mares, Alina; Smith, Ian E D; Ko, Eunhwa; Campos, Sebastien; Miah, Afjal H; Mulholland, Katie E; Routly, Natasha; Buckley, Dennis L; Gustafson, Jeffrey L; Zinn, Nico; Grandi, Paola; Shimamura, Satoko; Bergamini, Giovanna; Faelth-Savitski, Maria; Bantscheff, Marcus; Cox, Carly; Gordon, Deborah A; Willard, Ryan R; Flanagan, John J; Casillas, Linda N; Votta, Bartholomew J; den Besten, Willem; Famm, Kristoffer; Kruidenier, Laurens; Carter, Paul S; Harling, John D; Churcher, Ian; Crews, Craig M

    2015-01-01

    The current predominant theapeutic paradigm is based on maximizing drug-receptor occupancy to achieve clinical benefit. This strategy, however, generally requires excessive drug concentrations to ensure sufficient occupancy, often leading to adverse side effects. Here, we describe major improvements to the proteolysis targeting chimeras (PROTACs) method, a chemical knockdown strategy in which a heterobifunctional molecule recruits a specific protein target to an E3 ubiquitin ligase, resulting in the target’s ubiquitination and degradation. These compounds behave catalytically in their ability to induce the ubiquitination of super-stoichiometric quantities of proteins, providing efficacy that is not limited by equilibrium occupancy. We present two PROTACs that are capable of specifically reducing protein levels by >90% at nanomolar concentrations. In addition, mouse studies indicate that they provide broad tissue distribution and knockdown of the targeted protein in tumor xenografts. Together, these data demonstrate a protein knockdown system combining many of the favorable properties of small-molecule agents with the potent protein knockdown of RNAi and CRISPR. PMID:26075522

  7. The Role of RNA Interference (RNAi) in Arbovirus-Vector Interactions

    PubMed Central

    Blair, Carol D.; Olson, Ken E.

    2015-01-01

    RNA interference (RNAi) was shown over 18 years ago to be a mechanism by which arbovirus replication and transmission could be controlled in arthropod vectors. During the intervening period, research on RNAi has defined many of the components and mechanisms of this antiviral pathway in arthropods, yet a number of unexplored questions remain. RNAi refers to RNA-mediated regulation of gene expression. Originally, the term described silencing of endogenous genes by introduction of exogenous double-stranded (ds)RNA with the same sequence as the gene to be silenced. Further research has shown that RNAi comprises three gene regulation pathways that are mediated by small RNAs: the small interfering (si)RNA, micro (mi)RNA, and Piwi-interacting (pi)RNA pathways. The exogenous (exo-)siRNA pathway is now recognized as a major antiviral innate immune response of arthropods. More recent studies suggest that the piRNA and miRNA pathways might also have important roles in arbovirus-vector interactions. This review will focus on current knowledge of the role of the exo-siRNA pathway as an arthropod vector antiviral response and on emerging research into vector piRNA and miRNA pathway modulation of arbovirus-vector interactions. Although it is assumed that arboviruses must evade the vector’s antiviral RNAi response in order to maintain their natural transmission cycles, the strategies by which this is accomplished are not well defined. RNAi is also an important tool for arthropod gene knock-down in functional genomics studies and in development of arbovirus-resistant mosquito populations. Possible arbovirus strategies for evasion of RNAi and applications of RNAi in functional genomics analysis and arbovirus transmission control will also be reviewed. PMID:25690800

  8. Towards the elements of successful insect RNAi

    PubMed Central

    Scott, Jeffrey G.; Michel, Kristin; Bartholomay, Lyric; Siegfried, Blair D.; Hunter, Wayne B.; Smagghe, Guy; Zhu, Kun Yan; Douglas, Angela E.

    2013-01-01

    RNA interference (RNAi), the sequence-specific suppression of gene expression, offers great opportunities for insect science, especially to analyze gene function, manage pest populations, and reduce disease pathogens. The accumulating body of literature on insect RNAi has revealed that the efficiency of RNAi varies between different species, the mode of RNAi delivery, and the genes being targeted. There is also variation in the duration of transcript suppression. At present, we have a limited capacity to predict the ideal experimental strategy for RNAi of a particular gene/insect because of our incomplete understanding of whether and how the RNAi signal is amplified and spread among insect cells. Consequently, development of the optimal RNAi protocols is a highly empirical process. This limitation can be relieved by systematic analysis of the molecular physiological basis of RNAi mechanisms in insects. An enhanced conceptual understanding of RNAi function in insects will facilitate the application of RNAi for dissection of gene function, and to fast-track the application of RNAi to both control pests and develop effective methods to protect beneficial insects and non-insect arthropods, particularly the honey bee (Apis mellifera) and cultured Pacific white shrimp (Litopenaeus vannamei) from viral and parasitic diseases. PMID:24041495

  9. Nanolayered siRNA dressing for sustained localized knockdown.

    PubMed

    Castleberry, Steven; Wang, Mary; Hammond, Paula T

    2013-06-25

    The success of RNA interference (RNAi) in medicine relies on the development of technology capable of successfully delivering it to tissues of interest. Significant research has focused on the difficult task of systemic delivery of RNAi; however its local delivery could be a more easily realized approach. Localized delivery is of particular interest for many medical applications, including the treatment of localized diseases, the modulation of cellular response to implants or tissue engineering constructs, and the management of wound healing and regenerative medicine. In this work we present an ultrathin electrostatically assembled coating for localized and sustained delivery of short interfering RNA (siRNA). This film was applied to a commercially available woven nylon dressing commonly used for surgical applications and was demonstrated to sustain significant knockdown of protein expression in multiple cell types for more than one week in vitro. Significantly, this coating can be easily applied to a medically relevant device and requires no externally delivered transfection agents for effective delivery of siRNA. These results present promising opportunities for the localized administration of RNAi. PMID:23672676

  10. RNAi-assisted genome evolution in Saccharomyces cerevisiae for complex phenotype engineering.

    PubMed

    Si, Tong; Luo, Yunzi; Bao, Zehua; Zhao, Huimin

    2015-03-20

    A fundamental challenge in basic and applied biology is to reprogram cells with improved or novel traits on a genomic scale. However, the current ability to reprogram a cell on the genome scale is limited to bacterial cells. Here, we report RNA interference (RNAi)-assisted genome evolution (RAGE) as a generally applicable method for genome-scale engineering in the yeast Saccharomyces cerevisiae. Through iterative cycles of creating a library of RNAi induced reduction-of-function mutants coupled with high throughput screening or selection, RAGE can continuously improve target trait(s) by accumulating multiplex beneficial genetic modifications in an evolving yeast genome. To validate the RNAi library constructed with yeast genomic DNA and convergent-promoter expression cassette, we demonstrated RNAi screening in Saccharomyces cerevisiae for the first time by identifying two known and three novel suppressors of a telomerase-deficient mutation yku70Δ. We then showed the application of RAGE for improved acetic acid tolerance, a key trait for microbial production of chemicals and fuels. Three rounds of iterative RNAi screening led to the identification of three gene knockdown targets that acted synergistically to confer an engineered yeast strain with substantially improved acetic acid tolerance. RAGE should greatly accelerate the design and evolution of organisms with desired traits and provide new insights on genome structure, function, and evolution. PMID:24758359

  11. West Nile Virus Infection of Drosophila melanogaster Induces a Protective RNAi Response

    PubMed Central

    Chotkowski, Heather L.; Ciota, Alexander T.; Jia, Yongqing; Puig-Basagoiti, Francesc; Kramer, Laura D.; Shi, Pei-Yong; Glaser, Robert L.

    2008-01-01

    To determine if West Nile virus (WNV) infection of insect cells induces a protective RNAi response, Drosophila melanogaster S2 and Aedes albopictus C6/36 cells were infected with WNV, and the production of WNV-homologous small RNAs was assayed as an indicator of RNAi induction. A distinct population of ~25 nt WNV-homologous small RNAs was detected in infected S2 cells but not C6/36 cells. RNAi knockdown of Argonaute 2 in S2 cells resulted in slightly increased susceptibility to WNV infection, suggesting that some WNV-homologous small RNAs produced in infected S2 cells are functional small interfering RNAs. WNV was shown to infect adult D. melanogaster, and adult flies containing mutations in each of four different RNAi genes (Argonaute 2, spindle-E, piwi, and Dicer-2) were significantly more susceptible to WNV infection than wildtype flies. These results combined with the analysis of WNV infection of S2 and C6/36 cells support the conclusion that WNV infection of D. melanogaster, but perhaps not Ae. albopictus, induces a protective RNAi response. PMID:18501400

  12. A Simple Retroelement Based Knock-Down System in Dictyostelium: Further Insights into RNA Interference Mechanisms

    PubMed Central

    Schuster, Isabelle; Nellen, Wolfgang

    2015-01-01

    Characteristics of DIRS-1 Mediated Knock-Downs We have previously shown that the most abundant Dictyostelium discoideum retroelement DIRS-1 is suppressed by RNAi mechanisms. Here we provide evidence that both inverted terminal repeats have strong promoter activity and that bidirectional expression apparently generates a substrate for Dicer. A cassette containing the inverted terminal repeats and a fragment of a gene of interest was sufficient to activate the RNAi response, resulting in the generation of ~21 nt siRNAs, a reduction of mRNA and protein expression of the respective endogene. Surprisingly, no transitivity was observed on the endogene. This was in contrast to previous observations, where endogenous siRNAs caused spreading on an artificial transgene. Knock-down was successful on seven target genes that we examined. In three cases a phenotypic analysis proved the efficiency of the approach. One of the target genes was apparently essential because no knock-out could be obtained; the RNAi mediated knock-down, however, resulted in a very slow growing culture indicating a still viable reduction of gene expression. Advantages of the DIRS-1–RNAi System The knock-down system required a short DNA fragment (~400 bp) of the target gene as an initial trigger. Further siRNAs were generated by RdRPs since we have shown some siRNAs with a 5’-triphosphate group. Extrachromosomal vectors facilitate the procedure and allowed for molecular and phenotypic analysis within one week. The system provides an efficient and rapid method to reduce protein levels including those of essential genes. PMID:26110905

  13. RNAi in agriculturally-important arthropods

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Our aim in this chapter is to provide an overview of the profound knowledge accumulated in recent years from invertebrate RNAi studies, but with a focus on agriculturally important arthropods. We start with a brief discussion of the RNAi mechanism to introduce readers to key concepts that underlie t...

  14. Ultra-low gossypol cottonseed: generational stability of the seed-specific, RNAi mediated phenotype and resumption of terpenoid profile following seed germination

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Cottonseed, containing 22.5% protein, remains an under-utilized and under-valued resource due to the presence of toxic gossypol. RNAi-knockdown of beta-cadinene synthase gene(s) was used to engineer plants that produced ultra-low gossypol cottonseed(ULGCS). In the original study, we observed that RN...

  15. Ultra-low gossypol cottonseed: generational stability of the seed-specific, RNAi-mediated phenotype and resumption of terpenoid activity/profile following seed germination

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Cottonseed, containing 22.5% protein, remains an under-utilized and under-valued resource due to the presence of toxic gossypol. RNAi-knockdown of Beta-cadinene synthase gene(s) was used to engineer plants that produced ultra-low gossypol cottonseeds (ULGCS). In the original study, we observed tha...

  16. Ultra-low Gossypol cottonseed: Stability and specificity of the RNAi-mediated trait and performance of transgenic lines under field conditions

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Cottonseed, containing 22.5% protein, remains a low-value byproduct of lint production mainly due to the presence of toxic gossypol that makes it unfit for monogastrics. RNAi-knockdown of d-cadinene synthase gene(s) was used to engineer plants that produced ultra-low gossypol cottonseed (ULGCS). T...

  17. An Arabidopsis tissue-specific RNAi method for studying genes essential to mitosis.

    PubMed

    Burgos-Rivera, Brunilís; Dawe, R Kelly

    2012-01-01

    A large fraction of the genes in plants can be considered essential in the sense that when absent the plant fails to develop past the first few cell divisions. The fact that angiosperms pass through a haploid gametophyte stage can make it challenging to propagate such mutants even in the heterozygous condition. Here we describe a tissue-specific RNAi method that allows us to visualize cell division phenotypes in petals, which are large dispensable organs. Portions of the APETALA (AP3) and PISTILLATA (PI) promoters confer early petal-specific expression. We show that when either promoter is used to drive the expression of a beta-glucuronidase (GUS) RNAi transgene in plants uniformly expressing GUS, GUS expression is knocked down specifically in petals. We further tested the system by targeting the essential kinetochore protein CENPC and two different components of the Spindle Assembly Checkpoint (MAD2 and BUBR1). Plant lines expressing petal-specific RNAi hairpins targeting these genes exhibited an array of petal phenotypes. Cytological analyses of the affected flower buds confirmed that CENPC knockdown causes cell cycle arrest but provided no evidence that either MAD2 or BUBR1 are required for mitosis (although both genes are required for petal growth by this assay). A key benefit of the petal-specific RNAi method is that the phenotypes are not expressed in the lineages leading to germ cells, and the phenotypes are faithfully transmitted for at least four generations despite their pronounced effects on growth. PMID:23236491

  18. Robust RNAi enhancement via human Argonaute-2 overexpression from plasmids, viral vectors and cell lines

    PubMed Central

    Börner, Kathleen; Niopek, Dominik; Cotugno, Gabriella; Kaldenbach, Michaela; Pankert, Teresa; Willemsen, Joschka; Zhang, Xian; Schürmann, Nina; Mockenhaupt, Stefan; Serva, Andrius; Hiet, Marie-Sophie; Wiedtke, Ellen; Castoldi, Mirco; Starkuviene, Vytaute; Erfle, Holger; Gilbert, Daniel F.; Bartenschlager, Ralf; Boutros, Michael; Binder, Marco; Streetz, Konrad; Kräusslich, Hans-Georg; Grimm, Dirk

    2013-01-01

    As the only mammalian Argonaute protein capable of directly cleaving mRNAs in a small RNA-guided manner, Argonaute-2 (Ago2) is a keyplayer in RNA interference (RNAi) silencing via small interfering (si) or short hairpin (sh) RNAs. It is also a rate-limiting factor whose saturation by si/shRNAs limits RNAi efficiency and causes numerous adverse side effects. Here, we report a set of versatile tools and widely applicable strategies for transient or stable Ago2 co-expression, which overcome these concerns. Specifically, we engineered plasmids and viral vectors to co-encode a codon-optimized human Ago2 cDNA along with custom shRNAs. Furthermore, we stably integrated this Ago2 cDNA into a panel of standard human cell lines via plasmid transfection or lentiviral transduction. Using various endo- or exogenous targets, we demonstrate the potential of all three strategies to boost mRNA silencing efficiencies in cell culture by up to 10-fold, and to facilitate combinatorial knockdowns. Importantly, these robust improvements were reflected by augmented RNAi phenotypes and accompanied by reduced off-targeting effects. We moreover show that Ago2/shRNA-co-encoding vectors can enhance and prolong transgene silencing in livers of adult mice, while concurrently alleviating hepatotoxicity. Our customizable reagents and avenues should broadly improve future in vitro and in vivo RNAi experiments in mammalian systems. PMID:24049077

  19. RNAi-mediated plant protection against aphids.

    PubMed

    Yu, Xiu-Dao; Liu, Zong-Cai; Huang, Si-Liang; Chen, Zhi-Qin; Sun, Yong-Wei; Duan, Peng-Fei; Ma, You-Zhi; Xia, Lan-Qin

    2016-06-01

    Aphids (Aphididae) are major agricultural pests that cause significant yield losses of crop plants each year by inflicting damage both through the direct effects of feeding and by vectoring harmful plant viruses. Expression of double-stranded RNA (dsRNA) directed against suitable insect target genes in transgenic plants has been shown to give protection against pests through plant-mediated RNA interference (RNAi). Thus, as a potential alternative and effective strategy for insect pest management in agricultural practice, plant-mediated RNAi for aphid control has received close attention in recent years. In this review, the mechanism of RNAi in insects and the so far explored effective RNAi target genes in aphids, their potential applications in the development of transgenic plants for aphid control and the major challenges in this regard are reviewed, and the future prospects of using plant-mediated RNAi for aphid control are discussed. This review is intended to be a helpful insight into the generation of aphid-resistant plants through plant-mediated RNAi strategy. © 2016 Society of Chemical Industry. PMID:26888776

  20. Conditional RNAi Using the Lentiviral GLTR System.

    PubMed

    Pfeiffenberger, Elisabeth; Sigl, Reinhard; Geley, Stephan

    2016-01-01

    RNA interference (RNAi) has become an essential technology for functional gene analysis. Its success depends on the effective expression of target gene-specific RNAi-inducing small double-stranded interfering RNA molecules (siRNAs). Here, were describe the use of a recently developed lentiviral RNAi system that allows the rapid generation of stable cell lines with inducible RNAi based on conditional expression of double-stranded short hairpin RNA (shRNA). These lentiviral vectors can be generated rapidly using the GATEWAY recombination cloning technology. Conditional cell lines can be established by using either a two-vector system in which the regulator is encoded by a separate vector or by a one-vector system. The available different lentiviral vectors for conditional shRNA expression cassette delivery co-express additional genes that allow (1) the use of fluorescent proteins for color-coded combinatorial RNAi or monitoring RNAi induction (pGLTR-FP), (2) selection of transduced cells (pGLTR-S), and (3) the generation of conditional cell lines using a one-vector system (pGLTR-X). PMID:27317178

  1. Stable SET knockdown in breast cell carcinoma inhibits cell migration and invasion

    SciTech Connect

    Li, Jie; Yang, Xi-fei; Ren, Xiao-hu; Meng, Xiao-jing; Huang, Hai-yan; Zhao, Qiong-hui; Yuan, Jian-hui; Hong, Wen-xu; Xia, Bo; Huang, Xin-feng; Zhou, Li; Liu, Jian-jun; Zou, Fei

    2014-10-10

    Highlights: • We employed RNA interference to knockdown SET expression in breast cancer cells. • Knockdown of SET expression inhibits cell proliferation, migration and invasion. • Knockdown of SET expression increases the activity and expression of PP2A. • Knockdown of SET expression decreases the expression of MMP-9. - Abstract: Breast cancer is the most malignant tumor for women, however, the mechanisms underlying this devastating disease remain unclear. SET is an endogenous inhibitor of protein phosphatase 2A (PP2A) and involved in many physiological and pathological processes. SET could promote the occurrence of tumor through inhibiting PP2A. In this study, we explore the role of SET in the migration and invasion of breast cancer cells MDA-MB-231 and ZR-75-30. The stable suppression of SET expression through lentivirus-mediated RNA interference (RNAi) was shown to inhibit the growth, migration and invasion of breast cancer cells. Knockdown of SET increases the activity and expression of PP2Ac and decrease the expression of matrix metalloproteinase 9 (MMP-9). These data demonstrate that SET may be involved in the pathogenic processes of breast cancer, indicating that SET can serve as a potential therapeutic target for the treatment of breast cancer.

  2. Knockdown of the adipokinetic hormone receptor increases feeding frequency in the two-spotted cricket Gryllus bimaculatus.

    PubMed

    Konuma, Takahiro; Morooka, Nobukatsu; Nagasawa, Hiromichi; Nagata, Shinji

    2012-07-01

    Adipokinetic hormone (AKH) is a peptide hormone that regulates the nutritional state in insects by supporting the mobilization of lipids. In the present study, we manipulated AKH signaling to evaluate how metabolic state regulates feeding in an orthopteran insect, the two-spotted cricket, Gryllus bimaculatus. This was accomplished by RNA interference (RNAi) targeting the receptor gene for AKH [G. bimaculatus AKHR (GrybiAKHR)]. We found that the knockdown of GrybiAKHR by AKHR-double-stranded RNA treatment decreased the levels of 1,2-diacylglycerol and trehalose in the hemolymph, whereas it increased the level of triacylglycerol in the fat body. In addition, the knockdown of GrybiAKHR enhanced starvation resistance and increased food intake. Furthermore, direct observation of GrybiAKHR(RNAi) crickets revealed that the knockdown of GrybiAKHR increased feeding frequency but did not alter meal duration, whereas locomotor activity decreased. The increased frequency of feeding by GrybiAKHR(RNAi) crickets eventually resulted in an increase of food intake. These data demonstrate that the regulation of the metabolic state by AKH signaling affects feeding frequency, probably through nutritional control. PMID:22619358

  3. miRNA-embedded shRNAs for Lineage-specific BCL11A Knockdown and Hemoglobin F Induction

    PubMed Central

    Guda, Swaroopa; Brendel, Christian; Renella, Raffaele; Du, Peng; Bauer, Daniel E; Canver, Matthew C; Grenier, Jennifer K; Grimson, Andrew W; Kamran, Sophia C; Thornton, James; de Boer, Helen; Root, David E; Milsom, Michael D; Orkin, Stuart H; Gregory, Richard I; Williams, David A

    2015-01-01

    RNA interference (RNAi) technology using short hairpin RNAs (shRNAs) expressed via RNA polymerase (pol) III promoters has been widely exploited to modulate gene expression in a variety of mammalian cell types. For certain applications, such as lineage-specific knockdown, embedding targeting sequences into pol II-driven microRNA (miRNA) architecture is required. Here, using the potential therapeutic target BCL11A, we demonstrate that pol III-driven shRNAs lead to significantly increased knockdown but also increased cytotoxcity in comparison to pol II-driven miRNA adapted shRNAs (shRNAmiR) in multiple hematopoietic cell lines. We show that the two expression systems yield mature guide strand sequences that differ by a 4 bp shift. This results in alternate seed sequences and consequently influences the efficacy of target gene knockdown. Incorporating a corresponding 4 bp shift into the guide strand of shRNAmiRs resulted in improved knockdown efficiency of BCL11A. This was associated with a significant de-repression of the hemoglobin target of BCL11A, human γ-globin or the murine homolog Hbb-y. Our results suggest the requirement for optimization of shRNA sequences upon incorporation into a miRNA backbone. These findings have important implications in future design of shRNAmiRs for RNAi-based therapy in hemoglobinopathies and other diseases requiring lineage-specific expression of gene silencing sequences. PMID:26080908

  4. Morphogenesis defects are associated with abnormal nervous system regeneration following roboA RNAi in planarians.

    PubMed

    Cebrià, Francesc; Newmark, Phillip A

    2007-03-01

    The process by which the proper pattern is restored to newly formed tissues during metazoan regeneration remains an open question. Here, we provide evidence that the nervous system plays a role in regulating morphogenesis during anterior regeneration in the planarian Schmidtea mediterranea. RNA interference (RNAi) knockdown of a planarian ortholog of the axon-guidance receptor roundabout (robo) leads to unexpected phenotypes during anterior regeneration, including the development of a supernumerary pharynx (the feeding organ of the animal) and the production of ectopic, dorsal outgrowths with cephalic identity. We show that Smed-roboA RNAi knockdown disrupts nervous system structure during cephalic regeneration: the newly regenerated brain and ventral nerve cords do not re-establish proper connections. These neural defects precede, and are correlated with, the development of ectopic structures. We propose that, in the absence of proper connectivity between the cephalic ganglia and the ventral nerve cords, neurally derived signals promote the differentiation of pharyngeal and cephalic structures. Together with previous studies on regeneration in annelids and amphibians, these results suggest a conserved role of the nervous system in pattern formation during blastema-based regeneration. PMID:17251262

  5. A Tendon Cell Specific RNAi Screen Reveals Novel Candidates Essential for Muscle Tendon Interaction.

    PubMed

    Tiwari, Prabhat; Kumar, Arun; Das, Rudra Nayan; Malhotra, Vivek; VijayRaghavan, K

    2015-01-01

    Tendons are fibrous connective tissue which connect muscles to the skeletal elements thus acting as passive transmitters of force during locomotion and provide appropriate body posture. Tendon-derived cues, albeit poorly understood, are necessary for proper muscle guidance and attachment during development. In the present study, we used dorsal longitudinal muscles of Drosophila and their tendon attachment sites to unravel the molecular nature of interactions between muscles and tendons. We performed a genetic screen using RNAi-mediated knockdown in tendon cells to find out molecular players involved in the formation and maintenance of myotendinous junction and found 21 candidates out of 2507 RNAi lines screened. Of these, 19 were novel molecules in context of myotendinous system. Integrin-βPS and Talin, picked as candidates in this screen, are known to play important role in the cell-cell interaction and myotendinous junction formation validating our screen. We have found candidates with enzymatic function, transcription activity, cell adhesion, protein folding and intracellular transport function. Tango1, an ER exit protein involved in collagen secretion was identified as a candidate molecule involved in the formation of myotendinous junction. Tango1 knockdown was found to affect development of muscle attachment sites and formation of myotendinous junction. Tango1 was also found to be involved in secretion of Viking (Collagen type IV) and BM-40 from hemocytes and fat cells. PMID:26488612

  6. A Tendon Cell Specific RNAi Screen Reveals Novel Candidates Essential for Muscle Tendon Interaction

    PubMed Central

    Tiwari, Prabhat; Malhotra, Vivek; VijayRaghavan, K.

    2015-01-01

    Tendons are fibrous connective tissue which connect muscles to the skeletal elements thus acting as passive transmitters of force during locomotion and provide appropriate body posture. Tendon-derived cues, albeit poorly understood, are necessary for proper muscle guidance and attachment during development. In the present study, we used dorsal longitudinal muscles of Drosophila and their tendon attachment sites to unravel the molecular nature of interactions between muscles and tendons. We performed a genetic screen using RNAi-mediated knockdown in tendon cells to find out molecular players involved in the formation and maintenance of myotendinous junction and found 21 candidates out of 2507 RNAi lines screened. Of these, 19 were novel molecules in context of myotendinous system. Integrin-βPS and Talin, picked as candidates in this screen, are known to play important role in the cell-cell interaction and myotendinous junction formation validating our screen. We have found candidates with enzymatic function, transcription activity, cell adhesion, protein folding and intracellular transport function. Tango1, an ER exit protein involved in collagen secretion was identified as a candidate molecule involved in the formation of myotendinous junction. Tango1 knockdown was found to affect development of muscle attachment sites and formation of myotendinous junction. Tango1 was also found to be involved in secretion of Viking (Collagen type IV) and BM-40 from hemocytes and fat cells. PMID:26488612

  7. Short Hairpin RNA (shRNA): Design, Delivery, and Assessment of Gene Knockdown

    PubMed Central

    Moore, Chris B.; Guthrie, Elizabeth H.; Huang, Max Tze-Han; Taxman, Debra J.

    2013-01-01

    Shortly after the cellular mechanism of RNA interference (RNAi) was first described, scientists began using this powerful technique to study gene function. This included designing better methods for the successful delivery of small interfering RNAs (siRNAs) and short hairpin RNAs (shRNAs) into mammalian cells. While the simplest method for RNAi is the cytosolic delivery of siRNA oligonucleotides, this technique is limited to cells capable of transfection and is primarily utilized during transient in vitro studies. The introduction of shRNA into mammalian cells through infection with viral vectors allows for stable integration of shRNA and long-term knockdown of the targeted gene; however, several challenges exist with the implementation of this technology. Here we describe some well-tested protocols which should increase the chances of successful design, delivery, and assessment of gene knockdown by shRNA. We provide suggestions for designing shRNA targets and controls, a protocol for sequencing through the secondary structure of the shRNA hairpin structure, and protocols for packaging and delivery of shRNA lentiviral particles. Using real-time PCR and functional assays we demonstrate the successful knockdown of ASC, an inflammatory adaptor molecule. These studies demonstrate the practicality of including two shRNAs with different efficacies of knockdown to provide an additional level of control and to verify dose dependency of functional effects. Along with the methods described here, as new techniques and algorithms are designed in the future, shRNA is likely to include further promising application and continue to be a critical component of gene discovery. PMID:20387148

  8. Novel Methods for Mosquito Control using RNAi.

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The discovery and development of novel insecticides for vector control is a primary focus of toxicology research conducted at the Mosquito and Fly Research Unit, Gainesville, FL. Targeting critical genes/proteins in mosquitoes using RNA interference (RNAi) is being investigated as a method to devel...

  9. RNA interference (RNAi) patents and human health related applications of RNAi.

    PubMed

    Ebhardt, H Alexander

    2007-01-01

    The Nobel Prize in Physiology or Medicine in 2006 was shared by A.Z. Fire and C.C. Mello. The honour was given to these two principal investigators for demonstrating in the nematode Caenorhabditis elegans that double stranded RNA directs cleavage of messenger RNAs (mRNA) in a homologous manner. This process was termed RNA interference (RNAi) and was published in 1998. Since then, further research revealed that small 21-22 nts long RNAs guide an RNA-induced silencing complex (RISC) to a target mRNA causing translational inhibition or mRNA cleavage. This review will focus on RNAi patents, delivery of RNAi to combat human disease and reviewing some recent applications regarding detection and possible cure of human diseases using RNAi. PMID:19075926

  10. Are RNAi and miRNA therapeutics truly dead?

    PubMed

    Conde, João; Artzi, Natalie

    2015-03-01

    Only a few years ago pharmaceutical companies were excited about the potential of RNA interference (RNAi). Now, financial volatility and subsequent dissolutions of in-house facilities by pharmaceutical companies have had media channels pronouncing that RNAi therapeutics are dead. However, advances in nanomedicine may now help the vast potential of RNAi therapeutics to be fulfilled. PMID:25595555

  11. Multiple Renal Cyst Development but Not Situs Abnormalities in Transgenic RNAi Mice against Inv::GFP Rescue Gene

    PubMed Central

    Kamijho, Yuki; Shiozaki, Yayoi; Sakurai, Eiki; Hanaoka, Kazunori; Watanabe, Daisuke

    2014-01-01

    In this study we generated RNA interference (RNAi)-mediated gene knockdown transgenic mice (transgenic RNAi mice) against the functional Inv gene. Inv mutant mice show consistently reversed internal organs (situs inversus), multiple renal cysts and neonatal lethality. The Inv::GFP-rescue mice, which introduced the Inv::GFP fusion gene, can rescue inv mutant mice phenotypes. This indicates that the Inv::GFP gene is functional in vivo. To analyze the physiological functions of the Inv gene, and to demonstrate the availability of transgenic RNAi mice, we introduced a short hairpin RNA expression vector against GFP mRNA into Inv::GFP-rescue mice and analyzed the gene silencing effects and Inv functions by examining phenotypes. Transgenic RNAi mice with the Inv::GFP-rescue gene (Inv-KD mice) down-regulated Inv::GFP fusion protein and showed hypomorphic phenotypes of inv mutant mice, such as renal cyst development, but not situs abnormalities or postnatal lethality. This indicates that shRNAi-mediated gene silencing systems that target the tag sequence of the fusion gene work properly in vivo, and suggests that a relatively high level of Inv protein is required for kidney development in contrast to left/right axis determination. Inv::GFP protein was significantly down-regulated in the germ cells of Inv-KD mice testis compared with somatic cells, suggesting the existence of a testicular germ cell-specific enhanced RNAi system that regulates germ cell development. The Inv-KD mouse is useful for studying Inv gene functions in adult tissue that are unable to be analyzed in inv mutant mice showing postnatal lethality. In addition, the shRNA-based gene silencing system against the tag sequence of the fusion gene can be utilized as a new technique to regulate gene expression in either in vitro or in vivo experiments. PMID:24586938

  12. Antitumor activity and inhibitory effects on cancer stem cell-like properties of Adeno-associated virus (AAV) -mediated Bmi-1 interference driven by Bmi-1 promoter for gastric cancer

    PubMed Central

    Wang, Xiaofeng; Liu, Xinyang; Huang, Mingzhu; Gan, Lu; Cheng, Yufan; Li, Jin

    2016-01-01

    Bmi-1 is aberrantly activated in various cancers and plays a vital role in maintaining the self-renewal of stem cells. Our previous research revealed that Bmi-1 was overexpressed in gastric cancer (GC) and it's overexpression was an independent negative prognostic factor, suggesting it can be a therapeutic target. The main purpose of this investigation was to explore the antitumor activity of Bmi-1 interference driven by its own promoter (Ad-Bmi-1i) for GC. In this study, we used adenoviral vector to deliver Bmi-1 shRNA driven by its own promoter to treat GC. Our results revealed that Ad-Bmi-1i could selectively silence Bmi-1 in GC cells which overexpress Bmi-1 and suppress the malignant phenotypes and stem-like properties of GC cells in vitro and in vivo. Moreover, direct injection of Ad-Bmi-1i into xenografts suppressed tumor growth and destroyed cancer cells in vivo. Ad-Bmi-1i inhibited the proliferation of GC cells mainly via inducing senescence in vitro, but it suppressed tumor through inducing senescence and apoptosis, and inhibiting angiogenesis in vivo. Bmi-1 knockdown by Ad-Bmi-1i downregulated VEGF via inhibiting AKT activity. These results suggest that Ad-Bmi-1i not only inhibits tumor growth and stem cell-like phenotype by inducing cellular senescence directly, but also has an indirect anti-tumor activity by anti-angiogenesis effects via regulating PTEN/AKT/VEGF pathway. Transfer of gene interference guided by its own promoter by an adeno-associated virus (AAV) vector might be a potent antitumor approach for cancer therapy. PMID:27009837

  13. In Vivo GFP Knockdown by Cationic Nanogel-siRNA Polyplexes

    PubMed Central

    Shrivats, Arun R.; Mishina, Yuji; Averick, Saadyah; Matyjaszewski, Krzysztof; Hollinger, Jeffrey O.

    2016-01-01

    RNA interference (RNAi) is a powerful tool to treat diseases and elucidate target gene function. Prior to clinical implementation, however, challenges including the safe, efficient and targeted delivery of siRNA must be addressed. Here, we report cationic nanogel nanostructured polymers (NSPs) prepared by atom transfer radical polymerization (ATRP) for in vitro and in vivo siRNA delivery in mammalian models. Outcomes from siRNA protection studies suggested that nanogel NSPs reduce enzymatic degradation of siRNA within polyplexes. Further, the methylation of siRNA may enhance nuclease resistance without compromising gene knockdown potency. NSP-mediated RNAi treatments against Gapdh significantly reduced GAPDH enzyme activity in mammalian cell culture models supplemented with 10% serum. Moreover, nanogel NSP-mediated siRNA delivery significantly inhibited in vivo GFP expression in a mouse model. GFP knockdown was siRNA sequence-dependent and facilitated by nanogel NSP carriers. Continued testing of NSP/siRNA compositions in disease models may produce important new therapeutic options for patient care. PMID:27280121

  14. Nanoparticle-Based Delivery of RNAi Therapeutics: Progress and Challenges

    PubMed Central

    Zhou, Jiehua; Shum, Ka-To; Burnett, John C.; Rossi, John J.

    2013-01-01

    RNA interference (RNAi) is an evolutionarily conserved, endogenous process for post-transcriptional regulation of gene expression. Although RNAi therapeutics have recently progressed through the pipeline toward clinical trials, the application of these as ideal, clinical therapeutics requires the development of safe and effective delivery systems. Inspired by the immense progress with nanotechnology in drug delivery, efforts have been dedicated to the development of nanoparticle-based RNAi delivery systems. For example, a precisely engineered, multifunctional nanocarrier with combined passive and active targeting capabilities may address the delivery challenges for the widespread use of RNAi as a therapy. Therefore, in this review, we introduce the major hurdles in achieving efficient RNAi delivery and discuss the current advances in applying nanotechnology-based delivery systems to overcome the delivery hurdles of RNAi therapeutics. In particular, some representative examples of nanoparticle-based delivery formulations for targeted RNAi therapeutics are highlighted. PMID:23667320

  15. Emerging strategies for RNA interference (RNAi) applications in insects

    PubMed Central

    Nandety, Raja Sekhar; Kuo, Yen-Wen; Nouri, Shahideh; Falk, Bryce W

    2015-01-01

    RNA interference (RNAi) in insects is a gene regulatory process that also plays a vital role in the maintenance and in the regulation of host defenses against invading viruses. Small RNAs determine the specificity of the RNAi through precise recognition of their targets. These small RNAs in insects comprise small interfering RNAs (siRNAs), micro RNAs (miRNAs) and Piwi interacting RNAs (piRNAs) of various lengths. In this review, we have explored different forms of the RNAi inducers that are presently in use, and their applications for an effective and efficient fundamental and practical RNAi research with insects. Further, we reviewed trends in next generation sequencing (NGS) technologies and their importance for insect RNAi, including the identification of novel insect targets as well as insect viruses. Here we also describe a rapidly emerging trend of using plant viruses to deliver the RNAi inducer molecules into insects for an efficient RNAi response. PMID:25424593

  16. A genome scale RNAi screen identifies GLI1 as a novel gene regulating vorinostat sensitivity.

    PubMed

    Falkenberg, K J; Newbold, A; Gould, C M; Luu, J; Trapani, J A; Matthews, G M; Simpson, K J; Johnstone, R W

    2016-07-01

    Vorinostat is an FDA-approved histone deacetylase inhibitor (HDACi) that has proven clinical success in some patients; however, it remains unclear why certain patients remain unresponsive to this agent and other HDACis. Constitutive STAT (signal transducer and activator of transcription) activation, overexpression of prosurvival Bcl-2 proteins and loss of HR23B have been identified as potential biomarkers of HDACi resistance; however, none have yet been used to aid the clinical utility of HDACi. Herein, we aimed to further elucidate vorinostat-resistance mechanisms through a functional genomics screen to identify novel genes that when knocked down by RNA interference (RNAi) sensitized cells to vorinostat-induced apoptosis. A synthetic lethal functional screen using a whole-genome protein-coding RNAi library was used to identify genes that when knocked down cooperated with vorinostat to induce tumor cell apoptosis in otherwise resistant cells. Through iterative screening, we identified 10 vorinostat-resistance candidate genes that sensitized specifically to vorinostat. One of these vorinostat-resistance genes was GLI1, an oncogene not previously known to regulate the activity of HDACi. Treatment of vorinostat-resistant cells with the GLI1 small-molecule inhibitor, GANT61, phenocopied the effect of GLI1 knockdown. The mechanism by which GLI1 loss of function sensitized tumor cells to vorinostat-induced apoptosis is at least in part through interactions with vorinostat to alter gene expression in a manner that favored apoptosis. Upon GLI1 knockdown and vorinostat treatment, BCL2L1 expression was repressed and overexpression of BCL2L1 inhibited GLI1-knockdown-mediated vorinostat sensitization. Taken together, we present the identification and characterization of GLI1 as a new HDACi resistance gene, providing a strong rationale for development of GLI1 inhibitors for clinical use in combination with HDACi therapy. PMID:26868908

  17. Antiviral RNAi: Translating Science Toward Therapeutic Success

    PubMed Central

    2016-01-01

    Viruses continuously evolve to contend with an ever-changing environment that involves transmission between hosts and sometimes species, immune responses, and in some cases therapeutic interventions. Given the high mutation rate of viruses relative to the timescales of host evolution and drug development, novel drug classes that are readily screened and translated to the clinic are needed. RNA interference (RNAi) – a natural mechanism for specific degradation of target RNAs that is conserved from plants to invertebrates and vertebrates – can potentially be harnessed to yield therapies with extensive specificity, ease of design, and broad application. In this review, we discuss basic mechanisms of action and therapeutic applications of RNAi, including design considerations and areas for future development in the field. PMID:21826573

  18. siRNA and RNAi optimization.

    PubMed

    Alagia, Adele; Eritja, Ramon

    2016-05-01

    The discovery and examination of the posttranscriptional gene regulatory mechanism known as RNA interference (RNAi) contributed to the identification of small interfering RNA (siRNA) and the comprehension of its enormous potential for clinical purposes. Theoretically, the ability of specific target gene downregulation makes the RNAi pathway an appealing solution for several diseases. Despite numerous hurdles resulting from the inherent properties of siRNA molecule and proper delivery to the target tissue, more than 50 RNA-based drugs are currently under clinical testing. In this work, we analyze the recent literature in the optimization of siRNA molecules. In detail, we focused on describing the most recent advances of siRNA field aimed at optimize siRNA pharmacokinetic properties. Special attention has been given in describing the impact of RNA modifications in the potential off-target effects (OTEs) such as saturation of the RNAi machinery, passenger strand-mediated silencing, immunostimulation, and miRNA-like OTEs as well as to recent developments on the delivery issue. The novel delivery systems and modified siRNA provide significant steps toward the development of reliable siRNA molecules for therapeutic use. WIREs RNA 2016, 7:316-329. doi: 10.1002/wrna.1337 For further resources related to this article, please visit the WIREs website. PMID:26840434

  19. Recombinant AAV as a Platform for Translating the Therapeutic Potential of RNA Interference

    PubMed Central

    Borel, Florie; Kay, Mark A; Mueller, Christian

    2014-01-01

    RNA interference has become a ubiquitous biological tool, and is being harnessed for therapeutic purposes as well. Therapeutic posttranscriptional gene silencing takes advantage of the endogenous RNAi pathway through delivery of either chemically synthesized siRNAs, or transgenes expressing hairpin-based inhibitory RNAs (e.g., shRNAs and artificial miRNAs). RNAi has expanded the field of viral gene therapy from gene replacement to gene knockdown. Here, we review various noncoding RNAs such as shRNAs, miRNAs, and miRNA decoys which can be utilized for therapeutic applications when expressed from recombinant adeno-associated vectors (AAV), and present examples of their basic design. In addition the basis of exploiting cellular miRNA profiles for detargeting AAV expression from specific cells is described. Finally, an overview of AAV-mediated RNAi preclinical studies is presented, and current RNAi-based clinical trials are reviewed. PMID:24352214

  20. Knockdown of Inner Arm Protein IC138 in Trypanosoma brucei Causes Defective Motility and Flagellar Detachment

    PubMed Central

    Wilson, Corinne S.; Chang, Alex J.; Greene, Rebecca; Machado, Sulynn; Parsons, Matthew W.; Takats, Taylor A.; Zambetti, Luke J.; Springer, Amy L.

    2015-01-01

    Motility in the protozoan parasite Trypanosoma brucei is conferred by a single flagellum, attached alongside the cell, which moves the cell forward using a beat that is generated from tip-to-base. We are interested in characterizing components that regulate flagellar beating, in this study we extend the characterization of TbIC138, the ortholog of a dynein intermediate chain that regulates axonemal inner arm dynein f/I1. TbIC138 was tagged In situ-and shown to fractionate with the inner arm components of the flagellum. RNAi knockdown of TbIC138 resulted in significantly reduced protein levels, mild growth defect and significant motility defects. These cells tended to cluster, exhibited slow and abnormal motility and some cells had partially or fully detached flagella. Slight but significant increases were observed in the incidence of mis-localized or missing kinetoplasts. To document development of the TbIC138 knockdown phenotype over time, we performed a detailed analysis of flagellar detachment and motility changes over 108 hours following induction of RNAi. Abnormal motility, such as slow twitching or irregular beating, was observed early, and became progressively more severe such that by 72 hours-post-induction, approximately 80% of the cells were immotile. Progressively more cells exhibited flagellar detachment over time, but this phenotype was not as prevalent as immotility, affecting less than 60% of the population. Detached flagella had abnormal beating, but abnormal beating was also observed in cells with no flagellar detachment, suggesting that TbIC138 has a direct, or primary, effect on the flagellar beat, whereas detachment is a secondary phenotype of TbIC138 knockdown. Our results are consistent with the role of TbIC138 as a regulator of motility, and has a phenotype amenable to more extensive structure-function analyses to further elucidate its role in the control of flagellar beat in T. brucei. PMID:26555902

  1. MRP4 knockdown enhances migration, suppresses apoptosis, and produces aggregated morphology in human retinal vascular endothelial cells

    SciTech Connect

    Tagami, Mizuki; Kusuhara, Sentaro; Imai, Hisanori; Uemura, Akiyoshi; Honda, Shigeru; Tsukahara, Yasutomo; Negi, Akira

    2010-10-01

    Research highlights: {yields} Exogenous VEGF decreases MRP4 expression in a dose-dependent manner. {yields} MRP4 knockdown leads to enhanced cell migration. {yields} MRP4 knockdown suppresses caspase-3-mediated cell apoptosis. {yields} MRP4 knockdown produces cell assembly and cell aggregation. -- Abstract: The multidrug resistance protein (MRP) MRP4/ABCC4 is an ATP-binding cassette transporter that actively effluxes endogenous and xenobiotic substrates out of cells. In the rodent retina, Mrp4 mRNA and protein are exclusively expressed in vascular endothelial cells, but the angiogenic properties of Mrp4 are poorly understood so far. This study aims to explore the angiogenic properties of MRP4 in human retinal microvascular endothelial cells (HRECs) utilizing the RNA interference (RNAi) technique. MRP4 expression was decreased at the mRNA and protein levels after stimulation with exogenous vascular endothelial growth factor in a dose-dependent manner. RNAi-mediated MRP4 knockdown in HRECs do not affect cell proliferation but enhances cell migration. Moreover, cell apoptosis induced by serum starvation was less prominent in MRP4 siRNA-treated HRECs as compared to control siRNA-treated HRECs. In a Matrigel-based tube-formation assay, although MRP4 knockdown did not lead to a significant change in the total tube length, MRP4 siRNA-treated HRECs assembled and aggregated into a massive tube-like structure, which was not observed in control siRNA-treated HRECs. These results suggest that MRP4 is uniquely involved in retinal angiogenesis.

  2. Regulation of mammalian transcription and splicing by Nuclear RNAi

    PubMed Central

    Kalantari, Roya; Chiang, Cheng-Ming; Corey, David R.

    2016-01-01

    RNA interference (RNAi) is well known as a mechanism for controlling mammalian mRNA translation in the cytoplasm, but what would be the consequences if it also functions in cell nuclei? Although RNAi has also been found in nuclei of plants, yeast, and other organisms, there has been relatively little progress towards understanding the potential involvement of mammalian RNAi factors in nuclear processes including transcription and splicing. This review summarizes evidence for mammalian RNAi factors in cell nuclei and mechanisms that might contribute to the control of gene expression. When RNAi factors bind small RNAs, they form ribonucleoprotein complexes that can be selective for target sequences within different classes of nuclear RNA substrates. The versatility of nuclear RNAi may supply a previously underappreciated layer of regulation to transcription, splicing, and other nuclear processes. PMID:26612865

  3. Use of chromatin remodeling ATPases as RNAi targets for parental control of western corn rootworm (Diabrotica virgifera virgifera) and Neotropical brown stink bug (Euschistus heros).

    PubMed

    Fishilevich, Elane; Vélez, Ana M; Khajuria, Chitvan; Frey, Meghan L F; Hamm, Ronda L; Wang, Haichuan; Schulenberg, Greg A; Bowling, Andrew J; Pence, Heather E; Gandra, Premchand; Arora, Kanika; Storer, Nicholas P; Narva, Kenneth E; Siegfried, Blair D

    2016-04-01

    RNA interference (RNAi) is a gene silencing mechanism that is present in animals and plants and is triggered by double stranded RNA (dsRNA) or small interfering RNA (siRNA), depending on the organism. In the western corn rootworm (WCR), Diabrotica virgifera virgifera LeConte (Coleoptera: Chrysomelidae), RNAi can be achieved by feeding rootworms dsRNA added to artificial diet or plant tissues transformed to express dsRNA. The effect of RNAi depends on the targeted gene function and can range from an absence of phenotypic response to readily apparent responses, including lethality. Furthermore, RNAi can directly affect individuals that consume dsRNA or the effect may be transferred to the next generation. Our previous work described the potential use of genes involved in embryonic development as a parental RNAi technology for the control of WCR. In this study, we describe the use of chromatin-remodeling ATPases as target genes to achieve parental gene silencing in two insect pests, a coleopteran, WCR, and a hemipteran, the Neotropical brown stink bug, Euschistus heros Fabricius (Hemiptera: Pentatomidae). Our results show that dsRNA targeting chromatin-remodeling ATPase transcripts, brahma, mi-2, and iswi strongly reduced the fecundity of the exposed females in both insect species. Additionally, knockdown of chd1 reduced the fecundity of E. heros. PMID:26873291

  4. Data Analysis for High-Throughput RNAi Screening.

    PubMed

    Azorsa, David O; Turnidge, Megan A; Arora, Shilpi

    2016-01-01

    High-throughput RNA interference (HT-RNAi) screening is an effective technology to help identify important genes and pathways involved in a biological process. Analysis of high-throughput RNAi screening data is a critical part of this technology, and many analysis methods have been described. Here, we summarize the workflow and types of analyses commonly used in high-throughput RNAi screening. PMID:27581298

  5. The best control for the specificity of RNAi.

    PubMed

    Sarov, Mihail; Stewart, A Francis

    2005-09-01

    RNA interference (RNAi) is revolutionizing functional genomics. However, there are several reasons to be concerned about the specificity and off-target effects of this technique. A recent paper by Kittler et al. describes a straightforward way to validate RNAi specificity, which exploits the increasing availability of bacterial artificial chromosome (BAC) clone resources. Genetic rescue of the RNAi phenotype by BAC transgenesis is the best control yet described for specificity, and has further implications for reverse genetics. PMID:15979179

  6. Gene Silencing in Insect Cells Using RNAi.

    PubMed

    Wu, Hsuan-Chen; March, John C; Bentley, William E

    2016-01-01

    A technique is described for synthesizing and transfecting double stranded RNA (dsRNA) for RNA interference (RNAi) in Sf-21 cell culture. Transfection with dsRNA only requires an hour and the cells usually recover within 12 h. Suggestions for designing dsRNA are included in the methods. Furthermore, websites are provided for rapid and effective dsRNA design. Three kits are essential for using the described methods: RNAqueous®-4PCR, Megascript™ T7 kit, and the Superscript™ III kit from Life Technologies, Inc. PMID:26820874

  7. Toolkit for evaluating genes required for proliferation and survival using tetracycline-regulated RNAi

    PubMed Central

    Zuber, Johannes; McJunkin, Katherine; Fellmann, Christof; Dow, Lukas E; Taylor, Meredith J; Hannon, Gregory J; Lowe, Scott W

    2012-01-01

    Short hairpin RNAs (shRNAs) are versatile tools for analyzing loss-of-function phenotypes in vitro and in vivo1. However, their use for studying genes involved in proliferation and survival, which are potential therapeutic targets in cancer and other diseases, is confounded by the strong selective advantage of cells in which shRNA expression is inefficient. We therefore developed a toolkit that combines Tet-regulated miR30-shRNA technology, robust transactivator expression and two fluorescent reporters to track and isolate cells with potent target knockdown. We demonstrated that this system improves the study of essential genes and was sufficiently robust to eradicate aggressive cancer in mice by suppressing a single gene. Further, we applied this system for in vivo negative-selection screening with pooled shRNAs and propose a streamlined, inexpensive workflow that will facilitate the use of RNA interference (RNAi) for the identification and evaluation of essential therapeutic targets. PMID:21131983

  8. Effect of LYRM1 knockdown on proliferation, apoptosis, differentiation and mitochondrial function in the P19 cell model of cardiac differentiation in vitro.

    PubMed

    Chen, Yu-Mei; Li, Xing; Song, Gui-Xian; Liu, Ming; Fan, Yi; Wu, Li-Jie; Li, Hua; Zhang, Qi-Jun; Liu, Yao-Qiu; Qian, Ling-Mei

    2016-02-01

    To explore the effects of LYRM1 knockdown on proliferation, apoptosis, differentiation and mitochondrial function in the embryonic carcinoma (P19) cell model of cardiac differentiation. Knockdown of LYRM1 using small interfering RNA (siRNA) was confirmed by quantitative real-time PCR. Cell Counting Kit-8(CCK-8) proliferation assays and cell cycle analysis demonstrated that LYRM1 gene silencing significantly inhibited P19 cell proliferation. Flow cytometry and measurement of their caspase-3 activities revealed that knockdown of LYRM1 increased P19 cell apoptosis. Observation of morphological changes using an inverted microscope and expression analysis of specific differentiation marker genes using quantitative real-time PCR and Western blotting revealed that knockdown of LYRM1 significantly inhibited the differentiation of P19 cells into cardiomyocytes. Furthermore, real-time quantitative PCR applied to detect mitochondrial DNA (mtDNA) copy number implied that there was no significant difference in the LYRM1 knockdown group compared with the control group. Cellular ATP production investigated by luciferase-based luminescence assay was dramatically decreased in differentiated cells transfected with LYRM1 RNAi. Fluorescence microscopy and flow cytometery were used to detect the reactive oxygen species (ROS) and the mitochondrial membrane potential (MMP) showed that the level of ROS was dramatically increased and MMP was obviously decreased in differentiated cells transfected with LYRM1 RNAi. Collectively, knockdown of LYRM1 promoted apoptosis and suppressed proliferation and differentiation in P19 cells. In addition, knockdown of LYRM1 induced mitochondrial impairment in P19 cells during differentiation, which was reflected by decreased ATP synthesis, lower MMP and increased ROS levels. PMID:26759027

  9. Knockdown of Midgut Genes by dsRNA-Transgenic Plant-Mediated RNA Interference in the Hemipteran Insect Nilaparvata lugens

    PubMed Central

    Zha, Wenjun; Peng, Xinxin; Chen, Rongzhi; Du, Bo; Zhu, Lili; He, Guangcun

    2011-01-01

    Background RNA interference (RNAi) is a powerful technique for functional genomics research in insects. Transgenic plants producing double-stranded RNA (dsRNA) directed against insect genes have been reported for lepidopteran and coleopteran insects, showing potential for field-level control of insect pests, but this has not been reported for other insect orders. Methodology/Principal Findings The Hemipteran insect brown planthopper (Nilaparvata lugens Stål) is a typical phloem sap feeder specific to rice (Oryza sativa L.). To analyze the potential of exploiting RNAi-mediated effects in this insect, we identified genes (Nlsid-1 and Nlaub) encoding proteins that might be involved in the RNAi pathway in N. lugens. Both genes are expressed ubiquitously in nymphs and adult insects. Three genes (the hexose transporter gene NlHT1, the carboxypeptidase gene Nlcar and the trypsin-like serine protease gene Nltry) that are highly expressed in the N. lugens midgut were isolated and used to develop dsRNA constructs for transforming rice. RNA blot analysis showed that the dsRNAs were transcribed and some of them were processed to siRNAs in the transgenic lines. When nymphs were fed on rice plants expressing dsRNA, levels of transcripts of the targeted genes in the midgut were reduced; however, lethal phenotypic effects after dsRNA feeding were not observed. Conclusions Our study shows that genes for the RNAi pathway (Nlsid-1 and Nlaub) are present in N. lugens. When insects were fed on rice plant materials expressing dsRNAs, RNA interference was triggered and the target genes transcript levels were suppressed. The gene knockdown technique described here may prove to be a valuable tool for further investigations in N. lugens. The results demonstrate the potential of dsRNA-mediated RNAi for field-level control of planthoppers, but appropriate target genes must be selected when designing the dsRNA-transgenic plants. PMID:21655219

  10. CD22ΔE12 as a molecular target for RNAi therapy

    PubMed Central

    Uckun, Fatih M.; Ma, Hong; Cheng, Jianjun; Myers, Dorothea E.; Qazi, Sanjive

    2015-01-01

    B-precursor acute lymphoblastic leukemia (BPL) is the most common form of cancer in children and adolescents. Our recent studies have demonstrated that CD22ΔE12 is a characteristic genetic defect of therapy-refractory clones in pediatric BPL and implicated the CD22ΔE12 genetic defect in the aggressive biology of relapsed or therapy-refractory pediatric BPL. The purpose of the present study was to evaluate the biologic significance of the CD22ΔE12 molecular lesion in BPL and determine if it could serve as a molecular target for RNA interference (RNAi) therapy. Here we report a previously unrecognized causal link between CD22ΔE12 and aggressive biology of human BPL cells by demonstrating that siRNA-mediated knockdown of CD22ΔE12 in primary leukemic B-cell precursors is associated with a marked inhibition of their clonogenicity. Additionally, we report a nanoscale liposomal formulation of CD22ΔE12-specific siRNA with potent in vitro and in vivo anti-leukemic activity against primary human BPL cells as a first-in-class RNAi therapeutic candidate targeting CD22ΔE12. PMID:25659406

  11. RNAi delivery by exosome-mimetic nanovesicles - Implications for targeting c-Myc in cancer.

    PubMed

    Lunavat, Taral R; Jang, Su Chul; Nilsson, Lisa; Park, Hyun Taek; Repiska, Gabriela; Lässer, Cecilia; Nilsson, Jonas A; Gho, Yong Song; Lötvall, Jan

    2016-09-01

    To develop RNA-based therapeutics, it is crucial to create delivery vectors that transport the RNA molecule into the cell cytoplasm. Naturally released exosomes vesicles (also called "Extracellular Vesicles") have been proposed as possible RNAi carriers, but their yield is relatively small in any cell culture system. We have previously generated exosome-mimetic nanovesicles (NV) by serial extrusions of cells through nano-sized filters, which results in 100-times higher yield of extracellular vesicles. We here test 1) whether NV can be loaded with siRNA exogenously and endogenously, 2) whether the siRNA-loaded NV are taken up by recipient cells, and 3) whether the siRNA can induce functional knock-down responses in recipient cells. A siRNA against GFP was first loaded into NV by electroporation, or a c-Myc shRNA was expressed inside of the cells. The NV were efficiently loaded with siRNA with both techniques, were taken up by recipient cells, which resulted in attenuation of target gene expression. In conclusion, our study suggests that exosome-mimetic nanovesicles can be a platform for RNAi delivery to cell cytoplasm. PMID:27344366

  12. Expanding the Diversity of Imaging-Based RNAi Screen Applications Using Cell Spot Microarrays

    PubMed Central

    Rantala, Juha K.; Kwon, Sunjong; Korkola, James; Gray, Joe W.

    2013-01-01

    Over the past decade, great strides have been made in identifying gene aberrations and deregulated pathways that are associated with specific disease states. These association studies guide experimental studies aimed at identifying the aberrant genes and networks that cause the disease states. This requires functional manipulation of these genes and networks in laboratory models of normal and diseased cells. One approach is to assess molecular and biological responses to high-throughput RNA interference (RNAi)-induced gene knockdown. These responses can be revealed by immunofluorescent staining for a molecular or cellular process of interest and quantified using fluorescence image analysis. These applications are typically performed in multiwell format, but are limited by high reagent costs and long plate processing times. These limitations can be mitigated by analyzing cells grown in cell spot microarray (CSMA) format. CSMAs are produced by growing cells on small (~200 μm diameter) spots with each spot carrying an siRNA with transfection reagent. The spacing between spots is only a few hundred micrometers, thus thousands of cell spots can be arranged on a single cell culture surface. These high-density cell cultures can be immunofluorescently stained with minimal reagent consumption and analyzed quickly using automated fluorescence microscopy platforms. This review covers basic aspects of imaging-based CSMA technology, describes a wide range of immunofluorescence assays that have already been implemented successfully for CSMA screening and suggests future directions for advanced RNAi screening experiments.

  13. Specific in vivo knockdown of protein function by intrabodies

    PubMed Central

    Marschall, Andrea LJ; Dübel, Stefan; Böldicke, Thomas

    2015-01-01

    Intracellular antibodies (intrabodies) are recombinant antibody fragments that bind to target proteins expressed inside of the same living cell producing the antibodies. The molecules are commonly used to study the function of the target proteins (i.e., their antigens). The intrabody technology is an attractive alternative to the generation of gene-targeted knockout animals, and complements knockdown techniques such as RNAi, miRNA and small molecule inhibitors, by-passing various limitations and disadvantages of these methods. The advantages of intrabodies include very high specificity for the target, the possibility to knock down several protein isoforms by one intrabody and targeting of specific splice variants or even post-translational modifications. Different types of intrabodies must be designed to target proteins at different locations, typically either in the cytoplasm, in the nucleus or in the endoplasmic reticulum (ER). Most straightforward is the use of intrabodies retained in the ER (ER intrabodies) to knock down the function of proteins passing the ER, which disturbs the function of members of the membrane or plasma proteomes. More effort is needed to functionally knock down cytoplasmic or nuclear proteins because in this case antibodies need to provide an inhibitory effect and must be able to fold in the reducing milieu of the cytoplasm. In this review, we present a broad overview of intrabody technology, as well as applications both of ER and cytoplasmic intrabodies, which have yielded valuable insights in the biology of many targets relevant for drug development, including α-synuclein, TAU, BCR-ABL, ErbB-2, EGFR, HIV gp120, CCR5, IL-2, IL-6, β-amyloid protein and p75NTR. Strategies for the generation of intrabodies and various designs of their applications are also reviewed. PMID:26252565

  14. RNAi mediated, stable resistance to Triticum mosaic virus in wheat

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Triticum mosaic virus (TriMV), discovered in 2006, affects wheat production systems in the Great Plains of the United States. There are no available TriMV resistant commercial varieties. RNA interference (RNAi) was evaluated as an alternative strategy to generate resistance to TriMV. An RNAi pANDA...

  15. "Caenorhabditis Elegans" as an Undergraduate Educational Tool for Teaching RNAi

    ERIC Educational Resources Information Center

    Andersen, Janet; Krichevsky, Alexander; Leheste, Joerg R.; Moloney, Daniel J.

    2008-01-01

    Discovery of RNA-mediated interference (RNAi) is widely recognized as one of the most significant molecular biology breakthroughs in the past 10 years. There is a need for science educators to develop teaching tools and laboratory activities that demonstrate the power of this new technology and help students to better understand the RNAi process.…

  16. Imaging-guided delivery of RNAi for anticancer treatment.

    PubMed

    Wang, Junqing; Mi, Peng; Lin, Gan; Wáng, Yì Xiáng J; Liu, Gang; Chen, Xiaoyuan

    2016-09-01

    The RNA interference (RNAi) technique is a new modality for cancer therapy, and several candidates are being tested clinically. In the development of RNAi-based therapeutics, imaging methods can provide a visible and quantitative way to investigate the therapeutic effect at anatomical, cellular, and molecular level; to noninvasively trace the distribution; to and study the biological processes in preclinical and clinical stages. Their abilities are important not only for therapeutic optimization and evaluation but also for shortening of the time of drug development to market. Typically, imaging-functionalized RNAi therapeutics delivery that combines nanovehicles and imaging techniques to study and improve their biodistribution and accumulation in tumor site has been progressively integrated into anticancer drug discovery and development processes. This review presents an overview of the current status of translating the RNAi cancer therapeutics in the clinic, a brief description of the biological barriers in drug delivery, and the roles of imaging in aspects of administration route, systemic circulation, and cellular barriers for the clinical translation of RNAi cancer therapeutics, and with partial content for discussing the safety concerns. Finally, we focus on imaging-guided delivery of RNAi therapeutics in preclinical development, including the basic principles of different imaging modalities, and their advantages and limitations for biological imaging. With growing number of RNAi therapeutics entering the clinic, various imaging methods will play an important role in facilitating the translation of RNAi cancer therapeutics from bench to bedside. PMID:26805788

  17. RNAi control of aflatoxins in peanut plants, a multifactorial system

    Technology Transfer Automated Retrieval System (TEKTRAN)

    RNA-interference (RNAi)-mediated control of aflatoxin contamination in peanut plants is a multifactorial and hyper variable system. The use of RNAi biotechnology to silence single genes in plants has inherently high-variability among transgenic events. Also the level of expression of small interfe...

  18. Bringing RNA Interference (RNAi) into the High School Classroom

    ERIC Educational Resources Information Center

    Sengupta, Sibani

    2013-01-01

    RNA interference (abbreviated RNAi) is a relatively new discovery in the field of mechanisms that serve to regulate gene expression (a.k.a. protein synthesis). Gene expression can be regulated at the transcriptional level (mRNA production, processing, or stability) and at the translational level (protein synthesis). RNAi acts in a gene-specific…

  19. A library of MiMICs allows tagging of genes and reversible, spatial and temporal knockdown of proteins in Drosophila

    SciTech Connect

    Nagarkar-Jaiswal, Sonal; Lee, Pei-Tseng; Campbell, Megan E.; Chen, Kuchuan; Anguiano-Zarate, Stephanie; Cantu Gutierrez, Manuel; Busby, Theodore; Lin, Wen-Wen; He, Yuchun; Schulze, Karen L.; Booth, Benjamin W.; Evans-Holm, Martha; Venken, Koen J.T.; Levis, Robert W.; Spradling, Allan C.; Hoskins, Roger A.; Bellen, Hugo J.

    2015-03-31

    Here, we document a collection of ~7434 MiMIC (Minos Mediated Integration Cassette) insertions of which 2854 are inserted in coding introns. They allowed us to create a library of 400 GFP-tagged genes. We show that 72% of internally tagged proteins are functional, and that more than 90% can be imaged in unfixed tissues. Moreover, the tagged mRNAs can be knocked down by RNAi against GFP (iGFPi), and the tagged proteins can be efficiently knocked down by deGradFP technology. The phenotypes associated with RNA and protein knockdown typically correspond to severe loss of function or null mutant phenotypes. Finally, we demonstrate reversible, spatial, and temporal knockdown of tagged proteins in larvae and adult flies. This new strategy and collection of strains allows unprecedented in vivo manipulations in flies for many genes. These strategies will likely extend to vertebrates.

  20. A library of MiMICs allows tagging of genes and reversible, spatial and temporal knockdown of proteins in Drosophila

    DOE PAGESBeta

    Nagarkar-Jaiswal, Sonal; Lee, Pei-Tseng; Campbell, Megan E.; Chen, Kuchuan; Anguiano-Zarate, Stephanie; Cantu Gutierrez, Manuel; Busby, Theodore; Lin, Wen-Wen; He, Yuchun; Schulze, Karen L.; et al

    2015-03-31

    Here, we document a collection of ~7434 MiMIC (Minos Mediated Integration Cassette) insertions of which 2854 are inserted in coding introns. They allowed us to create a library of 400 GFP-tagged genes. We show that 72% of internally tagged proteins are functional, and that more than 90% can be imaged in unfixed tissues. Moreover, the tagged mRNAs can be knocked down by RNAi against GFP (iGFPi), and the tagged proteins can be efficiently knocked down by deGradFP technology. The phenotypes associated with RNA and protein knockdown typically correspond to severe loss of function or null mutant phenotypes. Finally, we demonstratemore » reversible, spatial, and temporal knockdown of tagged proteins in larvae and adult flies. This new strategy and collection of strains allows unprecedented in vivo manipulations in flies for many genes. These strategies will likely extend to vertebrates.« less

  1. RNAi-Mediated CCR5 Knockdown Provides HIV-1 Resistance to Memory T Cells in Humanized BLT Mice

    PubMed Central

    Shimizu, Saki; Ringpis, Gene-Errol; Marsden, Matthew D; Cortado, Ruth V; Wilhalme, Holly M; Elashoff, David; Zack, Jerome A; Chen, Irvin S Y; An, Dong Sung

    2015-01-01

    Transplantation of hematopoietic stem/progenitor cells (HSPC) modified with a lentiviral vector bearing a potent nontoxic short hairpin RNA (sh1005) directed to the HIV coreceptor CCR5 is capable of continuously producing CCR5 downregulated CD4+ T lymphocytes. Here, we characterized HIV-1 resistance of the sh1005-modified CD4+ T lymphocytes in vivo in humanized bone marrow/liver/thymus (hu BLT) mice. The sh1005-modified CD4+ T lymphocytes were positively selected in CCR5-tropic HIV-1–challenged mice. The sh1005-modified memory CD4+ T lymphocytes (the primary target of CCR5-tropic HIV-1) expressing sh1005 were maintained in lymphoid tissues in CCR5-tropic HIV-1–challenged mice. Frequencies of HIV-1 p24 expressing cells were significantly reduced in the sh1005-modified splenocytes by ex vivo cell stimulation confirming that CCR5 downregulated sh1005 modified cells are protected from viral infection. These results demonstrate that stable CCR5 downregulation through genetic modification of human HSPC by lentivirally delivered sh1005 is highly effective in providing HIV-1 resistance. Our results provide in vivo evidence in a relevant small animal model that sh1005 is a potent early-step anti-HIV reagent that has potential as a novel anti-HIV-1 HSPC gene therapeutic reagent for human applications. PMID:25689223

  2. Identification, RNAi Knockdown and Functional Analysis of an Ejaculate Protein that Mediates a Postmating, Prezgotic Phenotype in a cricket

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Male ejaculate proteins, including both sperm and seminal fluid proteins, play an important role in mediating reproductive biology. The function of ejaculate proteins can include enabling sperm-egg interactions, enhancing sperm storage, mediating female attractiveness, and even regulating female lif...

  3. Genome-wide RNAi Screen Identifies Cohesin Genes as Modifiers of Renewal and Differentiation in Human HSCs.

    PubMed

    Galeev, Roman; Baudet, Aurélie; Kumar, Praveen; Rundberg Nilsson, Alexandra; Nilsson, Björn; Soneji, Shamit; Törngren, Therese; Borg, Åke; Kvist, Anders; Larsson, Jonas

    2016-03-29

    To gain insights into the regulatory mechanisms of hematopoietic stem cells (HSCs), we employed a genome-wide RNAi screen in human cord-blood derived cells and identified candidate genes whose knockdown maintained the HSC phenotype during culture. A striking finding was the identification of members of the cohesin complex (STAG2, RAD21, STAG1, and SMC3) among the top 20 genes from the screen. Upon individual validation of these cohesin genes, we found that their knockdown led to an immediate expansion of cells with an HSC phenotype in vitro. A similar expansion was observed in vivo following transplantation to immunodeficient mice. Transcriptome analysis of cohesin-deficient CD34(+) cells showed an upregulation of HSC-specific genes, demonstrating an immediate shift toward a more stem-cell-like gene expression signature upon cohesin deficiency. Our findings implicate cohesin as a major regulator of HSCs and illustrate the power of global RNAi screens to identify modifiers of cell fate. PMID:26997282

  4. A Global In Vivo Drosophila RNAi Screen Identifies a Key Role of Ceramide Phosphoethanolamine for Glial Ensheathment of Axons

    PubMed Central

    Ghosh, Aniket; Kling, Tina; Snaidero, Nicolas; Sampaio, Julio L.; Shevchenko, Andrej; Gras, Heribert; Geurten, Bart; Göpfert, Martin C.; Schulz, Jörg B.; Voigt, Aaron; Simons, Mikael

    2013-01-01

    Glia are of vital importance for all complex nervous system. One of the many functions of glia is to insulate and provide trophic and metabolic support to axons. Here, using glial-specific RNAi knockdown in Drosophila, we silenced 6930 conserved genes in adult flies to identify essential genes and pathways. Among our screening hits, metabolic processes were highly represented, and genes involved in carbohydrate and lipid metabolic pathways appeared to be essential in glia. One critical pathway identified was de novo ceramide synthesis. Glial knockdown of lace, a subunit of the serine palmitoyltransferase associated with hereditary sensory and autonomic neuropathies in humans, resulted in ensheathment defects of peripheral nerves in Drosophila. A genetic dissection study combined with shotgun high-resolution mass spectrometry of lipids showed that levels of ceramide phosphoethanolamine are crucial for axonal ensheathment by glia. A detailed morphological and functional analysis demonstrated that the depletion of ceramide phosphoethanolamine resulted in axonal defasciculation, slowed spike propagation, and failure of wrapping glia to enwrap peripheral axons. Supplementing sphingosine into the diet rescued the neuropathy in flies. Thus, our RNAi study in Drosophila identifies a key role of ceramide phosphoethanolamine in wrapping of axons by glia. PMID:24348263

  5. Drosophila RNAi screening in a postgenomic world

    PubMed Central

    2011-01-01

    Drosophila melanogaster has a long history as a model organism with several unique features that make it an ideal research tool for the study of the relationship between genotype and phenotype. Importantly fundamental genetic principles as well as key human disease genes have been uncovered through the use of Drosophila. The contribution of the fruit fly to science and medicine continues in the postgenomic era as cell-based Drosophila RNAi screens are a cost-effective and scalable enabling technology that can be used to quantify the contribution of different genes to diverse cellular processes. Drosophila high-throughput screens can also be used as integral part of systems-level approaches to describe the architecture and dynamics of cellular networks. PMID:21752787

  6. RNAi Screening of Leukemia Cells Using Electroporation.

    PubMed

    Agarwal, Anupriya; Tyner, Jeffrey W

    2016-01-01

    RNAi-mediated screening has been an integral tool for biological discovery for the past 15 years. A variety of approaches have been employed for implementation of this technique, including pooled, depletion/enrichment screening with lentiviral shRNAs, and segregated screening of panels of individual siRNAs. The latter approach of siRNA panel screening requires efficient methods for transfection of siRNAs into the target cells. In the case of suspension leukemia cell lines and primary cells, many of the conventional transfection techniques using liposomal or calcium phosphate-mediated transfection provide very low efficiency. In this case, electroporation is the only transfection technique offering high efficiency transfection of siRNAs into the target leukemia cells. Here, we describe methods for optimization and implementation of siRNA electroporation into leukemia cell lines and primary patient specimens, and we further offer suggested electroporation settings for some commonly used leukemia cell lines. PMID:27581286

  7. AAV-mediated gene targeting methods for human cells

    PubMed Central

    Khan, Iram F; Hirata, Roli K; Russell, David W

    2013-01-01

    Gene targeting with adeno-associated virus (AAV) vectors has been demonstrated in multiple human cell types, with targeting frequencies ranging from 10−5 to 10−2 per infected cell. these targeting frequencies are 1–4 logs higher than those obtained by conventional transfection or electroporation approaches. a wide variety of different types of mutations can be introduced into chromosomal loci with high fidelity and without genotoxicity. Here we provide a detailed protocol for gene targeting in human cells with AAV vectors. We describe methods for vector design, stock preparation and titration. optimized transduction protocols are provided for human pluripotent stem cells, mesenchymal stem cells, fibroblasts and transformed cell lines, as well as a method for identifying targeted clones by southern blots. this protocol (from vector design through a single round of targeting and screening) can be completed in ~10 weeks; each subsequent round of targeting and screening should take an additional 7 weeks. PMID:21455185

  8. AAV-mediated gene transfer to the mouse CNS

    PubMed Central

    Stoica, Lorelei; Ahmed, Seemin S.

    2013-01-01

    Recombinant adeno associated virus (rAAV) vectors are great tools for gene transfer due to their ability to mediate long-term gene expression. Recombinant AAVs have been used at various ages of development with no apparent toxicity. There are multiple ways of delivering AAV vectors to the CNS, depending on the stage of development of the mouse. In neonates, intravascular injections into the facial vein are often used. In adults, direct injections into target regions of the brain are achieved with great spatiotemporal control through stereotaxic surgeries. Recently, discoveries of new AAV vectors with the ability to cross the blood brain barrier have made it possible to also target the adult CNS by intravascular injections. rAAVs have been successfully used as gene transfer vehicles in multiple animal models of CNS disorders, and several clinical trials are currently underway. PMID:23686825

  9. First knockdown gene expression in bat (Hipposideros armiger) brain mediated by lentivirus.

    PubMed

    Chen, Qi; Zhu, Tengteng; Jones, Gareth; Zhang, Junpeng; Sun, Yi

    2013-06-01

    Lentivirus-mediated RNA interference (RNAi) is a potent experimental tool for investigating gene functions in vitro and in vivo. It has advantages that transgenic technology lacks. However, in vivo applications are difficult to apply in the central nervous system of non-model organisms due to the lack of a standard brain atlas and genetic information. Here, we report the development of an in vivo gene delivery system used in bat brain tissue for the first time, based on lentivirus (LV) vectors expressing short hairpin RNA (shRNA) targeting Hipposideros armiger forkhead box P2 (FoxP2). In vitro transfection into HEK 293T cell with the vector bearing the cassettes encoding FoxP2 shRNA verified the knockdown efficiency. Pseudovirus particles were administered via stereotactic intracerebral microinjection into the anterior cingulate cortex of H. armiger. FoxP2 is of major interest because of its role in sensorimotor coordination and probably in echolocation. Subsequent in situ hybridization validated the in vivo silencing of the target gene. This report demonstrates that LV-mediated expression of RNAi could achieve effective gene silencing in bats, a non-model organism, and will assist in elucidating the functions of bat genes. PMID:22965420

  10. An optimized microRNA backbone for effective single-copy RNAi.

    PubMed

    Fellmann, Christof; Hoffmann, Thomas; Sridhar, Vaishali; Hopfgartner, Barbara; Muhar, Matthias; Roth, Mareike; Lai, Dan Yu; Barbosa, Inês A M; Kwon, Jung Shick; Guan, Yuanzhe; Sinha, Nishi; Zuber, Johannes

    2013-12-26

    Short hairpin RNA (shRNA) technology enables stable and regulated gene repression. For establishing experimentally versatile RNAi tools and minimizing toxicities, synthetic shRNAs can be embedded into endogenous microRNA contexts. However, due to our incomplete understanding of microRNA biogenesis, such "shRNAmirs" often fail to trigger potent knockdown, especially when expressed from a single genomic copy. Following recent advances in design of synthetic shRNAmir stems, here we take a systematic approach to optimize the experimental miR-30 backbone. Among several favorable features, we identify a conserved element 3' of the basal stem as critically required for optimal shRNAmir processing and implement it in an optimized backbone termed "miR-E", which strongly increases mature shRNA levels and knockdown efficacy. Existing miR-30 reagents can be easily converted to miR-E, and its combination with up-to-date design rules establishes a validated and accessible platform for generating effective single-copy shRNA libraries that will facilitate the functional annotation of the genome. PMID:24332856

  11. RNAi-based therapeutic nanostrategy: IL-8 gene silencing in pancreatic cancer cells using gold nanorods delivery vehicles

    NASA Astrophysics Data System (ADS)

    Panwar, Nishtha; Yang, Chengbin; Yin, Feng; Yoon, Ho Sup; Swee Chuan, Tjin; Yong, Ken-Tye

    2015-09-01

    RNA interference (RNAi)-based gene silencing possesses great ability for therapeutic intervention in pancreatic cancer. Among various oncogene mutations, Interleukin-8 (IL-8) gene mutations are found to be overexpressed in many pancreatic cell lines. In this work, we demonstrate IL-8 gene silencing by employing an RNAi-based gene therapy approach and this is achieved by using gold nanorods (AuNRs) for efficient delivery of IL-8 small interfering RNA (siRNA) to the pancreatic cell lines of MiaPaCa-2 and Panc-1. Upon comparing to Panc-1 cells, we found that the dominant expression of the IL-8 gene in MiaPaCa-2 cells resulted in an aggressive behavior towards the processes of cell invasion and metastasis. We have hence investigated the suitability of using AuNRs as novel non-viral nanocarriers for the efficient uptake and delivery of IL-8 siRNA in realizing gene knockdown of both MiaPaCa-2 and Panc-1 cells. Flow cytometry and fluorescence imaging techniques have been applied to confirm transfection and release of IL-8 siRNA. The ratio of AuNRs and siRNA has been optimized and transfection efficiencies as high as 88.40 ± 2.14% have been achieved. Upon successful delivery of IL-8 siRNA into cancer cells, the effects of IL-8 gene knockdown are quantified in terms of gene expression, cell invasion, cell migration and cell apoptosis assays. Statistical comparative studies for both MiaPaCa-2 and Panc-1 cells are presented in this work. IL-8 gene silencing has been demonstrated with knockdown efficiencies of 81.02 ± 10.14% and 75.73 ± 6.41% in MiaPaCa-2 and Panc-1 cells, respectively. Our results are then compared with a commercial transfection reagent, Oligofectamine, serving as positive control. The gene knockdown results illustrate the potential role of AuNRs as non-viral gene delivery vehicles for RNAi-based targeted cancer therapy applications.

  12. RNAi-based therapeutic nanostrategy: IL-8 gene silencing in pancreatic cancer cells using gold nanorods delivery vehicles.

    PubMed

    Panwar, Nishtha; Yang, Chengbin; Yin, Feng; Yoon, Ho Sup; Chuan, Tjin Swee; Yong, Ken-Tye

    2015-09-11

    RNA interference (RNAi)-based gene silencing possesses great ability for therapeutic intervention in pancreatic cancer. Among various oncogene mutations, Interleukin-8 (IL-8) gene mutations are found to be overexpressed in many pancreatic cell lines. In this work, we demonstrate IL-8 gene silencing by employing an RNAi-based gene therapy approach and this is achieved by using gold nanorods (AuNRs) for efficient delivery of IL-8 small interfering RNA (siRNA) to the pancreatic cell lines of MiaPaCa-2 and Panc-1. Upon comparing to Panc-1 cells, we found that the dominant expression of the IL-8 gene in MiaPaCa-2 cells resulted in an aggressive behavior towards the processes of cell invasion and metastasis. We have hence investigated the suitability of using AuNRs as novel non-viral nanocarriers for the efficient uptake and delivery of IL-8 siRNA in realizing gene knockdown of both MiaPaCa-2 and Panc-1 cells. Flow cytometry and fluorescence imaging techniques have been applied to confirm transfection and release of IL-8 siRNA. The ratio of AuNRs and siRNA has been optimized and transfection efficiencies as high as 88.40 ± 2.14% have been achieved. Upon successful delivery of IL-8 siRNA into cancer cells, the effects of IL-8 gene knockdown are quantified in terms of gene expression, cell invasion, cell migration and cell apoptosis assays. Statistical comparative studies for both MiaPaCa-2 and Panc-1 cells are presented in this work. IL-8 gene silencing has been demonstrated with knockdown efficiencies of 81.02 ± 10.14% and 75.73 ± 6.41% in MiaPaCa-2 and Panc-1 cells, respectively. Our results are then compared with a commercial transfection reagent, Oligofectamine, serving as positive control. The gene knockdown results illustrate the potential role of AuNRs as non-viral gene delivery vehicles for RNAi-based targeted cancer therapy applications. PMID:26291710

  13. CRISPR/Cas9-based generation of knockdown mice by intronic insertion of artificial microRNA using longer single-stranded DNA.

    PubMed

    Miura, Hiromi; Gurumurthy, Channabasavaiah B; Sato, Takehito; Sato, Masahiro; Ohtsuka, Masato

    2015-01-01

    Knockdown mouse models, where gene dosages can be modulated, provide valuable insights into gene function. Typically, such models are generated by embryonic stem (ES) cell-based targeted insertion, or pronuclear injection, of the knockdown expression cassette. However, these methods are associated with laborious and time-consuming steps, such as the generation of large constructs with elements needed for expression of a functional RNAi-cassette, ES-cell handling, or screening for mice with the desired knockdown effect. Here, we demonstrate that reliable knockdown models can be generated by targeted insertion of artificial microRNA (amiRNA) sequences into a specific locus in the genome [such as intronic regions of endogenous eukaryotic translation elongation factor 2 (eEF-2) gene] using the Clustered Regularly Interspaced Short Palindromic Repeats/Crispr associated 9 (CRISPR/Cas9) system. We used in vitro synthesized single-stranded DNAs (about 0.5-kb long) that code for amiRNA sequences as repair templates in CRISPR/Cas9 mutagenesis. Using this approach we demonstrate that amiRNA cassettes against exogenous (eGFP) or endogenous [orthodenticle homeobox 2 (Otx2)] genes can be efficiently targeted to a predetermined locus in the genome and result in knockdown of gene expression. We also provide a strategy to establish conditional knockdown models with this method. PMID:26242611

  14. Knockdown of the Rhipicephalus microplus Cytochrome c Oxidase Subunit III Gene Is Associated with a Failure of Anaplasma marginale Transmission

    PubMed Central

    Bifano, Thais D.; Ueti, Massaro W.; Esteves, Eliane; Reif, Kathryn E.; Braz, Glória R. C.; Scoles, Glen A.; Bastos, Reginaldo G.; White, Stephen N.; Daffre, Sirlei

    2014-01-01

    Rhipicephalus microplus is an obligate hematophagous ectoparasite of cattle and an important biological vector of Anaplasma marginale in tropical and subtropical regions. The primary determinants for A. marginale transmission are infection of the tick gut, followed by infection of salivary glands. Transmission of A. marginale to cattle occurs via infected saliva delivered during tick feeding. Interference in colonization of either the tick gut or salivary glands can affect transmission of A. marginale to naïve animals. In this study, we used the tick embryonic cell line BME26 to identify genes that are modulated in response to A. marginale infection. Suppression-subtractive hybridization libraries (SSH) were constructed, and five up-regulated genes {glutathione S-transferase (GST), cytochrome c oxidase sub III (COXIII), dynein (DYN), synaptobrevin (SYN) and phosphatidylinositol-3,4,5-triphosphate 3-phosphatase (PHOS)} were selected as targets for functional in vivo genomic analysis. RNA interference (RNAi) was used to determine the effect of tick gene knockdown on A. marginale acquisition and transmission. Although RNAi consistently knocked down all individually examined tick genes in infected tick guts and salivary glands, only the group of ticks injected with dsCOXIII failed to transmit A. marginale to naïve calves. To our knowledge, this is the first report demonstrating that RNAi of a tick gene is associated with a failure of A. marginale transmission. PMID:24878588

  15. Distinct RNA-dependent RNA polymerases are required for RNAi triggered by double-stranded RNA versus truncated transgenes in Paramecium tetraurelia

    PubMed Central

    Marker, Simone; Le Mouël, Anne; Meyer, Eric; Simon, Martin

    2010-01-01

    In many eukaryotes, RNA-dependent RNA polymerases (RdRPs) play key roles in the RNAi pathway. They have been implicated in the recognition and processing of aberrant transcripts triggering the process, and in amplification of the silencing response. We have tested the functions of RdRP genes from the ciliate Paramecium tetraurelia in experimentally induced and endogenous mechanisms of gene silencing. In this organism, RNAi can be triggered either by high-copy, truncated transgenes or by directly feeding cells with double-stranded RNA (dsRNA). Surprisingly, dsRNA-induced silencing depends on the putatively functional RDR1 and RDR2 genes, which are required for the accumulation of both primary siRNAs and a distinct class of small RNAs suggestive of secondary siRNAs. In contrast, a third gene with a highly divergent catalytic domain, RDR3, is required for siRNA accumulation when RNAi is triggered by truncated transgenes. Our data further implicate RDR3 in the accumulation of previously described endogenous siRNAs and in the regulation of the surface antigen gene family. While only one of these genes is normally expressed in any clonal cell line, the knockdown of RDR3 leads to co-expression of multiple antigens. These results provide evidence for a functional specialization of Paramecium RdRP genes in distinct RNAi pathways operating during vegetative growth. PMID:20200046

  16. Distinct RNA-dependent RNA polymerases are required for RNAi triggered by double-stranded RNA versus truncated transgenes in Paramecium tetraurelia.

    PubMed

    Marker, Simone; Le Mouël, Anne; Meyer, Eric; Simon, Martin

    2010-07-01

    In many eukaryotes, RNA-dependent RNA polymerases (RdRPs) play key roles in the RNAi pathway. They have been implicated in the recognition and processing of aberrant transcripts triggering the process, and in amplification of the silencing response. We have tested the functions of RdRP genes from the ciliate Paramecium tetraurelia in experimentally induced and endogenous mechanisms of gene silencing. In this organism, RNAi can be triggered either by high-copy, truncated transgenes or by directly feeding cells with double-stranded RNA (dsRNA). Surprisingly, dsRNA-induced silencing depends on the putatively functional RDR1 and RDR2 genes, which are required for the accumulation of both primary siRNAs and a distinct class of small RNAs suggestive of secondary siRNAs. In contrast, a third gene with a highly divergent catalytic domain, RDR3, is required for siRNA accumulation when RNAi is triggered by truncated transgenes. Our data further implicate RDR3 in the accumulation of previously described endogenous siRNAs and in the regulation of the surface antigen gene family. While only one of these genes is normally expressed in any clonal cell line, the knockdown of RDR3 leads to co-expression of multiple antigens. These results provide evidence for a functional specialization of Paramecium RdRP genes in distinct RNAi pathways operating during vegetative growth. PMID:20200046

  17. RNAi-Directed Downregulation of Vacuolar H+-ATPase Subunit A Results in Enhanced Stomatal Aperture and Density in Rice

    PubMed Central

    Xiao, Fangming; Cao, Shuqing; Liu, Yongsheng

    2013-01-01

    Stomatal movement plays a key role in plant development and response to drought and salt stress by regulating gas exchange and water loss. A number of genes have been demonstrated to be involved in the regulation of this process. Using inverse genetics approach, we characterized the function of a rice (Oryza sativa L.) vacuolar H+-ATPase subunit A (OsVHA-A) gene in stomatal conductance regulation and physiological response to salt and osmotic stress. OsVHA-A was constitutively expressed in different rice tissues, and the fusion protein of GFP-OsVHA-A was exclusively targeted to tonoplast when transiently expressed in the onion epidermal cells. Heterologous expression of OsVHA-A was able to rescue the yeast mutant vma1Δ (lacking subunit A activity) phenotype, suggesting that it partially restores the activity of V-ATPase. Meanwhile, RNAi-directed knockdown of OsVHA-A led to a reduction of vacuolar H+-ATPase activity and an enhancement of plasma membrane H+-ATPase activity, thereby increasing the concentrations of extracellular H+ and intracellular K+ and Na+ under stress conditions. Knockdown of OsVHA-A also resulted in the upregulation of PAM3 (plasma membrane H+-ATPase 3) and downregulation of CAM1 (calmodulin 1), CAM3 (calmodulin 3) and YDA1 (YODA, a MAPKK gene). Altered level of the ion concentration and the gene expression by knockdown of OsVHA-A probably resulted in expanded aperture of stomatal pores and increased stomatal density. In addition, OsVHA-A RNAi plants displayed significant growth inhibition under salt and osmotic stress conditions. Taken together, our results suggest that OsVHA-A takes part in regulating stomatal density and opening via interfering with pH value and ionic equilibrium in guard cells and thereby affects the growth of rice plants. PMID:23894405

  18. RNAi-directed downregulation of vacuolar H(+) -ATPase subunit a results in enhanced stomatal aperture and density in rice.

    PubMed

    Zhang, Huiying; Niu, Xiangli; Liu, Jia; Xiao, Fangming; Cao, Shuqing; Liu, Yongsheng

    2013-01-01

    Stomatal movement plays a key role in plant development and response to drought and salt stress by regulating gas exchange and water loss. A number of genes have been demonstrated to be involved in the regulation of this process. Using inverse genetics approach, we characterized the function of a rice (Oryza sativa L.) vacuolar H(+)-ATPase subunit A (OsVHA-A) gene in stomatal conductance regulation and physiological response to salt and osmotic stress. OsVHA-A was constitutively expressed in different rice tissues, and the fusion protein of GFP-OsVHA-A was exclusively targeted to tonoplast when transiently expressed in the onion epidermal cells. Heterologous expression of OsVHA-A was able to rescue the yeast mutant vma1Δ (lacking subunit A activity) phenotype, suggesting that it partially restores the activity of V-ATPase. Meanwhile, RNAi-directed knockdown of OsVHA-A led to a reduction of vacuolar H(+)-ATPase activity and an enhancement of plasma membrane H(+)-ATPase activity, thereby increasing the concentrations of extracellular H(+) and intracellular K(+) and Na(+) under stress conditions. Knockdown of OsVHA-A also resulted in the upregulation of PAM3 (plasma membrane H(+)-ATPase 3) and downregulation of CAM1 (calmodulin 1), CAM3 (calmodulin 3) and YDA1 (YODA, a MAPKK gene). Altered level of the ion concentration and the gene expression by knockdown of OsVHA-A probably resulted in expanded aperture of stomatal pores and increased stomatal density. In addition, OsVHA-A RNAi plants displayed significant growth inhibition under salt and osmotic stress conditions. Taken together, our results suggest that OsVHA-A takes part in regulating stomatal density and opening via interfering with pH value and ionic equilibrium in guard cells and thereby affects the growth of rice plants. PMID:23894405

  19. Knockdown of a nutrient amino acid transporter gene LdNAT1 reduces free neutral amino acid contents and impairs Leptinotarsa decemlineata pupation

    PubMed Central

    Fu, Kai-Yun; Guo, Wen-Chao; Ahmat, Tursun; Li, Guo-Qing

    2015-01-01

    A Leptinotarsa decemlineata SLC6 NAT gene (LdNAT1) was cloned. LdNAT1 was highly expressed in the larval alimentary canal especially midgut. LdNAT1 mRNA levels were high right after the molt and low just before the molt. JH and a JH analog pyriproxyfen activated LdNAT1 expression. RNAi of an allatostatin gene LdAS-C increased JH and upregulated LdNAT1 transcription. Conversely, silencing of a JH biosynthesis gene LdJHAMT decreased JH and reduced LdNAT1 expression. Moreover, 20E and an ecdysteroid agonist halofenozide repressed LdNAT1 expression, whereas a decrease in 20E by RNAi of an ecdysteroidogenesis gene LdSHD and disruption of 20E signaling by knockdown of LdE75 and LdFTZ-F1 activated LdNAT1 expression. Thus, LdNAT1 responded to both 20E and JH. Moreover, knockdown of LdNAT1 reduced the contents of cysteine, histidine, isoleucine, leucine, methionine, phenylalanine and serine in the larval bodies and increased the contents of these amino acids in the larval feces. Furthermore, RNAi of LdNAT1 inhibited insulin/target of rapamycin pathway, lowered 20E and JH titers, reduced 20E and JH signaling, retarded larval growth and impaired pupation. These data showed that LdNAT1 was involved in the absorption of several neutral amino acids critical for larval growth and metamorphosis. PMID:26657797

  20. An RNAi-based screen reveals PLK1, CDK1 and NDC80 as potential therapeutic targets in malignant pleural mesothelioma

    PubMed Central

    Linton, A; Cheng, Y Y; Griggs, K; Kirschner, M B; Gattani, S; Srikaran, S; Chuan-Hao Kao, S; McCaughan, B C; Klebe, S; van Zandwijk, N; Reid, G

    2014-01-01

    Background: Malignant pleural mesothelioma (MPM) is an aggressive tumour originating in the thoracic mesothelium. Prognosis remains poor with 9- to 12-month median survival, and new targets for treatments are desperately needed. Methods: Utilising an RNA interference (RNAi)-based screen of 40 genes overexpressed in tumours, including genes involved in the control of cell cycle, DNA replication and repair, we investigated potential therapeutic targets for MPM. Following in vitro characterisation of the effects of target silencing on MPM cells, candidates were assessed in tumour samples from 154 patients. Results: Gene knockdown in MPM cell lines identified growth inhibition following knockdown of NDC80, CDK1 and PLK1. Target knockdown induced cell-cycle arrest and increased apoptosis. Using small-molecule inhibitors specific for these three proteins also led to growth inhibition of MPM cell lines, and Roscovitine (inhibitor of CDK1) sensitised cells to cisplatin. Protein expression was also measured in tumour samples, with markedly variable levels of CDK1 and PLK1 noted. PLK1 expression in over 10% of cells correlated significantly with a poor prognosis. Conclusion: These results suggest that RNAi-based screening has utility in identifying new targets for MPM, and that inhibition of NDC80, CDK1 and PLK1 may hold promise for treatment of this disease. PMID:24327015

  1. Oral delivery of double-stranded RNAs and siRNAs induces RNAi effects in the potato/tomato psyllid, Bactericerca cockerelli.

    PubMed

    Wuriyanghan, Hada; Rosa, Cristina; Falk, Bryce W

    2011-01-01

    The potato/tomato psyllid, Bactericerca cockerelli (B. cockerelli), and the Asian citrus psyllid, Diaphorina citri (D. citri), are very important plant pests, but they are also vectors of phloem-limited bacteria that are associated with two devastating plant diseases. B. cockerelli is the vector of Candidatus Liberibacter psyllaurous (solanacearum), which is associated with zebra chip disease of potatoes, and D. citri is the vector of Ca. Liberibacter asiaticus, which is associated with the Huanglongbing (citrus greening) disease that currently threatens the entire Florida citrus industry. Here we used EST sequence information from D. citri to identify potential targets for RNA interference in B. cockerelli. We targeted ubiquitously expressed and gut-abundant mRNAs via injection and oral acquisition of double-stranded RNAs and siRNAs and were able to induce mortality in recipient psyllids. We also showed knockdown of target mRNAs, and that oral acquisition resulted primarily in mRNA knockdown in the psyllid gut. Concurrent with gene knockdown was the accumulation of target specific ∼ 21 nucleotide siRNAs for an abundant mRNA for BC-Actin. These results showed that RNAi can be a powerful tool for gene function studies in psyllids, and give support for continued efforts for investigating RNAi approaches as possible tools for psyllid and plant disease control. PMID:22110747

  2. Ribonucleic acid interference (RNAi) and control of citrus pests

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Ribonucleic acid interference, RNAi, applications and function are described for the non-scientist to bring a better understanding of how this emerging technology is providing environmentally friendly, non-transgenic, insect pest control. ...

  3. Biotechnological uses of RNAi in plants: risk assessment considerations.

    PubMed

    Casacuberta, Josep M; Devos, Yann; du Jardin, Patrick; Ramon, Matthew; Vaucheret, Hervé; Nogué, Fabien

    2015-03-01

    RNAi offers opportunities to generate new traits in genetically modified (GM) plants. Instead of expressing novel proteins, RNAi-based GM plants reduce target gene expression. Silencing of off-target genes may trigger unintended effects, and identifying these genes would facilitate risk assessment. However, using bioinformatics alone is not reliable, due to the lack of genomic data and insufficient knowledge of mechanisms governing mRNA-small (s)RNA interactions. PMID:25721261

  4. Novel Drosophila Viruses Encode Host-Specific Suppressors of RNAi

    PubMed Central

    van Mierlo, Joël T.; Overheul, Gijs J.; Obadia, Benjamin; van Cleef, Koen W. R.; Webster, Claire L.; Saleh, Maria-Carla; Obbard, Darren J.; van Rij, Ronald P.

    2014-01-01

    The ongoing conflict between viruses and their hosts can drive the co-evolution between host immune genes and viral suppressors of immunity. It has been suggested that an evolutionary ‘arms race’ may occur between rapidly evolving components of the antiviral RNAi pathway of Drosophila and viral genes that antagonize it. We have recently shown that viral protein 1 (VP1) of Drosophila melanogaster Nora virus (DmelNV) suppresses Argonaute-2 (AGO2)-mediated target RNA cleavage (slicer activity) to antagonize antiviral RNAi. Here we show that viral AGO2 antagonists of divergent Nora-like viruses can have host specific activities. We have identified novel Nora-like viruses in wild-caught populations of D. immigrans (DimmNV) and D. subobscura (DsubNV) that are 36% and 26% divergent from DmelNV at the amino acid level. We show that DimmNV and DsubNV VP1 are unable to suppress RNAi in D. melanogaster S2 cells, whereas DmelNV VP1 potently suppresses RNAi in this host species. Moreover, we show that the RNAi suppressor activity of DimmNV VP1 is restricted to its natural host species, D. immigrans. Specifically, we find that DimmNV VP1 interacts with D. immigrans AGO2, but not with D. melanogaster AGO2, and that it suppresses slicer activity in embryo lysates from D. immigrans, but not in lysates from D. melanogaster. This species-specific interaction is reflected in the ability of DimmNV VP1 to enhance RNA production by a recombinant Sindbis virus in a host-specific manner. Our results emphasize the importance of analyzing viral RNAi suppressor activity in the relevant host species. We suggest that rapid co-evolution between RNA viruses and their hosts may result in host species-specific activities of RNAi suppressor proteins, and therefore that viral RNAi suppressors could be host-specificity factors. PMID:25032815

  5. Knock-down of heat-shock protein 90 and isocitrate lyase gene expression reduced root-knot nematode reproduction.

    PubMed

    Lourenço-Tessutti, Isabela Tristan; Souza Junior, José Dijair Antonino; Martins-de-Sa, Diogo; Viana, Antônio Américo Barbosa; Carneiro, Regina Maria Dechechi Gomes; Togawa, Roberto Coiti; de Almeida-Engler, Janice; Batista, João Aguiar Nogueira; Silva, Maria Cristina Mattar; Fragoso, Rodrigo Rocha; Grossi-de-Sa, Maria Fatima

    2015-05-01

    Crop losses caused by nematode infections are estimated to be valued at USD 157 billion per year. Meloidogyne incognita, a root-knot nematode (RKN), is considered to be one of the most important plant pathogens due to its worldwide distribution and the austere damage it can cause to a large variety of agronomically important crops. RNA interference (RNAi), a gene silencing process, has proven to be a valuable biotechnology alternative method for RKN control. In this study, the RNAi approach was applied, using fragments of M. incognita genes that encode for two essential molecules, heat-shock protein 90 (HSP90) and isocitrate lyase (ICL). Plant-mediated RNAi of these genes led to a significant level of resistance against M. incognita in the transgenic Nicotiana tabacum plants. Bioassays of plants expressing HSP90 dsRNA demonstrated a delay in gall formation and up to 46% reduction in eggs compared with wild-type plants. A reduction in the level of HSP90 transcripts was observed in recovered eggs from plants expressing dsRNA, indicating that gene silencing persisted and was passed along to first progeny. The ICL knock-down had no clear effect on gall formation but resulted in up to 77% reduction in egg oviposition compared with wild-type plants. Our data suggest that both genes may be involved in RKN development and reproduction. Thus, in this paper, we describe essential candidate genes that could be applied to generate genetically modified crops, using the RNAi strategy to control RKN parasitism. PMID:26020830

  6. In Vivo RNAi-Based Screens: Studies in Model Organisms

    PubMed Central

    Yamamoto-Hino, Miki; Goto, Satoshi

    2013-01-01

    RNA interference (RNAi) is a technique widely used for gene silencing in organisms and cultured cells, and depends on sequence homology between double-stranded RNA (dsRNA) and target mRNA molecules. Numerous cell-based genome-wide screens have successfully identified novel genes involved in various biological processes, including signal transduction, cell viability/death, and cell morphology. However, cell-based screens cannot address cellular processes such as development, behavior, and immunity. Drosophila and Caenorhabditis elegans are two model organisms whose whole bodies and individual body parts have been subjected to RNAi-based genome-wide screening. Moreover, Drosophila RNAi allows the manipulation of gene function in a spatiotemporal manner when it is implemented using the Gal4/UAS system. Using this inducible RNAi technique, various large-scale screens have been performed in Drosophila, demonstrating that the method is straightforward and valuable. However, accumulated results reveal that the results of RNAi-based screens have relatively high levels of error, such as false positives and negatives. Here, we review in vivo RNAi screens in Drosophila and the methods that could be used to remove ambiguity from screening results. PMID:24705267

  7. RNAi screening to identify postembryonic phenotypes in C. elegans.

    PubMed

    Beifuss, Katherine K; Gumienny, Tina L

    2012-01-01

    C. elegans has proven to be a valuable model system for the discovery and functional characterization of many genes and gene pathways. More sophisticated tools and resources for studies in this system are facilitating continued discovery of genes with more subtle phenotypes or roles. Here we present a generalized protocol we adapted for identifying C. elegans genes with postembryonic phenotypes of interest using RNAi. This procedure is easily modified to assay the phenotype of choice, whether by light or fluorescence optics on a dissecting or compound microscope. This screening protocol capitalizes on the physical assets of the organism and molecular tools the C. elegans research community has produced. As an example, we demonstrate the use of an integrated transgene that expresses a fluorescent product in an RNAi screen to identify genes required for the normal localization of this product in late stage larvae and adults. First, we used a commercially available genomic RNAi library with full-length cDNA inserts. This library facilitates the rapid identification of multiple candidates by RNAi reduction of the candidate gene product. Second, we generated an integrated transgene that expresses our fluorecently tagged protein of interest in an RNAi-sensitive background. Third, by exposing hatched animals to RNAi, this screen permits identification of gene products that have a vital embryonic role that would otherwise mask a post-embryonic role in regulating the protein of interest. Lastly, this screen uses a compound microscope equipped for single cell resolution. PMID:22353760

  8. Inducible RNAi system and its application in novel therapeutics.

    PubMed

    Liao, Yi; Tang, Liling

    2016-08-01

    RNA interference (RNAi) was discovered as a cellular defense mechanism more than decade ago. It has been exploited as a powerful tool for genetic manipulation. Characterized with specifically silencing target gene expression, it has great potential application for disease treatment. Currently, there are human clinical trials in progress or planned. Despite the excitement regarding this prominent technology, there are many obstacles and concerns that prevent RNAi from being widely used in the therapeutic field. Among them, the non-spatial and non-temporal control is the most difficult challenge, as well as off-target effects and triggering type I immune responses. Inducible RNAi technology can effectively regulate target genes by inducer-mediated small hairpin RNA expression. Combination with inducible regulation systems this makes RNAi technology more sophisticated and may provide a wider application field. This review discusses approaches of inducible RNAi systems, the potential problem areas and solutions and their therapeutic applications. Given the limitations discussed herein being resolved, we believe that inducible RNAi will be a major therapeutic modality within the next several years. PMID:25697568

  9. Institutional Profile: The Sheffield RNAi screening facility: a service for high-throughput, genome-wide Drosophila RNAi screens.

    PubMed

    Brown, Stephen

    2010-12-01

    The Sheffield RNAi Screening Facility (SRSF) was established in November 2008, as Britain's first Drosophila RNAi screening centre, funded by the University of Sheffield, Biomedical Sciences Department and the Wellcome Trust. The SRSF was formed to service the needs of research groups wanting to carry out high-throughput RNAi screens with Drosophila cells. The rationale for the SRSF is to provide RNAi libraries and the specialist equipment and expertise to do such screens. The facility supports both plate reader assays, high-content microscopy as well as the equipment needed to process these samples in a high-throughput fashion. The SRSF can either be used to identify genes involved in disease representing future drug targets, or to identify genes involved in drug resistance and efficacy. PMID:21428803

  10. Lethality of PAK3 and SGK2 shRNAs to Human Papillomavirus Positive Cervical Cancer Cells Is Independent of PAK3 and SGK2 Knockdown

    PubMed Central

    Zhou, Nannan; Ding, Bo; Agler, Michele; Cockett, Mark; McPhee, Fiona

    2015-01-01

    The p21-activated kinase 3 (PAK3) and the serum and glucocorticoid-induced kinase 2 (SGK2) have been previously proposed as essential kinases for human papillomavirus positive (HPV+) cervical cancer cell survival. This was established using a shRNA knockdown approach. To validate PAK3 and SGK2 as potential targets for HPV+ cervical cancer therapy, the relationship between shRNA-induced phenotypes in HPV+ cervical cancer cells and PAK3 or SGK2 knockdown was carefully examined. We observed that the phenotypes of HPV+ cervical cancer cells induced by various PAK3 and SGK2 shRNAs could not be rescued by complement expression of respective cDNA constructs. A knockdown-deficient PAK3 shRNA with a single mismatch was sufficient to inhibit HeLa cell growth to a similar extent as wild-type PAK3 shRNA. The HPV+ cervical cancer cells were also susceptible to several non-human target shRNAs. The discrepancy between PAK3 and SGK2 shRNA-induced apoptosis and gene expression knockdown, as well as cell death stimulation, suggested that these shRNAs killed HeLa cells through different pathways that may not be target-specific. These data demonstrated that HPV+ cervical cancer cell death was not associated with RNAi-induced PAK3 and SGK2 knockdown but likely through off-target effects. PMID:25615606

  11. Superoxide dismutase 2 knockdown leads to defects in locomotor activity, sensitivity to paraquat, and increased cuticle pigmentation in Tribolium castaneum

    PubMed Central

    Tabunoki, Hiroko; Gorman, Maureen J.; Dittmer, Neal T.; Kanost, Michael R.

    2016-01-01

    Insects can rapidly adapt to environmental changes through physiological responses. The red flour beetle Tribolium castaneum is widely used as a model insect species. However, the stress–response system of this species remains unclear. Superoxide dismutase 2 (SOD2) is a crucial antioxidative enzyme that is found in mitochondria. T. castaneum SOD2 (TcSOD2) is composed of 215 amino acids, and has an iron/manganese superoxide dismutase domain. qRT-PCR experiments revealed that TcSOD2 was present through all developmental stages. To evaluate TcSOD2 function in T. castaneum, we performed RNAi and also assessed the phenotype and antioxidative tolerance of the knockdown of TcSOD2 by exposing larvae to paraquat. The administration of paraquat resulted in significantly higher 24-h mortality in TcSOD2 knockdown larval groups than in the control groups. The TcSOD2 knockdown adults moved significantly more slowly, had lower ATP content, and exhibited a different body color from the control groups. We found that TcSOD2 dsRNA treatment in larvae resulted in increased expression of tyrosinase and laccase2 mRNA after 10 days. This is the first report showing that TcSOD2 has an antioxidative function and demonstrates that T. castaneum may use an alternative antioxidative system when the SOD2-based system fails. PMID:27387523

  12. Superoxide dismutase 2 knockdown leads to defects in locomotor activity, sensitivity to paraquat, and increased cuticle pigmentation in Tribolium castaneum.

    PubMed

    Tabunoki, Hiroko; Gorman, Maureen J; Dittmer, Neal T; Kanost, Michael R

    2016-01-01

    Insects can rapidly adapt to environmental changes through physiological responses. The red flour beetle Tribolium castaneum is widely used as a model insect species. However, the stress-response system of this species remains unclear. Superoxide dismutase 2 (SOD2) is a crucial antioxidative enzyme that is found in mitochondria. T. castaneum SOD2 (TcSOD2) is composed of 215 amino acids, and has an iron/manganese superoxide dismutase domain. qRT-PCR experiments revealed that TcSOD2 was present through all developmental stages. To evaluate TcSOD2 function in T. castaneum, we performed RNAi and also assessed the phenotype and antioxidative tolerance of the knockdown of TcSOD2 by exposing larvae to paraquat. The administration of paraquat resulted in significantly higher 24-h mortality in TcSOD2 knockdown larval groups than in the control groups. The TcSOD2 knockdown adults moved significantly more slowly, had lower ATP content, and exhibited a different body color from the control groups. We found that TcSOD2 dsRNA treatment in larvae resulted in increased expression of tyrosinase and laccase2 mRNA after 10 days. This is the first report showing that TcSOD2 has an antioxidative function and demonstrates that T. castaneum may use an alternative antioxidative system when the SOD2-based system fails. PMID:27387523

  13. Knockdown of WHIRLY1 Affects Drought Stress-Induced Leaf Senescence and Histone Modifications of the Senescence-Associated Gene HvS40.

    PubMed

    Janack, Bianka; Sosoi, Paula; Krupinska, Karin; Humbeck, Klaus

    2016-01-01

    The plastid-nucleus located protein WHIRLY1 has been described as an upstream regulator of leaf senescence, binding to the promoter of senescence-associated genes like HvS40. To investigate the impact of WHIRLY1 on drought stress-induced, premature senescence, transgenic barley plants with an RNAi-mediated knockdown of the HvWHIRLY1 gene were grown under normal and drought stress conditions. The course of leaf senescence in these lines was monitored by physiological parameters and studies on the expression of senescence- and drought stress-related genes. Drought treatment accelerated leaf senescence in WT plants, whereas WHIRLY 1 knockdown lines (RNAi-W1) showed a stay-green phenotype. Expression of both senescence-associated and drought stress-responsive genes, was delayed in the transgenic plants. Notably, expression of transcription factors of the WRKY and NAC families, which are known to function in senescence- and stress-related signaling pathways, was affected in plants with impaired accumulation of WHIRLY1, indicating that WHIRLY1 acts as an upstream regulator of drought stress-induced senescence. To reveal the epigenetic indexing of HvS40 at the onset of drought-induced senescence in WT and RNAi-W1 lines, stress-responsive loading with histone modifications of promoter and coding sequences of HvS40 was analyzed by chromatin immunoprecipitation and quantified by qRT-PCR. In the wildtype, the euchromatic mark H3K9ac of the HvS40 gene was low under control conditions and was established in response to drought treatment, indicating the action of epigenetic mechanisms in response to drought stress. However, drought stress caused no significant increase in H3K9ac in plants impaired in accumulation of WHIRLY1. The results show that WHIRLY1 knockdown sets in motion a delay in senescence that involves all aspects of gene expression, including changes in chromatin structure. PMID:27608048

  14. Antiviral immunity in Drosophila requires systemic RNAi spread

    PubMed Central

    Saleh, Maria-Carla; Tassetto, Michel; van Rij, Ronald P.; Goic, Bertsy; Gausson, Valérie; Berry, Bassam; Jacquier, Caroline; Antoniewski, Christophe; Andino, Raul

    2014-01-01

    Multicellular organisms evolved sophisticated defense systems to confer protection against pathogens. An important characteristic of these immune systems is their ability to act both locally at the site of infection and at distal uninfected locations1-4. In insects, such as Drosophila melanogaster, RNA interference (RNAi) mediates antiviral immunity5-7. However, the antiviral RNAi defense in flies is thought to be a local, cell-autonomous process, since flies are considered unable to generate a systemic RNAi response8. Here we show that a recently defined double-stranded RNA (dsRNA) uptake pathway9 is essential for effective antiviral RNAi immunity in adult flies. Mutant flies defective in this dsRNA uptake pathway were hypersensitive to infection with Drosophila C virus (DCV) and Sindbis virus. Mortality in dsRNA-uptake defective flies was accompanied by 100-to 105-fold increases in viral titers and higher levels of viral RNA. Furthermore, inoculating naked dsRNA into flies elicited a sequence specific antiviral immune response that required an intact dsRNA uptake pathway. These findings suggest that spread of dsRNA to uninfected sites is essential for effective antiviral immunity. Strikingly, infection with Sindbis-GFP suppressed expression of host-encoded GFP at a distal site. Thus, similar to protein-based immunity in vertebrates, the antiviral RNAi-response in flies also relies on the systemic spread of a virus-specific immunity signal. PMID:19204732

  15. RNA Interference-Mediated Silencing of Atp6i Prevents Both Periapical Bone Erosion and Inflammation in the Mouse Model of Endodontic Disease

    PubMed Central

    Ma, Junqing; Zhang, Lijie; Tucker, Byron; Zhu, Guochun; Sasaki, Hajime; Hao, Liang; Wang, Lin; Ci, Hongliang; Jiang, Hongbing; Stashenko, Philip

    2013-01-01

    Dental caries is one of the most prevalent infectious diseases in the United States, affecting approximately 80% of children and the majority of adults. Dental caries may lead to endodontic disease, where the bacterial infection progresses to the root canal system of the tooth, leading to periapical inflammation, bone erosion, severe pain, and tooth loss. Periapical inflammation may also exacerbate inflammation in other parts of the body. Although conventional clinical therapies for this disease are successful in approximately 80% of cases, there is still an urgent need for increased efficacy of treatment. In this study, we applied a novel gene-therapeutic approach using recombinant adeno-associated virus (AAV)-mediated Atp6i RNA interference (RNAi) knockdown of Atp6i/TIRC7 gene expression to simultaneously target periapical bone resorption and periapical inflammation. We found that Atp6i inhibition impaired osteoclast function in vitro and in vivo and decreased the number of T cells in the periapical lesion. Notably, AAV-mediated Atp6i/TIRC7 knockdown gene therapy reduced bacterial infection-stimulated bone resorption by 80% in the mouse model of endodontic disease. Importantly, Atp6i+/− mice with haploinsufficiency of Atp6i exhibited protection similar to that in mice with bacterial infection-stimulated bone erosion and periapical inflammation, which confirms the potential therapeutic effect of AAV-small hairpin RNA (shRNA)-Atp6i/TIRC7. Our results demonstrate that AAV-mediated Atp6i/TIRC7 knockdown in periapical tissues can inhibit endodontic disease development, bone resorption, and inflammation, indicating for the first time that this potential gene therapy may significantly improve the health of those who suffer from endodontic disease. PMID:23166162

  16. RNA interference-mediated silencing of Atp6i prevents both periapical bone erosion and inflammation in the mouse model of endodontic disease.

    PubMed

    Ma, Junqing; Chen, Wei; Zhang, Lijie; Tucker, Byron; Zhu, Guochun; Sasaki, Hajime; Hao, Liang; Wang, Lin; Ci, Hongliang; Jiang, Hongbing; Stashenko, Philip; Li, Yi-Ping

    2013-04-01

    Dental caries is one of the most prevalent infectious diseases in the United States, affecting approximately 80% of children and the majority of adults. Dental caries may lead to endodontic disease, where the bacterial infection progresses to the root canal system of the tooth, leading to periapical inflammation, bone erosion, severe pain, and tooth loss. Periapical inflammation may also exacerbate inflammation in other parts of the body. Although conventional clinical therapies for this disease are successful in approximately 80% of cases, there is still an urgent need for increased efficacy of treatment. In this study, we applied a novel gene-therapeutic approach using recombinant adeno-associated virus (AAV)-mediated Atp6i RNA interference (RNAi) knockdown of Atp6i/TIRC7 gene expression to simultaneously target periapical bone resorption and periapical inflammation. We found that Atp6i inhibition impaired osteoclast function in vitro and in vivo and decreased the number of T cells in the periapical lesion. Notably, AAV-mediated Atp6i/TIRC7 knockdown gene therapy reduced bacterial infection-stimulated bone resorption by 80% in the mouse model of endodontic disease. Importantly, Atp6i(+/-) mice with haploinsufficiency of Atp6i exhibited protection similar to that in mice with bacterial infection-stimulated bone erosion and periapical inflammation, which confirms the potential therapeutic effect of AAV-small hairpin RNA (shRNA)-Atp6i/TIRC7. Our results demonstrate that AAV-mediated Atp6i/TIRC7 knockdown in periapical tissues can inhibit endodontic disease development, bone resorption, and inflammation, indicating for the first time that this potential gene therapy may significantly improve the health of those who suffer from endodontic disease. PMID:23166162

  17. Stable silencing of β-lactoglobulin (BLG) gene by lentivirus-mediated RNAi in goat fetal fibroblasts

    PubMed Central

    Zhang, Shumin; Xiong, Kai; Xie, Zhourui; Nan, Wenting; Liu, Honglin; Chen, Jie

    2012-01-01

    β-lactoglobulin (BLG), a dominant allergen in goat milk, is difficult to remove by traditional biochemical methods. Its elimination from goat milk by genetic modification therefore poses a major challenge for modern goat breeders. A shRNA targeting BLG mRNA with high interference efficiency was identified, with which lentiviral vectors were used for mediating stable shRNA interference in goat-fetal fibroblast cells. Apart from high efficiency in the knockdown of BLG expression in these cells, lentivector-mediated RNAi manifested stable integration into the goat genome itself. Consequently, an in vitro model for goat BLG-content control was compiled, and a goat-cell line for accompanying transgenetic goat production created. PMID:23055809

  18. Whole-animal genome-wide RNAi screen identifies networks regulating male germline stem cells in Drosophila

    PubMed Central

    Liu, Ying; Ge, Qinglan; Chan, Brian; Liu, Hanhan; Singh, Shree Ram; Manley, Jacob; Lee, Jae; Weideman, Ann Marie; Hou, Gerald; Hou, Steven X.

    2016-01-01

    Stem cells are regulated both intrinsically and externally, including by signals from the local environment and distant organs. To identify genes and pathways that regulate stem-cell fates in the whole organism, we perform a genome-wide transgenic RNAi screen through ubiquitous gene knockdowns, focusing on regulators of adult Drosophila testis germline stem cells (GSCs). Here we identify 530 genes that regulate GSC maintenance and differentiation. Of these, we further knock down 113 selected genes using cell-type-specific Gal4s and find that more than half were external regulators, that is, from the local microenvironment or more distal sources. Some genes, for example, versatile (vers), encoding a heterochromatin protein, regulates GSC fates differentially in different cell types and through multiple pathways. We also find that mitosis/cytokinesis proteins are especially important for male GSC maintenance. Our findings provide valuable insights and resources for studying stem cell regulation at the organismal level. PMID:27484291

  19. Whole-animal genome-wide RNAi screen identifies networks regulating male germline stem cells in Drosophila.

    PubMed

    Liu, Ying; Ge, Qinglan; Chan, Brian; Liu, Hanhan; Singh, Shree Ram; Manley, Jacob; Lee, Jae; Weideman, Ann Marie; Hou, Gerald; Hou, Steven X

    2016-01-01

    Stem cells are regulated both intrinsically and externally, including by signals from the local environment and distant organs. To identify genes and pathways that regulate stem-cell fates in the whole organism, we perform a genome-wide transgenic RNAi screen through ubiquitous gene knockdowns, focusing on regulators of adult Drosophila testis germline stem cells (GSCs). Here we identify 530 genes that regulate GSC maintenance and differentiation. Of these, we further knock down 113 selected genes using cell-type-specific Gal4s and find that more than half were external regulators, that is, from the local microenvironment or more distal sources. Some genes, for example, versatile (vers), encoding a heterochromatin protein, regulates GSC fates differentially in different cell types and through multiple pathways. We also find that mitosis/cytokinesis proteins are especially important for male GSC maintenance. Our findings provide valuable insights and resources for studying stem cell regulation at the organismal level. PMID:27484291

  20. RNA Viruses and RNAi: Quasispecies Implications for Viral Escape

    PubMed Central

    Presloid, John B.; Novella, Isabel S.

    2015-01-01

    Due to high mutation rates, populations of RNA viruses exist as a collection of closely related mutants known as a quasispecies. A consequence of error-prone replication is the potential for rapid adaptation of RNA viruses when a selective pressure is applied, including host immune systems and antiviral drugs. RNA interference (RNAi) acts to inhibit protein synthesis by targeting specific mRNAs for degradation and this process has been developed to target RNA viruses, exhibiting their potential as a therapeutic against infections. However, viruses containing mutations conferring resistance to RNAi were isolated in nearly all cases, underlining the problems of rapid viral evolution. Thus, while promising, the use of RNAi in treating or preventing viral diseases remains fraught with the typical complications that result from high specificity of the target, as seen in other antiviral regimens. PMID:26102581

  1. RNAi and functional genomics in plant parasitic nematodes.

    PubMed

    Rosso, M N; Jones, J T; Abad, P

    2009-01-01

    Plant nematology is currently undergoing a revolution with the availability of the first genome sequences as well as comprehensive expressed sequence tag (EST) libraries from a range of nematode species. Several strategies are being used to exploit this wealth of information. Comparative genomics is being used to explore the acquisition of novel genes associated with parasitic lifestyles. Functional analyses of nematode genes are moving toward larger scale studies including global transcriptome profiling. RNA interference (RNAi) has been shown to reduce expression of a range of plant parasitic nematode genes and is a powerful tool for functional analysis of nematode genes. RNAi-mediated suppression of genes essential for nematode development, survival, or parasitism is revealing new targets for nematode control. Plant nematology in the genomics era is now facing the challenge to develop RNAi screens adequate for high-throughput functional analyses. PMID:19400649

  2. The Nuclear Argonaute NRDE-3 Contributes to Transitive RNAi in Caenorhabditis elegans

    PubMed Central

    Zhuang, Jimmy J.; Banse, Stephen A.; Hunter, Craig P.

    2013-01-01

    The Caenorhabditis elegans nuclear RNA interference defective (Nrde) mutants were identified by their inability to silence polycistronic transcripts in enhanced RNAi (Eri) mutant backgrounds. Here, we report additional nrde-3-dependent RNAi phenomena that extend the mechanisms, roles, and functions of nuclear RNAi. We show that nrde-3 mutants are broadly RNAi deficient and that overexpressing NRDE-3 enhances RNAi. Consistent with NRDE-3 being a dose-dependent limiting resource for effective RNAi, we find that NRDE-3 is required for eri-dependent enhanced RNAi phenotypes, although only for a subset of target genes. We then identify pgl-1 as an additional limiting RNAi resource important for eri-dependent silencing of a nonoverlapping subset of target genes, so that an nrde-3; pgl-1; eri-1 triple mutant fails to show enhanced RNAi for any tested gene. These results suggest that nrde-3 and pgl-1 define separate and independent limiting RNAi resource pathways. Limiting RNAi resources are proposed to primarily act via endogenous RNA silencing pathways. Consistent with this, we find that nrde-3 mutants misexpress genes regulated by endogenous siRNAs and incompletely silence repetitive transgene arrays. Finally, we find that nrde-3 contributes to transitive RNAi, whereby amplified silencing triggers act in trans to silence sequence-similar genes. Because nrde-dependent silencing is thought to act in cis to limit the production of primary transcripts, this result reveals an unexpected role for nuclear processes in RNAi silencing. PMID:23457236

  3. The nuclear argonaute NRDE-3 contributes to transitive RNAi in Caenorhabditis elegans.

    PubMed

    Zhuang, Jimmy J; Banse, Stephen A; Hunter, Craig P

    2013-05-01

    The Caenorhabditis elegans nuclear RNA interference defective (Nrde) mutants were identified by their inability to silence polycistronic transcripts in enhanced RNAi (Eri) mutant backgrounds. Here, we report additional nrde-3-dependent RNAi phenomena that extend the mechanisms, roles, and functions of nuclear RNAi. We show that nrde-3 mutants are broadly RNAi deficient and that overexpressing NRDE-3 enhances RNAi. Consistent with NRDE-3 being a dose-dependent limiting resource for effective RNAi, we find that NRDE-3 is required for eri-dependent enhanced RNAi phenotypes, although only for a subset of target genes. We then identify pgl-1 as an additional limiting RNAi resource important for eri-dependent silencing of a nonoverlapping subset of target genes, so that an nrde-3; pgl-1; eri-1 triple mutant fails to show enhanced RNAi for any tested gene. These results suggest that nrde-3 and pgl-1 define separate and independent limiting RNAi resource pathways. Limiting RNAi resources are proposed to primarily act via endogenous RNA silencing pathways. Consistent with this, we find that nrde-3 mutants misexpress genes regulated by endogenous siRNAs and incompletely silence repetitive transgene arrays. Finally, we find that nrde-3 contributes to transitive RNAi, whereby amplified silencing triggers act in trans to silence sequence-similar genes. Because nrde-dependent silencing is thought to act in cis to limit the production of primary transcripts, this result reveals an unexpected role for nuclear processes in RNAi silencing. PMID:23457236

  4. Enhancement of Larval RNAi Efficiency by Over-expressing Argonaute2 in Bombyx mori

    PubMed Central

    Li, Zhiqian; Zeng, Baosheng; Ling, Lin; Xu, Jun; You, Lang; Aslam, Abu F.M.; Tan, Anjiang; Huang, Yongping

    2015-01-01

    RNA interference has been described as a powerful genetic tool for gene functional analysis and a promising approach for pest management. However, RNAi efficiency varies significantly among insect species due to distinct RNAi machineries. Lepidopteran insects include a large number of pests as well as model insects, such as the silkworm, Bombyx mori. However, only limited success of in vivo RNAi has been reported in lepidoptera, particularly during the larval stages when the worms feed the most and do the most harm to the host plant. Enhancing the efficiency of larval RNAi in lepidoptera is urgently needed to develop RNAi-based pest management strategies. In the present study, we investigate the function of the conserved RNAi core factor, Argonaute2 (Ago2), in mediating B. mori RNAi efficiency. We demonstrate that introducing BmAgo2 dsRNA inhibits the RNAi response in both BmN cells and embryos. Furthermore, we establish several transgenic silkworm lines to assess the roles of BmAgo2 in larval RNAi. Over-expressing BmAgo2 significantly facilitated both dsRNA-mediated larval RNAi when targeting DsRed using dsRNA injection and shRNA-mediated larval RNAi when targeting BmBlos2 using transgenic shRNA expression. Our results show that BmAgo2 is involved in RNAi in B. mori and provides a promising approach for improving larval RNAi efficiency in B. mori and in lepidopteran insects in general. PMID:25561900

  5. Transcription and RNAi in the formation of heterochromatin

    PubMed Central

    Grewal, Shiv S.; Elgin, Sarah C R

    2010-01-01

    Preface Our title appears to be an oxymoron – surely “silenced” chromatin should not be transcribed! But there have been frequent reports of low level transcription in heterochromatic domains, and in Drosophila several hundred genes are found within heterochromatic regions1. Most striking, recent investigations implicate RNAi (in the broadest sense) in targeting and maintaining heterochromatin, and RNAi-based mechanisms are inherently dependent on transcription. While this might involve trans-acting sources of the critical small RNAs, in some cases transcription of the domain to be silenced appears to be required at least initially, a seeming contradiction. PMID:17522672

  6. RNAi down-regulation of cinnamate-4-hydroxylase increases artemisinin biosynthesis in Artemisia annua

    PubMed Central

    Kumar, Ritesh; Vashisth, Divya; Misra, Amita; Akhtar, Md Qussen; Jalil, Syed Uzma; Shanker, Karuna; Gupta, Madan Mohan; Rout, Prashant Kumar; Gupta, Anil Kumar; Shasany, Ajit Kumar

    2016-01-01

    Cinnamate-4-hydroxylase (C4H) converts trans-cinnamic acid (CA) to p-coumaric acid (COA) in the phenylpropanoid/lignin biosynthesis pathway. Earlier we reported increased expression of AaCYP71AV1 (an important gene of artemisinin biosynthesis pathway) caused by CA treatment in Artemisia annua. Hence, AaC4H gene was identified, cloned, characterized and silenced in A. annua with the assumption that the elevated internal CA due to knock down may increase the artemisinin yield. Accumulation of trans-cinnamic acid in the plant due to AaC4H knockdown was accompanied with the reduction of p-coumaric acid, total phenolics, anthocyanin, cinnamate-4-hydroxylase (C4H) and phenylalanine ammonia lyase (PAL) activities but increase in salicylic acid (SA) and artemisinin. Interestingly, feeding trans-cinnamic acid to the RNAi line increased the level of artemisinin along with benzoic (BA) and SA with no effect on the downstream metabolites p-coumaric acid, coniferylaldehyde and sinapaldehyde, whereas p-coumaric acid feeding increased the content of downstream coniferylaldehyde and sinapaldehyde with no effect on BA, SA, trans-cinnamic acid or artemisinin. SA is reported earlier to be inducing the artemisinin yield. This report demonstrates the link between the phenylpropanoid/lignin pathway with artemisinin pathway through SA, triggered by accumulation of trans-cinnamic acid because of the blockage at C4H. PMID:27220407

  7. RNAi down-regulation of cinnamate-4-hydroxylase increases artemisinin biosynthesis in Artemisia annua.

    PubMed

    Kumar, Ritesh; Vashisth, Divya; Misra, Amita; Akhtar, Md Qussen; Jalil, Syed Uzma; Shanker, Karuna; Gupta, Madan Mohan; Rout, Prashant Kumar; Gupta, Anil Kumar; Shasany, Ajit Kumar

    2016-01-01

    Cinnamate-4-hydroxylase (C4H) converts trans-cinnamic acid (CA) to p-coumaric acid (COA) in the phenylpropanoid/lignin biosynthesis pathway. Earlier we reported increased expression of AaCYP71AV1 (an important gene of artemisinin biosynthesis pathway) caused by CA treatment in Artemisia annua. Hence, AaC4H gene was identified, cloned, characterized and silenced in A. annua with the assumption that the elevated internal CA due to knock down may increase the artemisinin yield. Accumulation of trans-cinnamic acid in the plant due to AaC4H knockdown was accompanied with the reduction of p-coumaric acid, total phenolics, anthocyanin, cinnamate-4-hydroxylase (C4H) and phenylalanine ammonia lyase (PAL) activities but increase in salicylic acid (SA) and artemisinin. Interestingly, feeding trans-cinnamic acid to the RNAi line increased the level of artemisinin along with benzoic (BA) and SA with no effect on the downstream metabolites p-coumaric acid, coniferylaldehyde and sinapaldehyde, whereas p-coumaric acid feeding increased the content of downstream coniferylaldehyde and sinapaldehyde with no effect on BA, SA, trans-cinnamic acid or artemisinin. SA is reported earlier to be inducing the artemisinin yield. This report demonstrates the link between the phenylpropanoid/lignin pathway with artemisinin pathway through SA, triggered by accumulation of trans-cinnamic acid because of the blockage at C4H. PMID:27220407

  8. RNAi Reveals Phase-Specific Global Regulators of Human Somatic Cell Reprogramming.

    PubMed

    Toh, Cheng-Xu Delon; Chan, Jun-Wei; Chong, Zheng-Shan; Wang, Hao Fei; Guo, Hong Chao; Satapathy, Sandeep; Ma, Dongrui; Goh, Germaine Yen Lin; Khattar, Ekta; Yang, Lin; Tergaonkar, Vinay; Chang, Young-Tae; Collins, James J; Daley, George Q; Wee, Keng Boon; Farran, Chadi A El; Li, Hu; Lim, Yoon-Pin; Bard, Frederic A; Loh, Yuin-Han

    2016-06-21

    Incomplete knowledge of the mechanisms at work continues to hamper efforts to maximize reprogramming efficiency. Here, we present a systematic genome-wide RNAi screen to determine the global regulators during the early stages of human reprogramming. Our screen identifies functional repressors and effectors that act to impede or promote the reprogramming process. Repressors and effectors form close interacting networks in pathways, including RNA processing, G protein signaling, protein ubiquitination, and chromatin modification. Combinatorial knockdown of five repressors (SMAD3, ZMYM2, SFRS11, SAE1, and ESET) synergistically resulted in ∼85% TRA-1-60-positive cells. Removal of the novel splicing factor SFRS11 during reprogramming is accompanied by rapid acquisition of pluripotency-specific spliced forms. Mechanistically, SFRS11 regulates exon skipping and mutually exclusive splicing of transcripts in genes involved in cell differentiation, mRNA splicing, and chromatin modification. Our study provides insights into the reprogramming process, which comprises comprehensive and multi-layered transcriptional, splicing, and epigenetic machineries. PMID:27292646

  9. A large-scale in vivo RNAi screen to identify genes involved in Notch-mediated follicle cell differentiation and cell cycle switches.

    PubMed

    Jia, Dongyu; Soylemez, Muhammed; Calvin, Gabriel; Bornmann, Randy; Bryant, Jamal; Hanna, Cameron; Huang, Yi-Chun; Deng, Wu-Min

    2015-01-01

    During Drosophila oogenesis, follicle cells sequentially undergo three distinct cell-cycle programs: the mitotic cycle, endocycle, and gene amplification. Notch signaling plays a central role in regulating follicle-cell differentiation and cell-cycle switches; its activation is essential for the mitotic cycle/endocycle (M/E) switch. Cut, a linker between Notch signaling and cell-cycle regulators, is specifically downregulated by Notch during the endocycle stage. To determine how signaling pathways coordinate during the M/E switch and to identify novel genes involved in follicle cell differentiation, we performed an in vivo RNAi screen through induced knockdown of gene expression and examination of Cut expression in follicle cells. We screened 2205 RNAi lines and found 33 genes regulating Cut expression during the M/E switch. These genes were confirmed with the staining of two other Notch signaling downstream factors, Hindsight and Broad, and validated with multiple independent RNAi lines. We applied gene ontology software to find enriched biological meaning and compared our results with other publications to find conserved genes across tissues. Specifically, we found earlier endocycle entry in anterior follicle cells than those in the posterior, identified that the insulin-PI3K pathway participates in the precise M/E switch, and suggested Nejire as a cofactor of Notch signaling during oogenesis. PMID:26205122

  10. RNAi-based functional elucidation of PtrPRP, a gene encoding a hybrid proline rich protein, in cold tolerance of Poncirus trifoliata

    PubMed Central

    Peng, Ting; Jia, Mao-Mao; Liu, Ji-Hong

    2015-01-01

    Hybrid proline-rich proteins (HyPRPs) have been suggested to play important roles in various plant development and stress response. In this study, we report the cloning and functional analysis of PtrPRP, a HyPRP-encoding gene of Poncirus trifoliata. PtrPRP contains 176 amino acids, among which 21% are proline residues, and has an 8-cysteine motif (8 CM) domain at the C terminal, a signal peptide and a proline-rich region at the N terminal. PtrPRP is constitutively expressed in root, stem and leaf, with the highest expression levels in leaf. It was progressively induced by cold, but transiently upregulated by salt and ABA. Transgenic P. trifoliata plants with knock-down PtrPRP by RNA interference (RNAi) were generated to investigate the role of PtrPRP in cold tolerance. When challenged by low temperature, the PtrPRP-RNAi plants displayed more sensitive performance compared with wild type (WT), as shown by higher electrolyte leakage and malondialdehyde content. In addition, the RNAi lines accumulated more reactive oxygen species (ROS) and lower levels of proline relative to WT. These results suggested that PtrPRP might be positively involved in cold tolerance by maintaining membrane integrity and ROS homeostasis. PMID:26483822

  11. A large-scale in vivo RNAi screen to identify genes involved in Notch-mediated follicle cell differentiation and cell cycle switches

    PubMed Central

    Jia, Dongyu; Soylemez, Muhammed; Calvin, Gabriel; Bornmann, Randy; Bryant, Jamal; Hanna, Cameron; Huang, Yi-Chun; Deng, Wu-Min

    2015-01-01

    During Drosophila oogenesis, follicle cells sequentially undergo three distinct cell-cycle programs: the mitotic cycle, endocycle, and gene amplification. Notch signaling plays a central role in regulating follicle-cell differentiation and cell-cycle switches; its activation is essential for the mitotic cycle/endocycle (M/E) switch. Cut, a linker between Notch signaling and cell-cycle regulators, is specifically downregulated by Notch during the endocycle stage. To determine how signaling pathways coordinate during the M/E switch and to identify novel genes involved in follicle cell differentiation, we performed an in vivo RNAi screen through induced knockdown of gene expression and examination of Cut expression in follicle cells. We screened 2205 RNAi lines and found 33 genes regulating Cut expression during the M/E switch. These genes were confirmed with the staining of two other Notch signaling downstream factors, Hindsight and Broad, and validated with multiple independent RNAi lines. We applied gene ontology software to find enriched biological meaning and compared our results with other publications to find conserved genes across tissues. Specifically, we found earlier endocycle entry in anterior follicle cells than those in the posterior, identified that the insulin-PI3K pathway participates in the precise M/E switch, and suggested Nejire as a cofactor of Notch signaling during oogenesis. PMID:26205122

  12. RNAi-based functional elucidation of PtrPRP, a gene encoding a hybrid proline rich protein, in cold tolerance of Poncirus trifoliata.

    PubMed

    Peng, Ting; Jia, Mao-Mao; Liu, Ji-Hong

    2015-01-01

    Hybrid proline-rich proteins (HyPRPs) have been suggested to play important roles in various plant development and stress response. In this study, we report the cloning and functional analysis of PtrPRP, a HyPRP-encoding gene of Poncirus trifoliata. PtrPRP contains 176 amino acids, among which 21% are proline residues, and has an 8-cysteine motif (8 CM) domain at the C terminal, a signal peptide and a proline-rich region at the N terminal. PtrPRP is constitutively expressed in root, stem and leaf, with the highest expression levels in leaf. It was progressively induced by cold, but transiently upregulated by salt and ABA. Transgenic P. trifoliata plants with knock-down PtrPRP by RNA interference (RNAi) were generated to investigate the role of PtrPRP in cold tolerance. When challenged by low temperature, the PtrPRP-RNAi plants displayed more sensitive performance compared with wild type (WT), as shown by higher electrolyte leakage and malondialdehyde content. In addition, the RNAi lines accumulated more reactive oxygen species (ROS) and lower levels of proline relative to WT. These results suggested that PtrPRP might be positively involved in cold tolerance by maintaining membrane integrity and ROS homeostasis. PMID:26483822

  13. Interference RNA (RNAi)-based silencing of endogenous thrombopoietin receptor (Mpl) in Dami cells resulted in decreased hNUDC-mediated megakaryocyte proliferation and differentiation

    SciTech Connect

    Pang, Shi-Feng; Li, Xiao-Kun; Zhang, Qiang; Yang, Fang; Xu, Peilin

    2009-12-10

    Recently our laboratory reported evidence showing that hNUDC acts as an additional cytokine for thrombopoietin receptor (Mpl). Previously known as the human homolog of a fungal nuclear migration protein, hNUDC plays a critical role in megakaryocyte differentiation and maturation. Here we sought to further clarify the hNUDC-Mpl ligand-receptor relationship by utilizing interference RNA (RNAi) to knockdown Mpl expression in a megakaryocyte cell line. We created U6 promoter driven constructs to express short hairpin RNAs (shRNA) with affinity for different sites on Mpl mRNA. By including Mpl-EGFP fusion protein in these constructs, we were able to effectively screen the shRNA that was most efficient in inhibiting Mpl mRNA expression. This shRNA was subsequently transferred into a lentivirus vector and transduced into Dami cells, a cell line which constitutively expresses endogenous Mpl. This lentiviral vector was also designed to simultaneously express EGFP to monitor transfection efficiency. Our results show that lentivirus can be used to effectively deliver shRNAs into Dami cells and cause specific inhibition of Mpl protein expression after transduction. Furthermore, we show the functional effects of shRNA-mediated Mpl silencing by demonstrating reduced hNUDC stimulated megakaryocyte proliferation and differentiation. Thus, the use of a RNAi knockdown strategy has allowed us to pinpoint the connection of hNUDC with Mpl in the regulation of megakaryocyte maturation.

  14. Non-transgenic RNAi technology to control insects on citrus

    Technology Transfer Automated Retrieval System (TEKTRAN)

    This research demonstrated a non-transgenic delivery method for ribonucleic acid interference, RNAi, that reduced fitness as measured in increased mortality over time, of two insect pests of citrus, ie. psyllids and leafhoppers. The Asian citrus psyllid transmits a deadly plant-infecting bacterium o...

  15. RNAi-Mediated Inactivation of Autophagy Genes in Caenorhabditis elegans.

    PubMed

    Palmisano, Nicholas J; Meléndez, Alicia

    2016-02-01

    RNA interference (RNAi) is a process that results in the sequence-specific silencing of endogenous mRNA through the introduction of double-stranded RNA (dsRNA). In the nematode Caenorhabditis elegans, RNA inactivation can be used at any specific developmental stage or during adulthood to inhibit a given target gene. Investigators can take advantage of the fact that, in C. elegans, RNAi is unusual in that it is systemic, meaning that dsRNA can spread throughout the animal and can affect virtually all tissues except neurons. Here, we describe a protocol for the most common method to achieve RNAi in C. elegans, which is to feed them bacteria that express dsRNA complementary to a specific target gene. This method has various advantages, including the availability of libraries that essentially cover the whole genome, the ability to treat animals at any developmental stage, and that it is relatively cost effective. We also discuss how RNAi specific to autophagy genes has proven to be an excellent method to study the role of these genes in autophagy, as well as other cellular and developmental processes, while also highlighting the caveats that must be applied. PMID:26832686

  16. The interaction of fungi with the environment orchestrated by RNAi.

    PubMed

    Villalobos-Escobedo, José Manuel; Herrera-Estrella, Alfredo; Carreras-Villaseñor, Nohemí

    2016-01-01

    The fungal kingdom has been key in the investigation of the biogenesis and function of small RNAs (sRNAs). The discovery of phenomena such as quelling in Neurospora crassa represents pioneering work in the identification of the main elements of the RNA interference (RNAi) machinery. Recent discoveries in the regulatory mechanisms in some yeast and filamentous fungi are helping us reach a deeper understanding of the transcriptional and post-transcriptional gene-silencing mechanisms involved in genome protection against viral infections, DNA damage and transposon activity. Although most of these mechanisms are reasonably well understood, their role in the physiology, response to the environment and interaction of fungi with other organisms had remained elusive. Nevertheless, studies in fungi such as Mucor circinelloides, Magnaporthe oryzae, Cryptococcus neoformans, Trichoderma atroviride, Botrytis cinerea and others have started to shed light on the relevance of the RNAi pathway. In these fungi gene regulation by RNAi is important for growth, reproduction, control of viral infections and transposon activity, as well as in the development of antibiotic resistance and interactions with their hosts. Moreover, the increasing number of reports of the discovery of microRNA-like RNAs in fungi under different conditions highlights the importance of fungi as models for understanding adaptation to the environment using regulation by sRNAs. The goal of this review is to provide the reader with an up-to-date overview of the importance of RNAi in the interaction of fungi with their environment. PMID:26932186

  17. RNAi at work: Targeting invertebrate pests and beneficial organisms' diseases

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Invertebrates present two types of large scale RNAi application opportunities: pest control and beneficial insect health. The former involves the introduction of sustainable applications to keep pest populations low, and the latter represents the challenge of keeping beneficial organisms healthy. RN...

  18. CERKL Knockdown Causes Retinal Degeneration in Zebrafish

    PubMed Central

    Riera, Marina; Burguera, Demian; Garcia-Fernàndez, Jordi; Gonzàlez-Duarte, Roser

    2013-01-01

    The human CERKL gene is responsible for common and severe forms of retinal dystrophies. Despite intense in vitro studies at the molecular and cellular level and in vivo analyses of the retina of murine knockout models, CERKL function remains unknown. In this study, we aimed to approach the developmental and functional features of cerkl in Danio rerio within an Evo-Devo framework. We show that gene expression increases from early developmental stages until the formation of the retina in the optic cup. Unlike the high mRNA-CERKL isoform multiplicity shown in mammals, the moderate transcriptional complexity in fish facilitates phenotypic studies derived from gene silencing. Moreover, of relevance to pathogenicity, teleost CERKL shares the two main human protein isoforms. Morpholino injection has been used to generate a cerkl knockdown zebrafish model. The morphant phenotype results in abnormal eye development with lamination defects, failure to develop photoreceptor outer segments, increased apoptosis of retinal cells and small eyes. Our data support that zebrafish Cerkl does not interfere with proliferation and neural differentiation during early developmental stages but is relevant for survival and protection of the retinal tissue. Overall, we propose that this zebrafish model is a powerful tool to unveil CERKL contribution to human retinal degeneration. PMID:23671706

  19. RNA interference knockdown of DNA methyl-transferase 3 affects gene alternative splicing in the honey bee

    PubMed Central

    Li-Byarlay, Hongmei; Li, Yang; Stroud, Hume; Feng, Suhua; Newman, Thomas C.; Kaneda, Megan; Hou, Kirk K.; Worley, Kim C.; Elsik, Christine G.; Wickline, Samuel A.; Jacobsen, Steven E.; Ma, Jian; Robinson, Gene E.

    2013-01-01

    Studies of DNA methylation from fungi, plants, and animals indicate that gene body methylation is ancient and highly conserved in eukaryotic genomes, but its role has not been clearly defined. It has been postulated that regulation of alternative splicing of transcripts was an original function of DNA methylation, but a direct experimental test of the effect of methylation on alternative slicing at the whole genome level has never been performed. To do this, we developed a unique method to administer RNA interference (RNAi) in a high-throughput and noninvasive manner and then used it to knock down the expression of DNA methyl-transferase 3 (dnmt3), which is required for de novo DNA methylation. We chose the honey bee (Apis mellifera) for this test because it has recently emerged as an important model organism for studying the effects of DNA methylation on development and social behavior, and DNA methylation in honey bees is predominantly on gene bodies. Here we show that dnmt3 RNAi decreased global genomic methylation level as expected and in addition caused widespread and diverse changes in alternative splicing in fat tissue. Four different types of splicing events were affected by dnmt3 gene knockdown, and change in two types, exon skipping and intron retention, was directly related to decreased methylation. These results demonstrate that one function of gene body DNA methylation is to regulate alternative splicing. PMID:23852726

  20. Role of RNA Interference (RNAi) in the Moss Physcomitrella patens

    PubMed Central

    Arif, Muhammad Asif; Frank, Wolfgang; Khraiwesh, Basel

    2013-01-01

    RNA interference (RNAi) is a mechanism that regulates genes by either transcriptional (TGS) or posttranscriptional gene silencing (PTGS), required for genome maintenance and proper development of an organism. Small non-coding RNAs are the key players in RNAi and have been intensively studied in eukaryotes. In plants, several classes of small RNAs with specific sizes and dedicated functions have evolved. The major classes of small RNAs include microRNAs (miRNAs) and small interfering RNAs (siRNAs), which differ in their biogenesis. miRNAs are synthesized from a short hairpin structure while siRNAs are derived from long double-stranded RNAs (dsRNA). Both miRNA and siRNAs control the expression of cognate target RNAs by binding to reverse complementary sequences mediating cleavage or translational inhibition of the target RNA. They also act on the DNA and cause epigenetic changes such as DNA methylation and histone modifications. In the last years, the analysis of plant RNAi pathways was extended to the bryophyte Physcomitrella patens, a non-flowering, non-vascular ancient land plant that diverged from the lineage of seed plants approximately 450 million years ago. Based on a number of characteristic features and its phylogenetic key position in land plant evolution P. patens emerged as a plant model species to address basic as well as applied topics in plant biology. Here we summarize the current knowledge on the role of RNAi in P. patens that shows functional overlap with RNAi pathways from seed plants, and also unique features specific to this species. PMID:23344055

  1. Antifungal drug resistance evokedvia RNAi-dependent epimutations

    PubMed Central

    Calo, Silvia; Shertz-Wall, Cecelia; Lee, Soo Chan; Bastidas, Robert J.; Nicolás, Francisco E.; Granek, Joshua A.; Mieczkowski, Piotr; Torres-Martinez, Santiago; Ruiz-Vazquez, Rosa M.; Cardenas, Maria E.; Heitman, Joseph

    2014-01-01

    Microorganisms evolve via mechanisms spanning sexual/parasexual reproduction, mutators, aneuploidy, Hsp90, and even prions. Mechanisms that may seem detrimental can be repurposed to generate diversity. Here we show the human fungal pathogen Mucor circinelloides develops spontaneous resistance to the antifungal drug FK506 (tacrolimus) via two distinct mechanisms. One involves Mendelian mutations that confer stable drug resistance; the other occurs via an epigenetic RNA interference (RNAi)-mediated pathway resulting in unstable drug resistance. The peptidyl-prolyl isomerase FKBP12 interacts with FK506 forming a complex that inhibits the protein phosphatase calcineurin1. Calcineurin inhibition by FK506 blocks M. circinelloides transition to hyphae and enforces yeast growth2. Mutations in the fkbA gene encoding FKBP12 or the calcineurin cnbR or cnaA genes confer FK506 resistance (FK506R) and restore hyphal growth. In parallel, RNAi is spontaneously triggered to silence the FKBP12 fkbA gene, giving rise to drug-resistant epimutants. FK506R epimutants readily reverted to the drug-sensitive wild-type (WT) phenotype when grown without drug. The establishment of these epimutants is accompanied by generation of abundant fkbA small RNA (sRNA) and requires the RNAi pathway as well as other factors that constrain or reverse the epimutant state. Silencing involves generation of a double-stranded RNA (dsRNA) trigger intermediate from the fkbA mature mRNA to produce antisense fkbA RNA. This study uncovers a novel epigenetic RNAi-based epimutation mechanism controlling phenotypic plasticity, with possible implications for antimicrobial drug resistance and RNAi-regulatory mechanisms in fungi and other eukaryotes. PMID:25079329

  2. Knockdown of the C. elegans Kinome identifies Kinases required for normal protein Homeostasis, Mitochondrial network structure, and Sarcomere structure in muscle

    PubMed Central

    2013-01-01

    Background Kinases are important signalling molecules for modulating cellular processes and major targets of drug discovery programs. However, functional information for roughly half the human kinome is lacking. We conducted three kinome wide, >90%, RNAi screens and epistasis testing of some identified kinases against known intramuscular signalling systems to increase the functional annotation of the C. elegans kinome and expand our understanding of kinome influence upon muscle protein degradation. Results 96 kinases were identified as required for normal protein homeostasis, 74 for normal mitochondrial networks and 50 for normal sarcomere structure. Knockdown of kinases required only for normal protein homeostasis and/or mitochondrial structure was significantly less likely to produce a developmental or behavioural phenotype than knockdown of kinases required for normal sarcomere structure and/or other sub-cellular processes. Lastly, assessment of kinases for which knockdown produced muscle protein degradation against the known regulatory pathways in C. elegans muscle revealed that close to half of kinase knockdowns activated autophagy in a MAPK dependent fashion. Conclusions Roughly 40% of kinases studied, 159 of 397, are important in establishing or maintaining muscle cell health, with most required for both. For kinases where decreased expression triggers protein degradation, autophagy is most commonly activated. These results increase the annotation of the C. elegans kinome to roughly 75% and enable future kinome research. As 33% of kinases identified have orthologues expressed in human muscle, our results also enable testing of whether identified kinases function similarly in maintaining human muscle homeostasis. PMID:24060339

  3. Genome-wide RNAi screens in human brain tumor isolates reveal a novel viability requirement for PHF5A

    PubMed Central

    Hubert, Christopher G.; Bradley, Robert K.; Ding, Yu; Toledo, Chad M.; Herman, Jacob; Skutt-Kakaria, Kyobi; Girard, Emily J.; Davison, Jerry; Berndt, Jason; Corrin, Philip; Hardcastle, Justin; Basom, Ryan; Delrow, Jeffery J.; Webb, Thomas; Pollard, Steven M.; Lee, Jeongwu; Olson, James M.; Paddison, Patrick J.

    2013-01-01

    To identify key regulators of human brain tumor maintenance and initiation, we performed multiple genome-wide RNAi screens in patient-derived glioblastoma multiforme (GBM) stem cells (GSCs). These screens identified the plant homeodomain (PHD)-finger domain protein PHF5A as differentially required for GSC expansion, as compared with untransformed neural stem cells (NSCs) and fibroblasts. Given PHF5A's known involvement in facilitating interactions between the U2 snRNP complex and ATP-dependent helicases, we examined cancer-specific roles in RNA splicing. We found that in GSCs, but not untransformed controls, PHF5A facilitates recognition of exons with unusual C-rich 3′ splice sites in thousands of essential genes. PHF5A knockdown in GSCs, but not untransformed NSCs, astrocytes, or fibroblasts, inhibited splicing of these genes, leading to cell cycle arrest and loss of viability. Notably, pharmacologic inhibition of U2 snRNP activity phenocopied PHF5A knockdown in GSCs and also in NSCs or fibroblasts overexpressing MYC. Furthermore, PHF5A inhibition compromised GSC tumor formation in vivo and inhibited growth of established GBM patient-derived xenograft tumors. Our results demonstrate a novel viability requirement for PHF5A to maintain proper exon recognition in brain tumor-initiating cells and may provide new inroads for novel anti-GBM therapeutic strategies. PMID:23651857

  4. Genome-wide RNAi screens in human brain tumor isolates reveal a novel viability requirement for PHF5A.

    PubMed

    Hubert, Christopher G; Bradley, Robert K; Ding, Yu; Toledo, Chad M; Herman, Jacob; Skutt-Kakaria, Kyobi; Girard, Emily J; Davison, Jerry; Berndt, Jason; Corrin, Philip; Hardcastle, Justin; Basom, Ryan; Delrow, Jeffery J; Webb, Thomas; Pollard, Steven M; Lee, Jeongwu; Olson, James M; Paddison, Patrick J

    2013-05-01

    To identify key regulators of human brain tumor maintenance and initiation, we performed multiple genome-wide RNAi screens in patient-derived glioblastoma multiforme (GBM) stem cells (GSCs). These screens identified the plant homeodomain (PHD)-finger domain protein PHF5A as differentially required for GSC expansion, as compared with untransformed neural stem cells (NSCs) and fibroblasts. Given PHF5A's known involvement in facilitating interactions between the U2 snRNP complex and ATP-dependent helicases, we examined cancer-specific roles in RNA splicing. We found that in GSCs, but not untransformed controls, PHF5A facilitates recognition of exons with unusual C-rich 3' splice sites in thousands of essential genes. PHF5A knockdown in GSCs, but not untransformed NSCs, astrocytes, or fibroblasts, inhibited splicing of these genes, leading to cell cycle arrest and loss of viability. Notably, pharmacologic inhibition of U2 snRNP activity phenocopied PHF5A knockdown in GSCs and also in NSCs or fibroblasts overexpressing MYC. Furthermore, PHF5A inhibition compromised GSC tumor formation in vivo and inhibited growth of established GBM patient-derived xenograft tumors. Our results demonstrate a novel viability requirement for PHF5A to maintain proper exon recognition in brain tumor-initiating cells and may provide new inroads for novel anti-GBM therapeutic strategies. PMID:23651857

  5. Exploring systemic RNA interference in insects: a genome-wide survey for RNAi genes in Tribolium

    PubMed Central

    Tomoyasu, Yoshinori; Miller, Sherry C; Tomita, Shuichiro; Schoppmeier, Michael; Grossmann, Daniela; Bucher, Gregor

    2008-01-01

    Background RNA interference (RNAi) is a highly conserved cellular mechanism. In some organisms, such as Caenorhabditis elegans, the RNAi response can be transmitted systemically. Some insects also exhibit a systemic RNAi response. However, Drosophila, the leading insect model organism, does not show a robust systemic RNAi response, necessitating another model system to study the molecular mechanism of systemic RNAi in insects. Results We used Tribolium, which exhibits robust systemic RNAi, as an alternative model system. We have identified the core RNAi genes, as well as genes potentially involved in systemic RNAi, from the Tribolium genome. Both phylogenetic and functional analyses suggest that Tribolium has a somewhat larger inventory of core component genes than Drosophila, perhaps allowing a more sensitive response to double-stranded RNA (dsRNA). We also identified three Tribolium homologs of C. elegans sid-1, which encodes a possible dsRNA channel. However, detailed sequence analysis has revealed that these Tribolium homologs share more identity with another C. elegans gene, tag-130. We analyzed tag-130 mutants, and found that this gene does not have a function in systemic RNAi in C. elegans. Likewise, the Tribolium sid-like genes do not seem to be required for systemic RNAi. These results suggest that insect sid-1-like genes have a different function than dsRNA uptake. Moreover, Tribolium lacks homologs of several genes important for RNAi in C. elegans. Conclusion Although both Tribolium and C. elegans show a robust systemic RNAi response, our genome-wide survey reveals significant differences between the RNAi mechanisms of these organisms. Thus, insects may use an alternative mechanism for the systemic RNAi response. Understanding this process would assist with rendering other insects amenable to systemic RNAi, and may influence pest control approaches. PMID:18201385

  6. Knockdown of proteins involved in iron metabolism limits tick reproduction and development

    PubMed Central

    Hajdusek, Ondrej; Sojka, Daniel; Kopacek, Petr; Buresova, Veronika; Franta, Zdenek; Sauman, Ivo; Winzerling, Joy; Grubhoffer, Libor

    2009-01-01

    Ticks are among the most important vectors of a wide range of human and animal diseases. During blood feeding, ticks are exposed to an enormous amount of free iron that must be appropriately used and detoxified. However, the mechanism of iron metabolism in ticks is poorly understood. Here, we show that ticks possess a complex system that efficiently utilizes, stores and transports non-heme iron within the tick body. We have characterized a new secreted ferritin (FER2) and an iron regulatory protein (IRP1) from the sheep tick, Ixodes ricinus, and have demonstrated their relationship to a previously described tick intracellular ferritin (FER1). By using RNA interference-mediated gene silencing in the tick, we show that synthesis of FER1, but not of FER2, is subject to IRP1-mediated translational control. Further, we find that depletion of FER2 from the tick plasma leads to a loss of FER1 expression in the salivary glands and ovaries that normally follows blood ingestion. We therefore suggest that secreted FER2 functions as the primary transporter of non-heme iron between the tick gut and the peripheral tissues. Silencing of the fer1, fer2, and irp1 genes by RNAi has an adverse impact on hatching rate and decreases postbloodmeal weight in tick females. Importantly, knockdown of fer2 dramatically impairs the ability of ticks to feed, thus making FER2 a promising candidate for development of an efficient anti-tick vaccine. PMID:19171899

  7. Systems-wide Analysis of a Phosphatase Knock-down by Quantitative Proteomics and Phosphoproteomics

    PubMed Central

    Hilger, Maximiliane; Bonaldi, Tiziana; Gnad, Florian; Mann, Matthias

    2009-01-01

    Signal transduction in metazoans regulates almost all aspects of biological function, and aberrant signaling is involved in many diseases. Perturbations in phosphorylation-based signaling networks are typically studied in a hypothesis-driven approach, using phospho-specific antibodies. Here we apply quantitative, high-resolution mass spectrometry to determine the systems response to the depletion of one signaling component. Drosophila cells were metabolically labeled using stable isotope labeling by amino acids in cell culture (SILAC) and the phosphatase Ptp61F, the ortholog of mammalian PTB1B, a drug target for diabetes, was knocked down by RNAi. In total we detected more than 10,000 phosphorylation sites in the phosphoproteome of Drosophila Schneider cells and trained a phosphorylation site predictor with this data. SILAC-based quantitation after phosphatase knock-down showed that apart from the phosphatase, the proteome was minimally affected whereas 288 of 6,478 high-confidence phosphorylation sites changed significantly. Responses at the phosphotyrosine level included the already described Ptp61F substrates Stat92E and Abi. Our analysis highlights a connection of Ptp61F to cytoskeletal regulation through GTPase regulating proteins and focal adhesion components. PMID:19429919

  8. Role of Neuronal NADPH Oxidase 1 in the Peri-Infarct Regions after Stroke

    PubMed Central

    Choi, Dong-Hee; Kim, Ji-Hye; Lee, Kyoung-Hee; Kim, Hahn-Young; Kim, Yoon-Seong; Choi, Wahn Soo; Lee, Jongmin

    2015-01-01

    The molecular mechanism underlying the selective vulnerability of neurons to oxidative damage caused by ischemia—reperfusion (I/R) injury remains unknown. We sought to determine the role of NADPH oxidase 1 (Nox1) in cerebral I/R-induced brain injury and survival of newborn cells in the ischemic injured region. Male Wistar rats were subjected to 90 min middle cerebral artery occlusion (MCAO) followed by reperfusion. After reperfusion, infarction size, level of superoxide and 8-hydroxy-2′-deoxyguanosine (8-oxo-2dG), and Nox1 immunoreactivity were determined. RNAi-mediated knockdown of Nox1 was used to investigate the role of Nox1 in I/R-induced oxidative damage, neuronal death, motor function recovery, and ischemic neurogenesis. After I/R, Nox1 expression and 8-oxo-2dG immunoreactivity was increased in cortical neurons of the peri-infarct regions. Both infarction size and neuronal death in I/R injury were significantly reduced by adeno-associated virus (AAV)-mediated transduction of Nox1 short hairpin RNA (shRNA). AAV-mediated Nox1 knockdown enhanced functional recovery after MCAO. The level of survival and differentiation of newborn cells in the peri-infarct regions were increased by Nox1 inhibition. Our data suggest that Nox-1 may be responsible for oxidative damage to DNA, subsequent cortical neuronal degeneration, functional recovery, and regulation of ischemic neurogenesis in the peri-infarct regions after stroke. PMID:25617620

  9. Knockdown of the coenzyme Q synthesis gene Smed-dlp1 affects planarian regeneration and tissue homeostasis

    PubMed Central

    Shiobara, Yumiko; Harada, Chiaki; Shiota, Takeshi; Sakamoto, Kimitoshi; Kita, Kiyoshi; Tanaka, Saeko; Tabata, Kenta; Sekie, Kiyoteru; Yamamoto, Yorihiro; Sugiyama, Tomoyasu

    2015-01-01

    The freshwater planarian is a model organism used to study tissue regeneration that occupies an important position among multicellular organisms. Planarian genomic databases have led to the identification of genes that are required for regeneration, with implications for their roles in its underlying mechanism. Coenzyme Q (CoQ) is a fundamental lipophilic molecule that is synthesized and expressed in every cell of every organism. Furthermore, CoQ levels affect development, life span, disease and aging in nematodes and mice. Because CoQ can be ingested in food, it has been used in preventive nutrition. In this study, we investigated the role of CoQ in planarian regeneration. Planarians synthesize both CoQ9 and rhodoquinone 9 (RQ9). Knockdown of Smed-dlp1, a trans-prenyltransferase gene that encodes an enzyme that synthesizes the CoQ side chain, led to a decrease in CoQ9 and RQ9 levels. However, ATP levels did not consistently decrease in these animals. Knockdown animals exhibited tissue regression and curling. The number of mitotic cells decreased in Smed-dlp1 (RNAi) animals. These results suggested a failure in physiological cell turnover and stem cell function. Accordingly, regenerating planarians died from lysis or exhibited delayed regeneration. Interestingly, the observed phenotypes were partially rescued by ingesting food supplemented with α-tocopherol. Taken together, our results suggest that oxidative stress induced by reduced CoQ9 levels affects planarian regeneration and tissue homeostasis. PMID:26516985

  10. Knockdown of the coenzyme Q synthesis gene Smed-dlp1 affects planarian regeneration and tissue homeostasis.

    PubMed

    Shiobara, Yumiko; Harada, Chiaki; Shiota, Takeshi; Sakamoto, Kimitoshi; Kita, Kiyoshi; Tanaka, Saeko; Tabata, Kenta; Sekie, Kiyoteru; Yamamoto, Yorihiro; Sugiyama, Tomoyasu

    2015-12-01

    The freshwater planarian is a model organism used to study tissue regeneration that occupies an important position among multicellular organisms. Planarian genomic databases have led to the identification of genes that are required for regeneration, with implications for their roles in its underlying mechanism. Coenzyme Q (CoQ) is a fundamental lipophilic molecule that is synthesized and expressed in every cell of every organism. Furthermore, CoQ levels affect development, life span, disease and aging in nematodes and mice. Because CoQ can be ingested in food, it has been used in preventive nutrition. In this study, we investigated the role of CoQ in planarian regeneration. Planarians synthesize both CoQ9 and rhodoquinone 9 (RQ9). Knockdown of Smed-dlp1, a trans-prenyltransferase gene that encodes an enzyme that synthesizes the CoQ side chain, led to a decrease in CoQ9 and RQ9 levels. However, ATP levels did not consistently decrease in these animals. Knockdown animals exhibited tissue regression and curling. The number of mitotic cells decreased in Smed-dlp1 (RNAi) animals. These results suggested a failure in physiological cell turnover and stem cell function. Accordingly, regenerating planarians died from lysis or exhibited delayed regeneration. Interestingly, the observed phenotypes were partially rescued by ingesting food supplemented with α-tocopherol. Taken together, our results suggest that oxidative stress induced by reduced CoQ9 levels affects planarian regeneration and tissue homeostasis. PMID:26516985

  11. Conditional knockdown of BCL2A1 reveals rate-limiting roles in BCR-dependent B-cell survival.

    PubMed

    Sochalska, M; Ottina, E; Tuzlak, S; Herzog, S; Herold, M; Villunger, A

    2016-04-01

    Bcl2 family proteins control mitochondrial apoptosis and its members exert critical cell type and differentiation stage-specific functions, acting as barriers against autoimmunity or transformation. Anti-apoptotic Bcl2a1/Bfl1/A1 is frequently deregulated in different types of blood cancers in humans but its physiological role is poorly understood as quadruplication of the Bcl2a1 gene locus in mice hampers conventional gene targeting strategies. Transgenic overexpression of A1, deletion of the A1-a paralogue or constitutive knockdown in the hematopoietic compartment of mice by RNAi suggested rate-limiting roles in lymphocyte development, granulopoiesis and mast cell activation. Here we report on the consequences of conditional knockdown of A1 protein expression using a reverse transactivator (rtTA)-driven approach that highlights a critical role for this Bcl2 family member in the maintenance of mature B-cell homeostasis. Furthermore, we define the A1/Bim (Bcl-2 interacting mediator of cell death) axis as a target of key kinases mediating B-cell receptor (BCR)-dependent survival signals, such as, spleen tyrosine kinase (Syk) and Brutons tyrosine kinase (Btk). As such, A1 represents a putative target for the treatment of B-cell-related pathologies depending on hyperactivation of BCR-emanating survival signals and loss of A1 expression accounts, in part, for the pro-apoptotic effects of Syk- or Btk inhibitors that rely on the 'BH3-only' protein Bim for cell killing. PMID:26450454

  12. A library of MiMICs allows tagging of genes and reversible, spatial and temporal knockdown of proteins in Drosophila

    PubMed Central

    Nagarkar-Jaiswal, Sonal; Lee, Pei-Tseng; Campbell, Megan E; Chen, Kuchuan; Anguiano-Zarate, Stephanie; Cantu Gutierrez, Manuel; Busby, Theodore; Lin, Wen-Wen; He, Yuchun; Schulze, Karen L; Booth, Benjamin W; Evans-Holm, Martha; Venken, Koen JT; Levis, Robert W; Spradling, Allan C; Hoskins, Roger A; Bellen, Hugo J

    2015-01-01

    Here, we document a collection of ∼7434 MiMIC (Minos Mediated Integration Cassette) insertions of which 2854 are inserted in coding introns. They allowed us to create a library of 400 GFP-tagged genes. We show that 72% of internally tagged proteins are functional, and that more than 90% can be imaged in unfixed tissues. Moreover, the tagged mRNAs can be knocked down by RNAi against GFP (iGFPi), and the tagged proteins can be efficiently knocked down by deGradFP technology. The phenotypes associated with RNA and protein knockdown typically correspond to severe loss of function or null mutant phenotypes. Finally, we demonstrate reversible, spatial, and temporal knockdown of tagged proteins in larvae and adult flies. This new strategy and collection of strains allows unprecedented in vivo manipulations in flies for many genes. These strategies will likely extend to vertebrates. DOI: http://dx.doi.org/10.7554/eLife.05338.001 PMID:25824290

  13. Rhox8 Ablation in the Sertoli Cells Using a Tissue-Specific RNAi Approach Results in Impaired Male Fertility in Mice.

    PubMed

    Welborn, Joshua P; Davis, Matthew G; Ebers, Steven D; Stodden, Genna R; Hayashi, Kanako; Cheatwood, Joseph L; Rao, Manjeet K; MacLean, James A

    2015-07-01

    The reproductive homeobox X-linked, Rhox, genes encode transcription factors that are selectively expressed in reproductive tissues. While there are 33 Rhox genes in mice, only Rhox and Rhox8 are expressed in Sertoli cells, suggesting that they may regulate the expression of somatic-cell gene products crucial for germ cell development. We previously characterized Rhox5-null mice, which are subfertile, exhibiting excessive germ cell apoptosis and compromised sperm motility. To assess the role of Rhox8 in Sertoli cells, we used a tissue-specific RNAi approach to knockdown RHOX8 in vivo, in which the Rhox5 promoter was used to drive Rhox8-siRNA transgene expression in the postnatal Sertoli cells. Western and immunohistochemical analysis confirmed Sertoli-specific knockdown of RHOX8. However, other Sertoli markers, Gata1 and Rhox5, maintained normal expression patterns, suggesting that the knockdown was specific. Interestingly, male RHOX8-knockdown animals showed significantly reduced spermatogenic output, increased germ cell apoptosis, and compromised sperm motility, leading to impaired fertility. Importantly, our results revealed that while some RHOX5-dependent factors were also misregulated in Sertoli cells of RHOX8-knockdown animals, the majority were not, and novel putative RHOX8-regulated genes were identified. This suggests that while reduction in levels of RHOX5 and RHOX8 in Sertoli cells elicits similar phenotypes, these genes are not entirely redundant. Taken together, our study underscores the importance of Rhox genes in male fertility and suggests that Sertoli cell-specific expression of Rhox5 and Rhox8 is critical for complete male fertility. PMID:25972016

  14. Rhox8 Ablation in the Sertoli Cells Using a Tissue-Specific RNAi Approach Results in Impaired Male Fertility in Mice1

    PubMed Central

    Welborn, Joshua P.; Davis, Matthew G.; Ebers, Steven D.; Stodden, Genna R.; Hayashi, Kanako; Cheatwood, Joseph L.; Rao, Manjeet K.; MacLean, James A.

    2015-01-01

    The reproductive homeobox X-linked, Rhox, genes encode transcription factors that are selectively expressed in reproductive tissues. While there are 33 Rhox genes in mice, only Rhox and Rhox8 are expressed in Sertoli cells, suggesting that they may regulate the expression of somatic-cell gene products crucial for germ cell development. We previously characterized Rhox5-null mice, which are subfertile, exhibiting excessive germ cell apoptosis and compromised sperm motility. To assess the role of Rhox8 in Sertoli cells, we used a tissue-specific RNAi approach to knockdown RHOX8 in vivo, in which the Rhox5 promoter was used to drive Rhox8-siRNA transgene expression in the postnatal Sertoli cells. Western and immunohistochemical analysis confirmed Sertoli-specific knockdown of RHOX8. However, other Sertoli markers, Gata1 and Rhox5, maintained normal expression patterns, suggesting that the knockdown was specific. Interestingly, male RHOX8-knockdown animals showed significantly reduced spermatogenic output, increased germ cell apoptosis, and compromised sperm motility, leading to impaired fertility. Importantly, our results revealed that while some RHOX5-dependent factors were also misregulated in Sertoli cells of RHOX8-knockdown animals, the majority were not, and novel putative RHOX8-regulated genes were identified. This suggests that while reduction in levels of RHOX5 and RHOX8 in Sertoli cells elicits similar phenotypes, these genes are not entirely redundant. Taken together, our study underscores the importance of Rhox genes in male fertility and suggests that Sertoli cell-specific expression of Rhox5 and Rhox8 is critical for complete male fertility. PMID:25972016

  15. RNAi-mediated resistance to viruses in genetically engineered plants.

    PubMed

    Ibrahim, Abdulrazak B; Aragão, Francisco J L

    2015-01-01

    RNA interference (RNAi) has emerged as a leading technology in designing genetically modified crops engineered to resist viral infection. The last decades have seen the development of a large number of crops whose inherent posttranscriptional gene silencing mechanism has been exploited to target essential viral genes through the production of dsRNA that triggers an endogenous RNA-induced silencing complex (RISC), leading to gene silencing in susceptible viruses conferring them with resistance even before the onset of infection. Selection and breeding events have allowed for establishing this highly important agronomic trait in diverse crops. With improved techniques and the availability of new data on genetic diversity among several viruses, significant progress is being made in engineering plants using RNAi with the release of a number of commercially available crops. Biosafety concerns with respect to consumption of RNAi crops, while relevant, have been addressed, given the fact that experimental evidence using miRNAs associated with the crops shows that they do not pose any health risk to humans and animals. PMID:25740357

  16. Optical imaging of RNAi-mediated silencing of cancer

    NASA Astrophysics Data System (ADS)

    Ochiya, Takahiro; Honma, Kimi; Takeshita, Fumitaka; Nagahara, Shunji

    2008-02-01

    RNAi has rapidly become a powerful tool for drug target discovery and validation in an in vitro culture system and, consequently, interest is rapidly growing for extension of its application to in vivo systems, such as animal disease models and human therapeutics. Cancer is one obvious application for RNAi therapeutics, because abnormal gene expression is thought to contribute to the pathogenesis and maintenance of the malignant phenotype of cancer and thereby many oncogenes and cell-signaling molecules present enticing drug target possibilities. RNAi, potent and specific, could silence tumor-related genes and would appear to be a rational approach to inhibit tumor growth. In subsequent in vivo studies, the appropriate cancer model must be developed for an evaluation of siRNA effects on tumors. How to evaluate the effect of siRNA in an in vivo therapeutic model is also important. Accelerating the analyses of these models and improving their predictive value through whole animal imaging methods, which provide cancer inhibition in real time and are sensitive to subtle changes, are crucial for rapid advancement of these approaches. Bioluminescent imaging is one of these optically based imaging methods that enable rapid in vivo analyses of a variety of cellular and molecular events with extreme sensitivity.

  17. Affinity approaches in RNAi-based therapeutics purification.

    PubMed

    Pereira, Patrícia; Queiroz, João A; Figueiras, Ana; Sousa, Fani

    2016-05-15

    The recent investigation on RNA interference (RNAi) related mechanisms and applications led to an increased awareness of the importance of RNA in biology. Nowadays, RNAi-based technology has emerged as a potentially powerful tool for silencing gene expression, being exploited to develop new therapeutics for treating a vast number of human disease conditions, as it is expected that this technology can be translated onto clinical applications in a near future. This approach makes use of a large number of small (namely short interfering RNAs, microRNAs and PIWI-interacting RNAs) and long non-coding RNAs (ncRNAs), which are likely to have a crucial role as the next generation therapeutics. The commercial and biomedical interest in these RNAi-based therapy applications have fostered the need to develop innovative procedures to easily and efficiently purify RNA, aiming to obtain the final product with high purity degree, good quality and biological activity. Recently, affinity chromatography has been applied to ncRNAs purification, in view of the high specificity. Therefore, this article intends to review the biogenesis pathways of regulatory ncRNAs and also to discuss the most significant and recent developments as well as applications of affinity chromatography in the challenging task of purifying ncRNAs. In addition, the importance of affinity chromatography in ncRNAs purification is addressed and prospects for what is forthcoming are presented. PMID:26830537

  18. Enhanced toxic cloud knockdown spray system for decontamination applications

    DOEpatents

    Betty, Rita G.; Tucker, Mark D.; Brockmann, John E.; Lucero, Daniel A.; Levin, Bruce L.; Leonard, Jonathan

    2011-09-06

    Methods and systems for knockdown and neutralization of toxic clouds of aerosolized chemical or biological warfare (CBW) agents and toxic industrial chemicals using a non-toxic, non-corrosive aqueous decontamination formulation.

  19. MiR-125a-5p decreases after long non-coding RNA HOTAIR knockdown to promote cancer cell apoptosis by releasing caspase 2

    PubMed Central

    Tang, L; Shen, H; Li, X; Li, Z; Liu, Z; Xu, J; Ma, S; Zhao, X; Bai, X; Li, M; Wang, Q; Ji, J

    2016-01-01

    HOTAIR (homeobox transcript antisense RNA), one of the prototypical long non-coding RNAs, has been verified overexpressed in multiple carcinomas and has emerged as a promising novel anticancer target. Its well-established role is acting as a predictor of poor prognosis and promoting cancer cell metastasis. Recently, another important mission of HOTAIR was uncovered that targeting HOTAIR caused cancer cell apoptosis. Nevertheless, so far there is no published data elaborating the mechanism. Here, we report that microRNA miR-125a-5p decreases and releases caspase 2 to promote cancer cell apoptosis after HOTAIR knockdown. We applied siRNAs targeting HOTAIR to various cancer cells, and observed apoptosis in all of these cell lines. RNA sequencing detected that miR-125a-5p was decreased after HOTAIR knockdown and miR-125a-5p mimics could rescue the apoptosis induced by HOTAIR deficiency. Luciferase assays identified caspase 2, an initiator caspase, to be a new target of miR-125a-5p. Elevated expression and subsequent cleavage of caspase 2 was observed after HOTAIR knockdown or inhibition of miR-125a-5p. RNAi of caspase 2 could attenuate the apoptosis induced by HOTAIR knockdown. In 80 clinical colon cancer tissues, HOTAIR and miR-125a-5p levels were higher than adjacent tissues, whereas caspase 2 was lower. MiR-125a-5p expression level was significantly correlated with colon tumor size, lymph node metastasis and clinical stage. These findings indicate that miR-125a-5p decreases after HOTAIR knockdown to promote cancer cell apoptosis by releasing caspase 2. Our work reveals a previously unidentified apoptotic mechanism, which might be exploitable in anticancer drug development. PMID:26962687

  20. Durable Knockdown and Protection From HIV Transmission in Humanized Mice Treated With Gel-formulated CD4 Aptamer-siRNA Chimeras

    PubMed Central

    Wheeler, Lee Adam; Vrbanac, Vladimir; Trifonova, Radiana; Brehm, Michael A; Gilboa-Geffen, Adi; Tanno, Serah; Greiner, Dale L; Luster, Andrew D; Tager, Andrew M; Lieberman, Judy

    2013-01-01

    The continued spread of HIV underscores the need to interrupt transmission. One attractive strategy, in the absence of an effective vaccine, is a topical microbicide, but the need for application around the time of sexual intercourse leads to poor patient compliance. Intravaginal (IVAG) application of CD4 aptamer-siRNA chimeras (CD4-AsiCs) targeting the HIV coreceptor CCR5, gag, and vif protected humanized mice from sexual transmission. In non-dividing cells and tissue, RNAi-mediated gene knockdown lasts for several weeks, providing an opportunity for infrequent dosing not temporally linked to sexual intercourse, when compliance is challenging. Here, we investigate the durability of gene knockdown and viral inhibition, protection afforded by CCR5 or HIV gene knockdown on their own, and effectiveness of CD4-AsiCs formulated in a gel in polarized human cervicovaginal explants and in humanized mice. CD4-AsiC–mediated gene knockdown persisted for several weeks. Cell-specific gene knockdown and protection were comparable in a hydroxyethylcellulose gel formulation. CD4-AsiCs against CCR5 or gag/vif performed as well as a cocktail in humanized mice. Transmission was completely blocked by CCR5 CD4-AsiCs applied 2 days before challenge. Significant, but incomplete, protection also occurred when exposure was delayed for 4 or 6 days. CD4-AsiCs targeting gag/vif provided some protection when administered only after exposure. These data suggest that CD4-AsiCs are a promising approach for developing an HIV microbicide. PMID:23629001

  1. Genetically Modifying the Insect Gut Microbiota to Control Chagas Disease Vectors through Systemic RNAi

    PubMed Central

    Taracena, Mabel L.; Oliveira, Pedro L.; Almendares, Olivia; Umaña, Claudia; Lowenberger, Carl; Dotson, Ellen M.; Paiva-Silva, Gabriela O.; Pennington, Pamela M.

    2015-01-01

    Technologies based on RNA interference may be used for insect control. Sustainable strategies are needed to control vectors of Chagas disease such as Rhodnius prolixus. The insect microbiota can be modified to deliver molecules to the gut. Here, Escherichia coli HT115(DE3) expressing dsRNA for the Rhodnius heme-binding protein (RHBP) and for catalase (CAT) were fed to nymphs and adult triatomine stages. RHBP is an egg protein and CAT is an antioxidant enzyme expressed in all tissues by all developmental stages. The RNA interference effect was systemic and temporal. Concentrations of E. coli HT115(DE3) above 3.35 × 107 CFU/mL produced a significant RHBP and CAT gene knockdown in nymphs and adults. RHBP expression in the fat body was reduced by 99% three days after feeding, returning to normal levels 10 days after feeding. CAT expression was reduced by 99% and 96% in the ovary and the posterior midgut, respectively, five days after ingestion. Mortality rates increased by 24-30% in first instars fed RHBP and CAT bacteria. Molting rates were reduced by 100% in first instars and 80% in third instars fed bacteria producing RHBP or CAT dsRNA. Oviposition was reduced by 43% (RHBP) and 84% (CAT). Embryogenesis was arrested in 16% (RHBP) and 20% (CAT) of laid eggs. Feeding females 105 CFU/mL of the natural symbiont, Rhodococcus rhodnii, transformed to express RHBP-specific hairpin RNA reduced RHBP expression by 89% and reduced oviposition. Modifying the insect microbiota to induce systemic RNAi in R. prolixus may result in a paratransgenic strategy for sustainable vector control. PMID:25675102

  2. A genome-wide RNAi screen identifies proteins modulating aberrant FLT3-ITD signaling

    PubMed Central

    Caldarelli, A; Müller, J P; Paskowski-Rogacz, M; Herrmann, K; Bauer, R; Koch, S; Heninger, A K; Krastev, D; Ding, L; Kasper, S; Fischer, T; Brodhun, M; Böhmer, F-D; Buchholz, F

    2013-01-01

    Fms-like tyrosine kinase-3 is a commonly mutated gene in acute myeloid leukemia, with about one-third of patients carrying an internal-tandem duplication of the juxtamembrane domain in the receptor (FLT3-ITD). FLT3-ITD exhibits altered signaling quality, including aberrant activation of STAT5. To identify genes affecting FLT3-ITD-mediated STAT5 signaling, we performed an esiRNA-based RNAi screen utilizing a STAT5-driven reporter assay. Knockdowns that caused reduced FLT3-ITD-mediated STAT5 signaling were enriched for genes encoding proteins involved in protein secretion and intracellular protein transport, indicating that modulation of protein transport processes could potentially be used to reduce constitutive STAT5 signaling in FLT3-ITD-positive cells. The relevance of KDELR1, a component involved in the Golgi-ER retrograde transport, was further analyzed. In FLT3-ITD-expressing leukemic MV4-11 cells, downregulation of KDELR1 resulted in reduced STAT5 activation, proliferation and colony-forming capacity. Stable shRNA-mediated depletion of KDELR1 in FLT3-ITD-expressing 32D cells likewise resulted in reduced STAT5 signaling and cell proliferation. Importantly, these cells also showed a reduced capacity to generate a leukemia-like disease in syngeneic C3H/HeJ mice. Together our data suggest intracellular protein transport as a potential target for FLT3-ITD driven leukemias, with KDELR1 emerging as a positive modulator of oncogenic FLT3-ITD activity. PMID:23508117

  3. Modulators of the microRNA biogenesis pathway via arrayed lentiviral enabled RNAi screening for drug and biomarker discovery

    PubMed Central

    Shum, David; Bhinder, Bhavneet; Djaballah, Hakim

    2013-01-01

    MicroRNAs (miRNAs) are small endogenous and conserved non-coding RNA molecules that regulate gene expression. Although the first miRNA was discovered well over sixteen years ago, little is known about their biogenesis and it is only recently that we have begun to understand their scope and diversity. For this purpose, we performed an RNAi screen aimed at identifying genes involved in their biogenesis pathway with a potential use as biomarkers. Using a previously developed miRNA 21 (miR-21) EGFP-based biosensor cell based assay monitoring green fluorescence enhancements, we performed an arrayed short hairpin RNA (shRNA) screen against a lentiviral particle ready TRC1 library covering 16,039 genes in 384-well plate format, and interrogating the genome one gene at a time building a panoramic view of endogenous miRNA activity. Using the BDA method for RNAi data analysis, we nominate 497 gene candidates the knockdown of which increased the EGFP fluorescence and yielding an initial hit rate of 3.09%; of which only 22, with reported validated clones, are deemed high-confidence gene candidates. An unexpected and surprising result was that only DROSHA was identified as a hit out of the seven core essential miRNA biogenesis genes; suggesting that perhaps intracellular shRNA processing into the correct duplex may be cell dependent and with differential outcome. Biological classification revealed several major control junctions among them genes involved in transport and vesicular trafficking. In summary, we report on 22 high confidence gene candidate regulators of miRNA biogenesis with potential use in drug and biomarker discovery. PMID:23977983

  4. Knockdown of Selenocysteine-Specific Elongation Factor in Amblyomma maculatum Alters the Pathogen Burden of Rickettsia parkeri with Epigenetic Control by the Sin3 Histone Deacetylase Corepressor Complex

    PubMed Central

    Adamson, Steven W.; Browning, Rebecca E.; Budachetri, Khemraj; Ribeiro, José M. C.; Karim, Shahid

    2013-01-01

    Selenocysteine is the 21st naturally-occurring amino acid. Selenoproteins have diverse functions and many remain uncharacterized, but they are typically associated with antioxidant activity. The incorporation of selenocysteine into the nascent polypeptide chain recodes the TGA stop codon and this process depends upon a number of essential factors including the selenocysteine elongation factor (SEF). The transcriptional expression of SEF did not change significantly in tick midguts throughout the blood meal, but decreased in salivary glands to 20% at the end of the fast feeding phase. Since selenoprotein translation requires this specialized elongation factor, we targeted this gene for knockdown by RNAi to gain a global view of the role selenoproteins play in tick physiology. We found no significant differences in tick engorgement and embryogenesis but detected no antioxidant capacity in tick saliva. The transcriptional profile of selenoproteins in R. parkeri-infected Amblyomma maculatum revealed declined activity of selenoprotein M and catalase and increased activity of selenoprotein O, selenoprotein S, and selenoprotein T. Furthermore, the pathogen burden was significantly altered in SEF-knockdowns. We then determined the global impact of SEF-knockdown by RNA-seq, and mapped huge shifts in secretory gene expression that could be the result of downregulation of the Sin3 histone deacetylase corepressor complex. PMID:24282621

  5. Dmp53, basket and drICE gene knockdown and polyphenol gallic acid increase life span and locomotor activity in a Drosophila Parkinson’s disease model

    PubMed Central

    Ortega-Arellano, Hector Flavio; Jimenez-Del-Rio, Marlene; Velez-Pardo, Carlos

    2013-01-01

    Understanding the mechanism(s) by which dopaminergic (DAergic) neurons are eroded in Parkinson’s disease (PD) is critical for effective therapeutic strategies. By using the binary tyrosine hydroxylase (TH)-Gal4/UAS-X RNAi Drosophila melanogaster system, we report that Dmp53, basket and drICE gene knockdown in dopaminergic neurons prolong life span (p < 0.05; log-rank test) and locomotor activity (p < 0.05; χ2 test) in D. melanogaster lines chronically exposed to (1 mM) paraquat (PQ, oxidative stress (OS) generator) compared to untreated transgenic fly lines. Likewise, knockdown flies displayed higher climbing performance than control flies. Amazingly, gallic acid (GA) significantly protected DAergic neurons, ameliorated life span, and climbing abilities in knockdown fly lines treated with PQ compared to flies treated with PQ only. Therefore, silencing specific gene(s) involved in neuronal death might constitute an excellent tool to study the response of DAergic neurons to OS stimuli. We propose that a therapy with antioxidants and selectively “switching off” death genes in DAergic neurons could provide a means for pre-clinical PD individuals to significantly ameliorate their disease condition. PMID:24385865

  6. RNAi mediates post-transcriptional repression of gene expression in fission yeast Schizosaccharomyces pombe

    SciTech Connect

    Smialowska, Agata; Djupedal, Ingela; Wang, Jingwen; Kylsten, Per; Swoboda, Peter; Ekwall, Karl

    2014-02-07

    Highlights: • Protein coding genes accumulate anti-sense sRNAs in fission yeast S. pombe. • RNAi represses protein-coding genes in S. pombe. • RNAi-mediated gene repression is post-transcriptional. - Abstract: RNA interference (RNAi) is a gene silencing mechanism conserved from fungi to mammals. Small interfering RNAs are products and mediators of the RNAi pathway and act as specificity factors in recruiting effector complexes. The Schizosaccharomyces pombe genome encodes one of each of the core RNAi proteins, Dicer, Argonaute and RNA-dependent RNA polymerase (dcr1, ago1, rdp1). Even though the function of RNAi in heterochromatin assembly in S. pombe is established, its role in controlling gene expression is elusive. Here, we report the identification of small RNAs mapped anti-sense to protein coding genes in fission yeast. We demonstrate that these genes are up-regulated at the protein level in RNAi mutants, while their mRNA levels are not significantly changed. We show that the repression by RNAi is not a result of heterochromatin formation. Thus, we conclude that RNAi is involved in post-transcriptional gene silencing in S. pombe.

  7. RNAi technologies in agricultural biotechnology: The Toxicology Forum 40th Annual Summer Meeting.

    PubMed

    Sherman, James H; Munyikwa, Tichafa; Chan, Stephen Y; Petrick, Jay S; Witwer, Kenneth W; Choudhuri, Supratim

    2015-11-01

    During the 40th Annual Meeting of The Toxicology Forum, the current and potential future science, regulations, and politics of agricultural biotechnology were presented and discussed. The meeting session described herein focused on the technology of RNA interference (RNAi) in agriculture. The general process by which RNAi works, currently registered RNAi-based plant traits, example RNAi-based traits in development, potential use of double stranded RNA (dsRNA) as topically applied pesticide active ingredients, research related to the safety of RNAi, biological barriers to ingested dsRNA, recent regulatory RNAi science reviews, and regulatory considerations related to the use of RNAi in agriculture were discussed. Participants generally agreed that the current regulatory framework is robust and appropriate for evaluating the safety of RNAi employed in agricultural biotechnology and were also supportive of the use of RNAi to develop improved crop traits. However, as with any emerging technology, the potential range of future products, potential future regulatory frameworks, and public acceptance of the technology will continue to evolve. As such, continuing dialogue was encouraged to promote education of consumers and science-based regulations. PMID:26361858

  8. Live Cell Microscopy-Based RNAi Screening in the Moss Physcomitrella patens.

    PubMed

    Miki, Tomohiro; Nakaoka, Yuki; Goshima, Gohta

    2016-01-01

    RNA interference (RNAi) is a powerful technique enabling the identification of the genes involved in a certain cellular process. Here, we discuss protocols for microscopy-based RNAi screening in protonemal cells of the moss Physcomitrella patens, an emerging model system for plant cell biology. Our method is characterized by the use of conditional (inducible) RNAi vectors, transgenic moss lines in which the RNAi vector is integrated, and time-lapse fluorescent microscopy. This method allows for effective and efficient screening of >100 genes involved in various cellular processes such as mitotic cell division, organelle distribution, or cell growth. PMID:27581297

  9. Baculovirus-mediated Gene Delivery and RNAi Applications

    PubMed Central

    Makkonen, Kaisa-Emilia; Airenne, Kari; Ylä-Herttulala, Seppo

    2015-01-01

    Baculoviruses are widely encountered in nature and a great deal of data is available about their safety and biology. Recently, these versatile, insect-specific viruses have demonstrated their usefulness in various biotechnological applications including protein production and gene transfer. Multiple in vitro and in vivo studies exist and support their use as gene delivery vehicles in vertebrate cells. Recently, baculoviruses have also demonstrated high potential in RNAi applications in which several advantages of the virus make it a promising tool for RNA gene transfer with high safety and wide tropism. PMID:25912715

  10. Nonionic surfactant vesicles for delivery of RNAi therapeutics

    PubMed Central

    Paecharoenchai, Orapan; Teng, Lesheng; Yung, Bryant C; Teng, Lirong; Opanasopit, Praneet; Lee, Robert J

    2014-01-01

    RNAi is a promising potential therapeutic approach for many diseases. A major barrier to its clinical translation is the lack of efficient delivery systems for siRNA. Among nonviral vectors, nonionic surfactant vesicles (niosomes) have shown a great deal of promise in terms of their efficacy and toxicity profiles. Nonionic surfactants have been shown to be a superior alternative to phospholipids in several studies. There is a large selection of surfactants with various properties that have been incorporated into niosomes. Therefore, there is great potential for innovation in terms of nisome composition. This article summarizes recent advancements in niosome technology for the delivery of siRNA. PMID:24156490

  11. Chitosan-based nanoparticles for mucosal delivery of RNAi therapeutics.

    PubMed

    Martirosyan, Alina; Olesen, Morten Jarlstad; Howard, Kenneth A

    2014-01-01

    RNA interference (RNAi) gene silencing by small interfering RNAs (siRNAs) offers a potent and highly specific therapeutic strategy; however, enabling technologies that overcome extracellular and intracellular barriers are required. Polycation-based nanoparticles (termed polyplexes) composed of the polysaccharide chitosan have been used to facilitate delivery of siRNA across mucosal surfaces following local administration. This chapter describes the mucosal barriers that need to be addressed in order to design an effective mucosal delivery strategy and the utilization of the mucoadhesive properties of chitosan. Focus is given to preparation methods and the preclinical application of chitosan nanoparticles for respiratory and oral delivery of siRNA. PMID:25409611

  12. PCTAIRE1-knockdown sensitizes cancer cells to TNF family cytokines.

    PubMed

    Yanagi, Teruki; Shi, Ranxin; Aza-Blanc, Pedro; Reed, John C; Matsuzawa, Shu-ichi

    2015-01-01

    While PCTAIRE1/PCTK1/Cdk16 is overexpressed in malignant cells and is crucial in tumorigenesis, its function in apoptosis remains unclear. Here we investigated the role of PCTAIRE1 in apoptosis, especially in the extrinsic cell death pathway. Gene-knockdown of PCTAIRE1 sensitized prostate cancer PPC1 and Du145 cells, and breast cancer MDA-MB-468 cells to TNF-family cytokines, including TNF-related apoptosis-inducing ligand (TRAIL). Meanwhile, PCTAIRE1-knockdown did not sensitize non-malignant cells, including diploid fibroblasts IMR-90 and the immortalized prostate epithelial cell line 267B1. PCTAIRE1-knockdown did not up-regulate death receptor expression on the cell surface or affect caspase-8, FADD and FLIP expression levels. PCTAIRE1-knockdown did promote caspase-8 cleavage and RIPK1 degradation, while RIPK1 mRNA knockdown sensitized PPC1 cells to TNF-family cytokines. Furthermore, the kinase inhibitor SNS-032, which inhibits PCTAIRE1 kinase activity, sensitized PPC1 cells to TRAIL-induced apoptosis. Together these results suggest that PCTAIRE1 contributes to the resistance of cancer cell lines to apoptosis induced by TNF-family cytokines, which implies that PCTAIRE1 inhibitors could have synergistic effects with TNF-family cytokines for cytodestruction of cancer cells. PMID:25790448

  13. PCTAIRE1-Knockdown Sensitizes Cancer Cells to TNF Family Cytokines

    PubMed Central

    Yanagi, Teruki; Shi, Ranxin; Aza-Blanc, Pedro; Reed, John C.; Matsuzawa, Shu-ichi

    2015-01-01

    While PCTAIRE1/PCTK1/Cdk16 is overexpressed in malignant cells and is crucial in tumorigenesis, its function in apoptosis remains unclear. Here we investigated the role of PCTAIRE1 in apoptosis, especially in the extrinsic cell death pathway. Gene-knockdown of PCTAIRE1 sensitized prostate cancer PPC1 and Du145 cells, and breast cancer MDA-MB-468 cells to TNF-family cytokines, including TNF-related apoptosis-inducing ligand (TRAIL). Meanwhile, PCTAIRE1-knockdown did not sensitize non-malignant cells, including diploid fibroblasts IMR-90 and the immortalized prostate epithelial cell line 267B1. PCTAIRE1-knockdown did not up-regulate death receptor expression on the cell surface or affect caspase-8, FADD and FLIP expression levels. PCTAIRE1-knockdown did promote caspase-8 cleavage and RIPK1 degradation, while RIPK1 mRNA knockdown sensitized PPC1 cells to TNF-family cytokines. Furthermore, the kinase inhibitor SNS-032, which inhibits PCTAIRE1 kinase activity, sensitized PPC1 cells to TRAIL-induced apoptosis. Together these results suggest that PCTAIRE1 contributes to the resistance of cancer cell lines to apoptosis induced by TNF-family cytokines, which implies that PCTAIRE1 inhibitors could have synergistic effects with TNF-family cytokines for cytodestruction of cancer cells. PMID:25790448

  14. Akt3 knockdown induces mitochondrial dysfunction in human cancer cells.

    PubMed

    Kim, Minjee; Kim, Young Yeon; Jee, Hye Jin; Bae, Sun Sik; Jeong, Na Young; Um, Jee-Hyun; Yun, Jeanho

    2016-05-01

    Akt/PKB plays a pivotal role in cell proliferation and survival. However, the isotype-specific roles of Akt in mitochondrial function have not been fully addressed. In this study, we explored the role of Akt in mitochondrial function after stable knockdown of the Akt isoforms in EJ human bladder cancer cells. We found that the mitochondrial mass was significantly increased in the Akt1- and Akt3-knockdown cells, and this increase was accompanied by an increase in TFAM and NRF1. Akt2 knockdown did not cause a similar effect. Interestingly, Akt3 knockdown also led to severe structural defects in the mitochondria, an increase in doxorubicin-induced senescence, and impairment of cell proliferation in galactose medium. Consistent with these observations, the mitochondrial oxygen consumption rate was significantly reduced in the Akt3-knockdown cells. An Akt3 deficiency-induced decrease in mitochondrial respiration was also observed in A549 lung cancer cells. Collectively, these results suggest that the Akt isoforms play distinct roles in mitochondrial function and that Akt3 is critical for proper mitochondrial respiration in human cancer cells. PMID:26972278

  15. NLRC5 knockdown in chicken macrophages alters response to LPS and poly (I:C) stimulation

    PubMed Central

    2012-01-01

    Background NLRC5 is a member of the CARD domain containing, nucleotide-binding oligomerization (NOD)-like receptor (NLR) family, which recognizes pathogen-associated molecular patterns (PAMPs) and initiates an innate immune response leading to inflammation and/or cell death. However, the specific role of NLRC5 as a modulator of the inflammatory immune response remains controversial. It has been reported to be a mediator of type I IFNs, NF-kB, and MHC class I gene. But no study on NLRC5 function has been reported to date in chickens. In the current study, we investigated the role of NLRC5 in the regulation of IFNA, IFNB, IL-6, and MHC class I in the chicken HD11 macrophage cell line, by using RNAi technology. HD11 cells were transfected with one of five siRNAs (s1, s2, s3, negative-siRNA, or a mixture of s1, s2, s3-siRNAs). After 24 hours, cells were exposed to LPS or poly (I:C) or a vehicle control. Gene expression of NLRC5, IFNA, IFNB, IL-6, and MHC class I at 2, 4, 6, and 8 hours post stimulation (hps) was quantified by qPCR. Results The expression of NLRC5, IFNA, IFNB, and IL-6 genes in negative irrelevant transfection controls was up-regulated at 2 hps after LPS treatment compared to the vehicle controls. S3-siRNA effectively knocked down NLRC5 expression at 4 hps, and the expression of IFNA and IFNB (but not IL-6 and MHC class I) was also down-regulated at 4 hps in s3-siRNA transfected cells, compared to negative irrelevant transfection controls. Stimulation by LPS appeared to relatively restore the decrease in NLRC5, IFNA, and IFNB expression, but the difference is not significant. Conclusions Functional characterization of chicken NLRC5 in an in vitro system demonstrated its importance in regulating intracellular molecules involved in inflammatory response. The knockdown of NLRC5 expression negatively mediates gene expression of IFNA and IFNB in the chicken HD11 cell line; therefore, NLRC5 likely has a role in positive regulation of IFNA and IFNB expression

  16. Knockdown of Expression of Cdk5 or p35 (a Cdk5 Activator) Results in Podocyte Apoptosis

    PubMed Central

    Zheng, Ya-Li; Zhang, Xia; Fu, Hai-Xia; Guo, Mei; Shukla, Varsha; Amin, Niranjana D.; E, Jing; Bao, Li; Luo, Hong-Yan; Li, Bo; Lu, Xiao-Hua; Gao, Yong-Cai

    2016-01-01

    Podocytes are terminally differentiated glomerular epithelial cells. Podocyte loss has been found in many renal diseases. Cdk5 is a cyclin-dependent protein kinase which is predominantly regulated by p35. To study the role of Cdk5/p35 in podocyte survival, we first applied western blotting (WB) analysis to confirm the time-course expression of Cdk5 and p35 during kidney development and in cultured immortalized mouse podocytes. We also demonstrated that p35 plays an important role in promoting podocyte differentiation by overexpression of p35 in podocytes. To deregulate the expression of Cdk5 or p35 in mouse podocytes, we used RNAi and analyzed cell function and apoptosis assaying for podocyte specific marker Wilms Tumor 1 (WT1) and cleaved caspase 3, respectively. We also counted viable cells using cell counting kit-8. We found that depletion of Cdk5 causes decreased expression of WT1 and apoptosis. It is noteworthy, however, that downregulation of p35 reduced Cdk5 activity, but had no effect on cleaved caspase 3 expression. It did, however, reduce expression of WT1, a transcription factor, and produced podocyte dysmorphism. On the other hand increased apoptosis could be detected in p35-deregulated podocytes using the TUNEL analysis and immunofluorescent staining with cleaved caspase3 antibody. Viability of podocytes was decreased in both Cdk5 and p35 knockdown cells. Knocking down Cdk5 or p35 gene by RNAi does not affect the cycline I expression, another Cdk5 activator in podocyes. We conclude that Cdk5 and p35 play a crucial role in maintaining podocyte differentiation and survival, and suggest these proteins as targets for therapeutic intervention in podocyte-damaged kidney diseases. PMID:27479491

  17. Knockdown of Expression of Cdk5 or p35 (a Cdk5 Activator) Results in Podocyte Apoptosis.

    PubMed

    Zheng, Ya-Li; Zhang, Xia; Fu, Hai-Xia; Guo, Mei; Shukla, Varsha; Amin, Niranjana D; E, Jing; Bao, Li; Luo, Hong-Yan; Li, Bo; Lu, Xiao-Hua; Gao, Yong-Cai

    2016-01-01

    Podocytes are terminally differentiated glomerular epithelial cells. Podocyte loss has been found in many renal diseases. Cdk5 is a cyclin-dependent protein kinase which is predominantly regulated by p35. To study the role of Cdk5/p35 in podocyte survival, we first applied western blotting (WB) analysis to confirm the time-course expression of Cdk5 and p35 during kidney development and in cultured immortalized mouse podocytes. We also demonstrated that p35 plays an important role in promoting podocyte differentiation by overexpression of p35 in podocytes. To deregulate the expression of Cdk5 or p35 in mouse podocytes, we used RNAi and analyzed cell function and apoptosis assaying for podocyte specific marker Wilms Tumor 1 (WT1) and cleaved caspase 3, respectively. We also counted viable cells using cell counting kit-8. We found that depletion of Cdk5 causes decreased expression of WT1 and apoptosis. It is noteworthy, however, that downregulation of p35 reduced Cdk5 activity, but had no effect on cleaved caspase 3 expression. It did, however, reduce expression of WT1, a transcription factor, and produced podocyte dysmorphism. On the other hand increased apoptosis could be detected in p35-deregulated podocytes using the TUNEL analysis and immunofluorescent staining with cleaved caspase3 antibody. Viability of podocytes was decreased in both Cdk5 and p35 knockdown cells. Knocking down Cdk5 or p35 gene by RNAi does not affect the cycline I expression, another Cdk5 activator in podocyes. We conclude that Cdk5 and p35 play a crucial role in maintaining podocyte differentiation and survival, and suggest these proteins as targets for therapeutic intervention in podocyte-damaged kidney diseases. PMID:27479491

  18. Differential effects of RNAi treatments on field populations of the western corn rootworm.

    PubMed

    Chu, Chia-Ching; Sun, Weilin; Spencer, Joseph L; Pittendrigh, Barry R; Seufferheld, Manfredo J

    2014-03-01

    RNA interference (RNAi) mediated crop protection against insect pests is a technology that is greatly anticipated by the academic and industrial pest control communities. Prior to commercialization, factors influencing the potential for evolution of insect resistance to RNAi should be evaluated. While mutations in genes encoding the RNAi machinery or the sequences targeted for interference may serve as a prominent mechanism of resistance evolution, differential effects of RNAi on target pests may also facilitate such evolution. However, to date, little is known about how variation of field insect populations could influence the effectiveness of RNAi treatments. To approach this question, we evaluated the effects of RNAi treatments on adults of three western corn rootworm (WCR; Diabrotica virgifera virgifera LeConte) populations exhibiting different levels of gut cysteine protease activity, tolerance of soybean herbivory, and immune gene expression; two populations were collected from crop rotation-resistant (RR) problem areas and one from a location where RR was not observed (wild type; WT). Our results demonstrated that RNAi targeting DvRS5 (a highly expressed cysteine protease gene) reduced gut cysteine protease activity in all three WCR populations. However, the proportion of the cysteine protease activity that was inhibited varied across populations. When WCR adults were treated with double-stranded RNA of an immune gene att1, different changes in survival among WT and RR populations on soybean diets occurred. Notably, for both genes, the sequences targeted for RNAi were the same across all populations examined. These findings indicate that the effectiveness of RNAi treatments could vary among field populations depending on their physiological and genetic backgrounds and that the consistency of an RNAi trait's effectiveness on phenotypically different populations should be considered or tested prior to wide deployment. Also, genes that are potentially subjected

  19. The protein kinase TOUSLED facilitates RNAi in Arabidopsis

    PubMed Central

    Uddin, Mohammad Nazim; Dunoyer, Patrice; Schott, Gregory; Akhter, Salina; Shi, Chunlin; Lucas, William J.; Voinnet, Olivier; Kim, Jae-Yean

    2014-01-01

    RNA silencing is an evolutionarily conserved mechanism triggered by double-stranded RNA that is processed into 21- to 24-nt small interfering (si)RNA or micro (mi)RNA by RNaseIII-like enzymes called Dicers. Gene regulations by RNA silencing have fundamental implications in a large number of biological processes that include antiviral defense, maintenance of genome integrity and the orchestration of cell fates. Although most generic or core components of the various plant small RNA pathways have been likely identified over the past 15 years, factors involved in RNAi regulation through post-translational modifications are just starting to emerge, mostly through forward genetic studies. A genetic screen designed to identify factors required for RNAi in Arabidopsis identified the serine/threonine protein kinase, TOUSLED (TSL). Mutations in TSL affect exogenous and virus-derived siRNA activity in a manner dependent upon its kinase activity. By contrast, despite their pleiotropic developmental phenotype, tsl mutants show no defect in biogenesis or activity of miRNA or endogenous trans-acting siRNA. These data suggest a possible role for TSL phosphorylation in the specific regulation of exogenous and antiviral RNA silencing in Arabidopsis and identify TSL as an intrinsic regulator of RNA interference. PMID:24920830

  20. RNAi and Antiviral Defense in the Honey Bee

    PubMed Central

    Brutscher, Laura M.; Flenniken, Michelle L.

    2015-01-01

    Honey bees play an important agricultural and ecological role as pollinators of numerous agricultural crops and other plant species. Therefore, investigating the factors associated with high annual losses of honey bee colonies in the US is an important and active area of research. Pathogen incidence and abundance correlate with Colony Collapse Disorder- (CCD-) affected colonies in the US and colony losses in the US and in some European countries. Honey bees are readily infected by single-stranded positive sense RNA viruses. Largely dependent on the host immune response, virus infections can either remain asymptomatic or result in deformities, paralysis, or death of adults or larvae. RNA interference (RNAi) is an important antiviral defense mechanism in insects, including honey bees. Herein, we review the role of RNAi in honey bee antiviral defense and highlight some parallels between insect and mammalian immune systems. A more thorough understanding of the role of pathogens on honey bee health and the immune mechanisms bees utilize to combat infectious agents may lead to the development of strategies that enhance honey bee health and result in the discovery of additional mechanisms of immunity in metazoans. PMID:26798663

  1. Comparative analysis of RNAi screening technologies at genome-scale reveals an inherent processing inefficiency of the plasmid-based shRNA hairpin.

    PubMed

    Bhinder, Bhavneet; Shum, David; Djaballah, Hakim

    2014-02-01

    RNAi screening in combination with the genome-sequencing projects would constitute the Holy Grail of modern genetics; enabling discovery and validation towards a better understanding of fundamental biology leading to novel targets to combat disease. Hit discordance at inter-screen level together with the lack of reproducibility is emerging as the technology's main pitfalls. To examine some of the underlining factors leading to such discrepancies, we reasoned that perhaps there is an inherent difference in knockdown efficiency of the various RNAi technologies. For this purpose, we utilized the two most popular ones, chemically synthesized siRNA duplex and plasmid-based shRNA hairpin, in order to perform a head to head comparison. Using a previously developed gain-of-function assay probing modulators of the miRNA biogenesis pathway, we first executed on a siRNA screen against the Silencer Select V4.0 library (AMB) nominating 1,273, followed by an shRNA screen against the TRC1 library (TRC1) nominating 497 gene candidates. We observed a poor overlap of only 29 hits given that there are 15,068 overlapping genes between the two libraries; with DROSHA as the only common hit out of the seven known core miRNA biogenesis genes. Distinct genes interacting with the same biogenesis regulators were observed in both screens, with a dismal cross-network overlap of only 3 genes (DROSHA, TGFBR1, and DIS3). Taken together, our study demonstrates differential knockdown activities between the two technologies, possibly due to the inefficient intracellular processing and potential cell-type specificity determinants in generating intended targeting sequences for the plasmid-based shRNA hairpins; and suggests this observed inefficiency as potential culprit in addressing the lack of reproducibility. PMID:24433414

  2. Comparative analysis of RNAi screening technologies at genome-scale reveals an inherent processing inefficiency of the plasmid-based shRNA hairpin

    PubMed Central

    Bhinder, Bhavneet; Shum, David; Djaballah, Hakim

    2014-01-01

    RNAi screening in combination with the genome-sequencing projects would constitute the Holy Grail of modern genetics; enabling discovery and validation towards a better understanding of fundamental biology leading to novel targets to combat disease. Hit discordance at inter-screen level together with the lack of reproducibility is emerging as the technology's main pitfalls. To examine some of the underlining factors leading to such discrepancies, we reasoned that perhaps there is an inherent difference in knockdown efficiency of the various RNAi technologies. For this purpose, we utilized the two most popular ones, chemically synthesized siRNA duplex and plasmid-based shRNA hairpin, in order to perform a head to head comparison. Using a previously developed gain-of-function assay probing modulators of the miRNA biogenesis pathway, we first executed on a siRNA screen against the Silencer Select V4.0 library (AMB) nominating 1,273, followed by an shRNA screen against the TRC1 library (TRC1) nominating 497 gene candidates. We observed a poor overlap of only 29 hits given that there are 15,068 overlapping genes between the two libraries; with DROSHA as the only common hit out of the seven known core miRNA biogenesis genes. Distinct genes interacting with the same biogenesis regulators were observed in both screens, with a dismal cross-network overlap of only 3 genes (DROSHA, TGFBR1, and DIS3). Taken together, our study demonstrates differential knockdown activities between the two technologies, possibly due to the inefficient intracellular processing and potential cell-type specificity determinants in generating intended targeting sequences for the plasmid-based shRNA hairpins; and suggests this observed inefficiency as potential culprit in addressing the lack of reproducibility. PMID:24433414

  3. Unbiased RNAi screen for hepcidin regulators links hepcidin suppression to proliferative Ras/RAF and nutrient-dependent mTOR signaling.

    PubMed

    Mleczko-Sanecka, Katarzyna; Roche, Franziska; da Silva, Ana Rita; Call, Debora; D'Alessio, Flavia; Ragab, Anan; Lapinski, Philip E; Ummanni, Ramesh; Korf, Ulrike; Oakes, Christopher; Damm, Georg; D'Alessandro, Lorenza A; Klingmüller, Ursula; King, Philip D; Boutros, Michael; Hentze, Matthias W; Muckenthaler, Martina U

    2014-03-01

    The hepatic hormone hepcidin is a key regulator of systemic iron metabolism. Its expression is largely regulated by 2 signaling pathways: the "iron-regulated" bone morphogenetic protein (BMP) and the inflammatory JAK-STAT pathways. To obtain broader insights into cellular processes that modulate hepcidin transcription and to provide a resource to identify novel genetic modifiers of systemic iron homeostasis, we designed an RNA interference (RNAi) screen that monitors hepcidin promoter activity after the knockdown of 19 599 genes in hepatocarcinoma cells. Interestingly, many of the putative hepcidin activators play roles in signal transduction, inflammation, or transcription, and affect hepcidin transcription through BMP-responsive elements. Furthermore, our work sheds light on new components of the transcriptional machinery that maintain steady-state levels of hepcidin expression and its responses to the BMP- and interleukin-6-triggered signals. Notably, we discover hepcidin suppression mediated via components of Ras/RAF MAPK and mTOR signaling, linking hepcidin transcriptional control to the pathways that respond to mitogen stimulation and nutrient status. Thus using a combination of RNAi screening, reverse phase protein arrays, and small molecules testing, we identify links between the control of systemic iron homeostasis and critical liver processes such as regeneration, response to injury, carcinogenesis, and nutrient metabolism. PMID:24385536

  4. RNA Interference in the Age of CRISPR: Will CRISPR Interfere with RNAi?

    PubMed

    Unniyampurath, Unnikrishnan; Pilankatta, Rajendra; Krishnan, Manoj N

    2016-01-01

    The recent emergence of multiple technologies for modifying gene structure has revolutionized mammalian biomedical research and enhanced the promises of gene therapy. Over the past decade, RNA interference (RNAi) based technologies widely dominated various research applications involving experimental modulation of gene expression at the post-transcriptional level. Recently, a new gene editing technology, Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and the CRISPR-associated protein 9 (Cas9) (CRISPR/Cas9) system, has received unprecedented acceptance in the scientific community for a variety of genetic applications. Unlike RNAi, the CRISPR/Cas9 system is bestowed with the ability to introduce heritable precision insertions and deletions in the eukaryotic genome. The combination of popularity and superior capabilities of CRISPR/Cas9 system raises the possibility that this technology may occupy the roles currently served by RNAi and may even make RNAi obsolete. We performed a comparative analysis of the technical aspects and applications of the CRISPR/Cas9 system and RNAi in mammalian systems, with the purpose of charting out a predictive picture on whether the CRISPR/Cas9 system will eclipse the existence and future of RNAi. The conclusion drawn from this analysis is that RNAi will still occupy specific domains of biomedical research and clinical applications, under the current state of development of these technologies. However, further improvements in CRISPR/Cas9 based technology may ultimately enable it to dominate RNAi in the long term. PMID:26927085

  5. HIV Develops Indirect Cross-resistance to Combinatorial RNAi Targeting Two Distinct and Spatially Distant Sites

    PubMed Central

    Shah, Priya S; Pham, Nhung P; Schaffer, David V

    2012-01-01

    Resistance to existing HIV therapies is an increasing problem, and alternative treatments are urgently needed. RNA interference (RNAi), an innate mechanism for sequence-specific gene silencing, can be harnessed therapeutically to treat viral infections, yet viral resistance can still emerge. Here, we demonstrate that HIV can develop indirect resistance to individual and combinatorial RNAi-targeting protein-coding regions up to 5,500 nucleotides (nt) downstream of the viral promoter. We identify several variants harboring mutations in the HIV promoter, and not within the RNAi targets, that produce more fully elongated transcripts. Furthermore, these variants are resistant to the RNAi, potentially by stoichiometrically overwhelming this cellular mechanism. Alarmingly, virus resistant to one short hairpin RNA (shRNA) also exhibits cross-resistance to a different shRNA, which targets a distinct and spatially distant region to which the virus has not been previously exposed. To our knowledge, this is the first example of HIV “cross-resistance” to viral inhibitors targeting different loci. Finally, combining anti-HIV RNAi with a small molecule enhancer of RNAi can inhibit the replication of an indirectly resistant mutant. These results suggest that indirect resistance to RNAi is a general mechanism that should be considered when investigating viral resistance and designing combinatorial RNAi therapies. PMID:22294151

  6. High-efficiency generation of RNAi mutants of apple by use of multi-vector transformation

    Technology Transfer Automated Retrieval System (TEKTRAN)

    A high-efficiency transformation and selection system was used to create apple RNAi mutants for determination of function of candidate genes in resistance of apple to Erwinia amylovora (fire blight). The M.26 apple genotype was transformed with a mixture of five RNAi EST-silencing vectors in each t...

  7. Analysis of Nuclear RNA Interference (RNAi) in Human Cells by Subcellular Fractionation and Argonaute Loading

    PubMed Central

    Gagnon, Keith T.; Li, Liande; Janowski, Bethany A.; Corey, David R.

    2014-01-01

    RNA interference (RNAi) is well known for its ability to regulate gene expression in the cytoplasm of mammalian cells. In mammalian cell nuclei, however, the impact of RNAi has remained more controversial. A key technical hurdle has been a lack of optimized protocols for the isolation and analysis of cell nuclei. Here we describe a simplified protocol for nuclei isolation from cultured cells that incorporates a method for obtaining nucleoplasmic and chromatin fractions and removing cytoplasmic contamination. Cell fractions can then be used to detect the presence and activity of RNAi factors in the nucleus. We present a protocol for investigating an early step in RNAi, Argonaute protein loading with small RNAs, which is enabled by our improved extract preparations. These protocols facilitate characterization of nuclear RNAi and can be applied to the analysis of other nuclear proteins and pathways. From cellular fractionation to analysis of Argonaute loading results, this protocol takes 4–6 d to complete. PMID:25079428

  8. RNAi effects on actin mRNAs in Homalodisca vitripennis cells

    PubMed Central

    Rosa, Cristina; Kamita, Shizuo G; Dequine, Haley; Wuriyanghan, Ha; Lindbo, John A; Falk, Bryce W

    2010-01-01

    The xylem feeding leafhopper Homalodisaca vitripennis (H. vitripennis) is an unusually robust and efficient vector of Xylella fastidiosa, a Gram-negative bacterium which causes several very important plant diseases. Here we investigated RNA interference (RNAi) to target actin, a key component of insect cells and whole bodies, in H. vitripennis cells. RNAi effectors were delivered via lipid based transfection and real-time RT-PCR, RNA hybridization, and microscopic analyses were employed to verify RNAi effects. When actin dsRNAs were used, a 10-fold decrease in the target H. vitripennis actin mRNA level was seen in cells. Altered phenotypic effects also were evident in transfected cells, as were small interfering RNAs, hallmarks of RNAi. The use of H. vitripennis cells and RNAi offers new opportunities to research hemipterans, the most important insect vectors of plant pathogens. PMID:20628496

  9. A Drosophila RNAi library modulates Hippo pathway-dependent tissue growth.

    PubMed

    Vissers, Joseph H A; Manning, Samuel A; Kulkarni, Aishwarya; Harvey, Kieran F

    2016-01-01

    Libraries of transgenic Drosophila melanogaster carrying RNA interference (RNAi) constructs have been used extensively to perform large-scale functional genetic screens in vivo. For example, RNAi screens have facilitated the discovery of multiple components of the Hippo pathway, an evolutionarily conserved growth-regulatory network. Here we investigate an important technical limitation with the widely used VDRC KK RNAi collection. We find that approximately 25% of VDRC KK RNAi lines cause false-positive enhancement of the Hippo pathway, owing to ectopic expression of the Tiptop transcription factor. Of relevance to the broader Drosophila community, ectopic tiptop (tio) expression can also cause organ malformations and mask phenotypes such as organ overgrowth. To enhance the use of the VDRC KK RNAi library, we have generated a D. melanogaster strain that will allow researchers to test, in a single cross, whether their genetic screen of interest will be affected by ectopic tio expression. PMID:26758424

  10. A Drosophila RNAi library modulates Hippo pathway-dependent tissue growth

    PubMed Central

    Vissers, Joseph H.A.; Manning, Samuel A.; Kulkarni, Aishwarya; Harvey, Kieran F.

    2016-01-01

    Libraries of transgenic Drosophila melanogaster carrying RNA interference (RNAi) constructs have been used extensively to perform large-scale functional genetic screens in vivo. For example, RNAi screens have facilitated the discovery of multiple components of the Hippo pathway, an evolutionarily conserved growth-regulatory network. Here we investigate an important technical limitation with the widely used VDRC KK RNAi collection. We find that approximately 25% of VDRC KK RNAi lines cause false-positive enhancement of the Hippo pathway, owing to ectopic expression of the Tiptop transcription factor. Of relevance to the broader Drosophila community, ectopic tiptop (tio) expression can also cause organ malformations and mask phenotypes such as organ overgrowth. To enhance the use of the VDRC KK RNAi library, we have generated a D. melanogaster strain that will allow researchers to test, in a single cross, whether their genetic screen of interest will be affected by ectopic tio expression. PMID:26758424

  11. RNAi-induced silencing of embryonic tryptophan oxygenase in the Pyralid moth, Plodia interpunctella

    PubMed Central

    Fabrick, Jeffrey A.; Kanost, Michael R.; Baker, James E.

    2004-01-01

    Gene silencing through the introduction of double-stranded RNA (RNA interference, RNAi) provides a powerful tool for the elucidation of gene function in many systems, including those where genomics and proteomics are incomplete. The use of RNAi technology for gene silencing in Lepidoptera has lacked significant attention compared to other systems. To demonstrate that RNAi can be utilized in the lepidopteran, Plodia interpunctella, we cloned a cDNA for tryptophan oxygenase, and showed that silencing of tryptophan oxygenase through RNAi during embryonic development resulted in loss of eye-color pigmentation. The complete amino acid sequence of Plodia tryptophan oxygenase can be accessed through NCBI Protein Database under NCBI Accession # AY427951. Abbreviation RNAi RNA interference PCR polymerase chain reaction RT-PCR reverse transcription-PCR PMID:15861231

  12. Cardiac-Restricted Expression of VCP/TER94 RNAi or Disease Alleles Perturbs Drosophila Heart Structure and Impairs Function

    PubMed Central

    Viswanathan, Meera C.; Blice-Baum, Anna C.; Sang, Tzu-Kang; Cammarato, Anthony

    2016-01-01

    Valosin-containing protein (VCP) is a highly conserved mechanoenzyme that helps maintain protein homeostasis in all cells and serves specialized functions in distinct cell types. In skeletal muscle, it is critical for myofibrillogenesis and atrophy. However, little is known about VCP's role(s) in the heart. Its functional diversity is determined by differential binding of distinct cofactors/adapters, which is likely disrupted during disease. VCP mutations cause multisystem proteinopathy (MSP), a pleiotropic degenerative disorder that involves inclusion body myopathy. MSP patients display progressive muscle weakness. They also exhibit cardiomyopathy and die from cardiac and respiratory failure, which are consistent with critical myocardial roles for the enzyme. Nonetheless, efficient models to interrogate VCP in cardiac muscle remain underdeveloped and poorly studied. Here, we investigated the significance of VCP and mutant VCP in the Drosophila heart. Cardiac-restricted RNAi-mediated knockdown of TER94, the Drosophila VCP homolog, severely perturbed myofibrillar organization and heart function in adult flies. Furthermore, expression of MSP disease-causing alleles engendered cardiomyopathy in adults and structural defects in embryonic hearts. Drosophila may therefore serve as a valuable model for examining role(s) of VCP in cardiogenesis and for identifying novel heart-specific VCP interactions, which when disrupted via mutation, contribute to or elicit cardiac pathology. PMID:27500162

  13. RNAi validation of resistance genes and their interactions in the highly DDT-resistant 91-R strain of Drosophila melanogaster.

    PubMed

    Gellatly, Kyle J; Yoon, Kyong Sup; Doherty, Jeffery J; Sun, Weilin; Pittendrigh, Barry R; Clark, J Marshall

    2015-06-01

    4,4'-dichlorodiphenyltrichloroethane (DDT) has been re-recommended by the World Health Organization for malaria mosquito control. Previous DDT use has resulted in resistance, and with continued use resistance will increase in terms of level and extent. Drosophila melanogaster is a model dipteran that has many available genetic tools, numerous studies done on insecticide resistance mechanisms, and is related to malaria mosquitoes allowing for extrapolation. The 91-R strain of D. melanogaster is highly resistant to DDT (>1500-fold), however, there is no mechanistic scheme that accounts for this level of resistance. Recently, reduced penetration, increased detoxification, and direct excretion have been identified as resistance mechanisms in the 91-R strain. Their interactions, however, remain unclear. Use of UAS-RNAi transgenic lines of D. melanogaster allowed for the targeted knockdown of genes putatively involved in DDT resistance and has validated the role of several cuticular proteins (Cyp4g1 and Lcp1), cytochrome P450 monooxygenases (Cyp6g1 and Cyp12d1), and ATP binding cassette transporters (Mdr50, Mdr65, and Mrp1) involved in DDT resistance. Further, increased sensitivity to DDT in the 91-R strain after intra-abdominal dsRNA injection for Mdr50, Mdr65, and Mrp1 was determined by a DDT contact bioassay, directly implicating these genes in DDT efflux and resistance. PMID:26047118

  14. Pooled RNAi screen identifies ubiquitin ligase Itch as crucial for influenza A virus release from the endosome during virus entry

    PubMed Central

    Su, Wen-Chi; Chen, Yung-Chia; Tseng, Chung-Hsin; Hsu, Paul Wei-Che; Tung, Kuo-Feng; Jeng, King-Song; Lai, Michael M. C.

    2013-01-01

    Influenza viruses, like other viruses, rely on host factors to support their life cycle as viral proteins usually “hijack,” or collaborate with, cellular proteins to execute their functions. Identification and understanding of these factors can increase the knowledge of molecular mechanisms manipulated by the viruses and facilitate development of antiviral drugs. To this end, we developed a unique genome-wide pooled shRNA screen to search for cellular factors important for influenza A virus (IAV) replication. We identified an E3 ubiquitin ligase, Itch, as an essential factor for an early step in the viral life cycle. In Itch knockdown cells, the incorporation of viral ribonucleoprotein complex into endosomes was normal, but its subsequent release from endosomes and transport to the nucleus was retarded. In addition, upon virus infection, Itch was phosphorylated and recruited to the endosomes, where virus particles were located. Furthermore, Itch interacted with viral M1 protein and ubiquitinated M1 protein. Collectively, our findings unravel a critical role of Itch in mediating IAV release from the endosome and offer insights into the mechanism for IAV uncoating during virus entry. These findings also highlight the feasibility of pooled RNAi screening for exploring the cellular cofactors of lytic viruses. PMID:24101521

  15. Comprehensive RNAi-based screening of human and mouse TLR pathways identifies species-specific preferences in signaling protein use.

    PubMed

    Sun, Jing; Li, Ning; Oh, Kyu-Seon; Dutta, Bhaskar; Vayttaden, Sharat J; Lin, Bin; Ebert, Thomas S; De Nardo, Dominic; Davis, Joie; Bagirzadeh, Rustam; Lounsbury, Nicolas W; Pasare, Chandrashekhar; Latz, Eicke; Hornung, Veit; Fraser, Iain D C

    2016-01-01

    Toll-like receptors (TLRs) are a major class of pattern recognition receptors, which mediate the responses of innate immune cells to microbial stimuli. To systematically determine the roles of proteins in canonical TLR signaling pathways, we conducted an RNA interference (RNAi)-based screen in human and mouse macrophages. We observed a pattern of conserved signaling module dependencies across species, but found notable species-specific requirements at the level of individual proteins. Among these, we identified unexpected differences in the involvement of members of the interleukin-1 receptor-associated kinase (IRAK) family between the human and mouse TLR pathways. Whereas TLR signaling in mouse macrophages depended primarily on IRAK4 and IRAK2, with little or no role for IRAK1, TLR signaling and proinflammatory cytokine production in human macrophages depended on IRAK1, with knockdown of IRAK4 or IRAK2 having less of an effect. Consistent with species-specific roles for these kinases, IRAK4 orthologs failed to rescue signaling in IRAK4-deficient macrophages from the other species, and only mouse macrophages required the kinase activity of IRAK4 to mediate TLR responses. The identification of a critical role for IRAK1 in TLR signaling in humans could potentially explain the association of IRAK1 with several autoimmune diseases. Furthermore, this study demonstrated how systematic screening can be used to identify important characteristics of innate immune responses across species, which could optimize therapeutic targeting to manipulate human TLR-dependent outputs. PMID:26732763

  16. Pooled RNAi screen identifies ubiquitin ligase Itch as crucial for influenza A virus release from the endosome during virus entry.

    PubMed

    Su, Wen-Chi; Chen, Yung-Chia; Tseng, Chung-Hsin; Hsu, Paul Wei-Che; Tung, Kuo-Feng; Jeng, King-Song; Lai, Michael M C

    2013-10-22

    Influenza viruses, like other viruses, rely on host factors to support their life cycle as viral proteins usually "hijack," or collaborate with, cellular proteins to execute their functions. Identification and understanding of these factors can increase the knowledge of molecular mechanisms manipulated by the viruses and facilitate development of antiviral drugs. To this end, we developed a unique genome-wide pooled shRNA screen to search for cellular factors important for influenza A virus (IAV) replication. We identified an E3 ubiquitin ligase, Itch, as an essential factor for an early step in the viral life cycle. In Itch knockdown cells, the incorporation of viral ribonucleoprotein complex into endosomes was normal, but its subsequent release from endosomes and transport to the nucleus was retarded. In addition, upon virus infection, Itch was phosphorylated and recruited to the endosomes, where virus particles were located. Furthermore, Itch interacted with viral M1 protein and ubiquitinated M1 protein. Collectively, our findings unravel a critical role of Itch in mediating IAV release from the endosome and offer insights into the mechanism for IAV uncoating during virus entry. These findings also highlight the feasibility of pooled RNAi screening for exploring the cellular cofactors of lytic viruses. PMID:24101521

  17. RNAi-Mediated Gene Silencing in a Gonad Organ Culture to Study Sex Determination Mechanisms in Sea Turtle

    PubMed Central

    Sifuentes-Romero, Itzel; Merchant-Larios, Horacio; Milton, Sarah L.; Moreno-Mendoza, Norma; Díaz-Hernández, Verónica; García-Gasca, Alejandra

    2013-01-01

    The autosomal Sry-related gene, Sox9, encodes a transcription factor, which performs an important role in testis differentiation in mammals. In several reptiles, Sox9 is differentially expressed in gonads, showing a significant upregulation during the thermo-sensitive period (TSP) at the male-promoting temperature, consistent with the idea that SOX9 plays a central role in the male pathway. However, in spite of numerous studies, it remains unclear how SOX9 functions during this event. In the present work, we developed an RNAi-based method for silencing Sox9 in an in vitro gonad culture system for the sea turtle, Lepidochelys olivacea. Gonads were dissected as soon as the embryos entered the TSP and were maintained in organ culture. Transfection of siRNA resulted in the decrease of both Sox9 mRNA and protein. Furthermore, we found coordinated expression patterns for Sox9 and the anti-Müllerian hormone gene, Amh, suggesting that SOX9 could directly or indirectly regulate Amh expression, as it occurs in mammals. These results demonstrate an in vitro method to knockdown endogenous genes in gonads from a sea turtle, which represents a novel approach to investigate the roles of important genes involved in sex determination or differentiation pathways in species with temperature-dependent sex determination. PMID:24705165

  18. The Effectiveness of RNAi in Caenorhabditis elegans Is Maintained during Spaceflight

    PubMed Central

    Hashizume, Toko; Mori, Chihiro; Sugimoto, Tomoko; Suzuki, Hiromi; Fukui, Keiji; Yamazaki, Takashi; Higashibata, Akira; Szewczyk, Nathaniel J.; Higashitani, Atsushi

    2011-01-01

    Background Overcoming spaceflight-induced (patho)physiologic adaptations is a major challenge preventing long-term deep space exploration. RNA interference (RNAi) has emerged as a promising therapeutic for combating diseases on Earth; however the efficacy of RNAi in space is currently unknown. Methods Caenorhabditis elegans were prepared in liquid media on Earth using standard techniques and treated acutely with RNAi or a vector control upon arrival in Low Earth Orbit. After culturing during 4 and 8 d spaceflight, experiments were stopped by freezing at −80°C until analysis by mRNA and microRNA array chips, microscopy and Western blot on return to Earth. Ground controls (GC) on Earth were simultaneously grown under identical conditions. Results After 8 d spaceflight, mRNA expression levels of components of the RNAi machinery were not different from that in GC (e.g., Dicer, Argonaute, Piwi; P>0.05). The expression of 228 microRNAs, of the 232 analysed, were also unaffected during 4 and 8 d spaceflight (P>0.05). In spaceflight, RNAi against green fluorescent protein (gfp) reduced chromosomal gfp expression in gonad tissue, which was not different from GC. RNAi against rbx-1 also induced abnormal chromosome segregation in the gonad during spaceflight as on Earth. Finally, culture in RNAi against lysosomal cathepsins prevented degradation of the muscle-specific α-actin protein in both spaceflight and GC conditions. Conclusions Treatment with RNAi works as effectively in the space environment as on Earth within multiple tissues, suggesting RNAi may provide an effective tool for combating spaceflight-induced pathologies aboard future long-duration space missions. Furthermore, this is the first demonstration that RNAi can be utilised to block muscle protein degradation, both on Earth and in space. PMID:21673804

  19. C. elegans rrf-1 mutations maintain RNAi efficiency in the soma in addition to the germline.

    PubMed

    Kumsta, Caroline; Hansen, Malene

    2012-01-01

    Gene inactivation through RNA interference (RNAi) has proven to be a valuable tool for studying gene function in C. elegans. When combined with tissue-specific gene inactivation methods, RNAi has the potential to shed light on the function of a gene in distinct tissues. In this study we characterized C. elegans rrf-1 mutants to determine their ability to process RNAi in various tissues. These mutants have been widely used in RNAi studies to assess the function of genes specifically in the C. elegans germline. Upon closer analysis, we found that two rrf-1 mutants carrying different loss-of-function alleles were capable of processing RNAi targeting several somatically expressed genes. Specifically, we observed that the intestine was able to process RNAi triggers efficiently, whereas cells in the hypodermis showed partial susceptibility to RNAi in rrf-1 mutants. Other somatic tissues in rrf-1 mutants, such as the muscles and the somatic gonad, appeared resistant to RNAi. In addition to these observations, we found that the rrf-1(pk1417) mutation induced the expression of several transgenic arrays, including the FOXO transcription factor DAF-16. Unexpectedly, rrf-1(pk1417) mutants showed increased endogenous expression of the DAF-16 target gene sod-3; however, the lifespan and thermo-tolerance of rrf-1(pk1417) mutants were similar to those of wild-type animals. In sum, these data show that rrf-1 mutants display several phenotypes not previously appreciated, including broader tissue-specific RNAi-processing capabilities, and our results underscore the need for careful characterization of tissue-specific RNAi tools. PMID:22574120

  20. Knock-down of transcript abundance of a family of Kunitz proteinase inhibitor genes in white clover (Trifolium repens) reveals a redundancy and diversity of gene function.

    PubMed

    Islam, Afsana; Leung, Susanna; Burgess, Elisabeth P J; Laing, William A; Richardson, Kim A; Hofmann, Rainer W; Dijkwel, Paul P; McManus, Michael T

    2015-12-01

    The transcriptional regulation of four phylogenetically distinct members of a family of Kunitz proteinase inhibitor (KPI) genes isolated from white clover (Trifolium repens; designated Tr-KPI1, Tr-KPI2, Tr-KPI4 and Tr-KPI5) has been investigated to determine their wider functional role. The four genes displayed differential transcription during seed germination, and in different tissues of the mature plant, and transcription was also ontogenetically regulated. Heterologous over-expression of Tr-KPI1, Tr-KPI2, Tr-KPI4 and Tr-KPI5 in Nicotiana tabacum retarded larval growth of the herbivore Spodoptera litura, and an increase in the transcription of the pathogenesis-related genes PR1 and PR4 was observed in the Tr-KPI1 and Tr-KPI4 over-expressing lines. RNA interference (RNAi) knock-down lines in white clover displayed significantly altered vegetative growth phenotypes with inhibition of shoot growth and a stimulation of root growth, while knock-down of Tr-KPI1, Tr-KPI2 and Tr-KPI5 transcript abundance also retarded larval growth of S. litura. Examination of these RNAi lines revealed constitutive stress-associated phenotypes as well as altered transcription of cellular signalling genes. These results reveal a functional redundancy across members of the KPI gene family. Further, the regulation of transcription of at least one member of the family, Tr-KPI2, may occupy a central role in the maintenance of a cellular homeostasis. PMID:26377591

  1. Knockdown of SKN-1 and the Wnt effector TCF/POP-1 reveals differences in endomesoderm specification in C. briggsae as compared with C. elegans

    PubMed Central

    Lin, Katy Tan-Hui; Broitman-Maduro, Gina; Hung, Wendy W. K.; Cervantes, Serena; Maduro, Morris F.

    2009-01-01

    In the nematode, C. elegans, the bZIP/homeodomain transcription factor SKN-1 and the Wnt effector TCF/POP-1 are central to the maternal specification of the endomesoderm prior to gastrulation. The 8-cell stage blastomere MS is primarily a mesodermal precursor, giving rise to cells of the pharynx and body muscle among others, while its sister E clonally generates the entire endoderm (gut). In C. elegans, loss of SKN-1 results in the absence of MS-derived tissues all of the time, and loss of gut most of the time, while loss of POP-1 results in a mis-specification of MS as an E-like cell, resulting in ectopic gut. We show that in C. briggsae, RNAi of skn-1 results in a stronger E defect but no apparent MS defect, while RNAi of pop-1 results in loss of gut and an apparent E to MS transformation, the opposite of the pop-1 knockdown phenotype seen in C. elegans. The difference in pop-1(−) phenotypes correlates with changes in how the endogenous endoderm-specifying end genes are regulated by POP-1 in the two species. Our results suggest that integration of Wnt-dependent and Wnt-independent cell fate specification pathways within the Caenorhabditis genus can occur in different ways. PMID:18977344

  2. Amastin Knockdown in Leishmania braziliensis Affects Parasite-Macrophage Interaction and Results in Impaired Viability of Intracellular Amastigotes.

    PubMed

    de Paiva, Rita Marcia Cardoso; Grazielle-Silva, Viviane; Cardoso, Mariana Santos; Nakagaki, Brenda Naemi; Mendonça-Neto, Rondon Pessoa; Canavaci, Adriana Monte Cassiano; Souza Melo, Normanda; Martinelli, Patrícia Massara; Fernandes, Ana Paula; daRocha, Wanderson Duarte; Teixeira, Santuza M R

    2015-12-01

    Leishmaniasis, a human parasitic disease with manifestations ranging from cutaneous ulcerations to fatal visceral infection, is caused by several Leishmania species. These protozoan parasites replicate as extracellular, flagellated promastigotes in the gut of a sandfly vector and as amastigotes inside the parasitophorous vacuole of vertebrate host macrophages. Amastins are surface glycoproteins encoded by large gene families present in the genomes of several trypanosomatids and highly expressed in the intracellular amastigote stages of Trypanosoma cruzi and Leishmania spp. Here, we showed that the genome of L. braziliensis contains 52 amastin genes belonging to all four previously described amastin subfamilies and that the expression of members of all subfamilies is upregulated in L. braziliensis amastigotes. Although primary sequence alignments showed no homology to any known protein sequence, homology searches based on secondary structure predictions indicate that amastins are related to claudins, a group of proteins that are components of eukaryotic tight junction complexes. By knocking-down the expression of δ-amastins in L. braziliensis, their essential role during infection became evident. δ-amastin knockdown parasites showed impaired growth after in vitro infection of mouse macrophages and completely failed to produce infection when inoculated in BALB/c mice, an attenuated phenotype that was reverted by the re-expression of an RNAi-resistant amastin gene. Further highlighting their essential role in host-parasite interactions, electron microscopy analyses of macrophages infected with amastin knockdown parasites showed significant alterations in the tight contact that is normally observed between the surface of wild type amastigotes and the membrane of the parasitophorous vacuole. PMID:26641088

  3. Amastin Knockdown in Leishmania braziliensis Affects Parasite-Macrophage Interaction and Results in Impaired Viability of Intracellular Amastigotes

    PubMed Central

    Nakagaki, Brenda Naemi; Mendonça-Neto, Rondon Pessoa; Canavaci, Adriana Monte Cassiano; Souza Melo, Normanda; Martinelli, Patrícia Massara; Fernandes, Ana Paula; daRocha, Wanderson Duarte; Teixeira, Santuza M. R.

    2015-01-01

    Leishmaniasis, a human parasitic disease with manifestations ranging from cutaneous ulcerations to fatal visceral infection, is caused by several Leishmania species. These protozoan parasites replicate as extracellular, flagellated promastigotes in the gut of a sandfly vector and as amastigotes inside the parasitophorous vacuole of vertebrate host macrophages. Amastins are surface glycoproteins encoded by large gene families present in the genomes of several trypanosomatids and highly expressed in the intracellular amastigote stages of Trypanosoma cruzi and Leishmania spp. Here, we showed that the genome of L. braziliensis contains 52 amastin genes belonging to all four previously described amastin subfamilies and that the expression of members of all subfamilies is upregulated in L. braziliensis amastigotes. Although primary sequence alignments showed no homology to any known protein sequence, homology searches based on secondary structure predictions indicate that amastins are related to claudins, a group of proteins that are components of eukaryotic tight junction complexes. By knocking-down the expression of δ-amastins in L. braziliensis, their essential role during infection became evident. δ-amastin knockdown parasites showed impaired growth after in vitro infection of mouse macrophages and completely failed to produce infection when inoculated in BALB/c mice, an attenuated phenotype that was reverted by the re-expression of an RNAi-resistant amastin gene. Further highlighting their essential role in host-parasite interactions, electron microscopy analyses of macrophages infected with amastin knockdown parasites showed significant alterations in the tight contact that is normally observed between the surface of wild type amastigotes and the membrane of the parasitophorous vacuole. PMID:26641088

  4. Discovery of a Dicer-Independent, Cell-Type Dependent Alternate Targeting Sequence Generator: Implications in Gene Silencing & Pooled RNAi Screens

    PubMed Central

    Bhinder, Bhavneet; Shum, David; Li, Mu; Ibáñez, Glorymar; Vlassov, Alexander V.; Magdaleno, Susan; Djaballah, Hakim

    2014-01-01

    There is an acceptance that plasmid-based delivery of interfering RNA always generates the intended targeting sequences in cells, making it as specific as its synthetic counterpart. However, recent studies have reported on cellular inefficiencies of the former, especially in light of emerging gene discordance at inter-screen level and across formats. Focusing primarily on the TRC plasmid-based shRNA hairpins, we reasoned that alleged specificities were perhaps compromised due to altered processing; resulting in a multitude of random interfering sequences. For this purpose, we opted to study the processing of hairpin TRCN#40273 targeting CTTN; which showed activity in a miRNA-21 gain-of-function shRNA screen, but inactive when used as an siRNA duplex. Using a previously described walk-through method, we identified 36 theoretical cleavage variants resulting in 78 potential siRNA duplexes targeting 53 genes. We synthesized and tested all of them. Surprisingly, six duplexes targeting ASH1L, DROSHA, GNG7, PRKCH, THEM4, and WDR92 scored as active. QRT-PCR analysis on hairpin transduced reporter cells confirmed knockdown of all six genes, besides CTTN; revealing a surprising 7 gene-signature perturbation by this one single hairpin. We expanded our qRT-PCR studies to 26 additional cell lines and observed unique knockdown profiles associated with each cell line tested; even for those lacking functional DICER1 gene suggesting no obvious dependence on dicer for shRNA hairpin processing; contrary to published models. Taken together, we report on a novel dicer independent, cell-type dependent mechanism for non-specific RNAi gene silencing we coin Alternate Targeting Sequence Generator (ATSG). In summary, ATSG adds another dimension to the already complex interpretation of RNAi screening data, and provides for the first time strong evidence in support of arrayed screening, and questions the scientific merits of performing pooled RNAi screens, where deconvolution of up to genome

  5. RNA therapeutics: RNAi and antisense mechanisms and clinical applications

    PubMed Central

    Chery, Jessica; Näär, Anders

    2016-01-01

    RNA therapeutics refers to the use of oligonucleotides to target primarily ribonucleic acids (RNA) for therapeutic efforts or in research studies to elucidate functions of genes. Oligonucleotides are distinct from other pharmacological modalities, such as small molecules and antibodies that target mainly proteins, due to their mechanisms of action and chemical properties. Nucleic acids come in two forms: deoxyribonucleic acids (DNA) and ribonucleic acids (RNA). Although DNA is more stable, RNA offers more structural variety ranging from messenger RNA (mRNA) that codes for protein to non-coding RNAs, microRNA (miRNA), transfer RNA (tRNA), short interfering RNAs (siRNAs), ribosomal RNA (rRNA), and long-noncoding RNAs (lncRNAs). As our understanding of the wide variety of RNAs deepens, researchers have sought to target RNA since >80% of the genome is estimated to be transcribed. These transcripts include non-coding RNAs such as miRNAs and siRNAs that function in gene regulation by playing key roles in the transfer of genetic information from DNA to protein, the final product of the central dogma in biology1. Currently there are two main approaches used to target RNA: double stranded RNA-mediated interference (RNAi) and antisense oligonucleotides (ASO). Both approaches are currently in clinical trials for targeting of RNAs involved in various diseases, such as cancer and neurodegeneration. In fact, ASOs targeting spinal muscular atrophy and amyotrophic lateral sclerosis have shown positive results in clinical trials2. Advantages of ASOs include higher affinity due to the development of chemical modifications that increase affinity, selectivity while decreasing toxicity due to off-target effects. This review will highlight the major therapeutic approaches of RNA medicine currently being applied with a focus on RNAi and ASOs. PMID:27570789

  6. RNAi in Arthropods: Insight into the Machinery and Applications for Understanding the Pathogen-Vector Interface

    PubMed Central

    Barnard, Annette-Christi; Nijhof, Ard M.; Fick, Wilma; Stutzer, Christian; Maritz-Olivier, Christine

    2012-01-01

    The availability of genome sequencing data in combination with knowledge of expressed genes via transcriptome and proteome data has greatly advanced our understanding of arthropod vectors of disease. Not only have we gained insight into vector biology, but also into their respective vector-pathogen interactions. By combining the strengths of postgenomic databases and reverse genetic approaches such as RNAi, the numbers of available drug and vaccine targets, as well as number of transgenes for subsequent transgenic or paratransgenic approaches, have expanded. These are now paving the way for in-field control strategies of vectors and their pathogens. Basic scientific questions, such as understanding the basic components of the vector RNAi machinery, is vital, as this allows for the transfer of basic RNAi machinery components into RNAi-deficient vectors, thereby expanding the genetic toolbox of these RNAi-deficient vectors and pathogens. In this review, we focus on the current knowledge of arthropod vector RNAi machinery and the impact of RNAi on understanding vector biology and vector-pathogen interactions for which vector genomic data is available on VectorBase. PMID:24705082

  7. ATARiS: Computational quantification of gene suppression phenotypes from multisample RNAi screens

    PubMed Central

    Shao, Diane D.; Tsherniak, Aviad; Gopal, Shuba; Weir, Barbara A.; Tamayo, Pablo; Stransky, Nicolas; Schumacher, Steven E.; Zack, Travis I.; Beroukhim, Rameen; Garraway, Levi A.; Margolin, Adam A.; Root, David E.; Hahn, William C.; Mesirov, Jill P.

    2013-01-01

    Genome-scale RNAi libraries enable the systematic interrogation of gene function. However, the interpretation of RNAi screens is complicated by the observation that RNAi reagents designed to suppress the mRNA transcripts of the same gene often produce a spectrum of phenotypic outcomes due to differential on-target gene suppression or perturbation of off-target transcripts. Here we present a computational method, Analytic Technique for Assessment of RNAi by Similarity (ATARiS), that takes advantage of patterns in RNAi data across multiple samples in order to enrich for RNAi reagents whose phenotypic effects relate to suppression of their intended targets. By summarizing only such reagent effects for each gene, ATARiS produces quantitative, gene-level phenotype values, which provide an intuitive measure of the effect of gene suppression in each sample. This method is robust for data sets that contain as few as 10 samples and can be used to analyze screens of any number of targeted genes. We used this analytic approach to interrogate RNAi data derived from screening more than 100 human cancer cell lines and identified HNF1B as a transforming oncogene required for the survival of cancer cells that harbor HNF1B amplifications. ATARiS is publicly available at http://broadinstitute.org/ataris. PMID:23269662

  8. Incomplete and Inaccurate Vocal Imitation after Knockdown of FoxP2 in Songbird Basal Ganglia Nucleus Area X

    PubMed Central

    Haesler, Sebastian; Rochefort, Christelle; Georgi, Benjamin; Licznerski, Pawel; Osten, Pavel; Scharff, Constance

    2007-01-01

    The gene encoding the forkhead box transcription factor, FOXP2, is essential for developing the full articulatory power of human language. Mutations of FOXP2 cause developmental verbal dyspraxia (DVD), a speech and language disorder that compromises the fluent production of words and the correct use and comprehension of grammar. FOXP2 patients have structural and functional abnormalities in the striatum of the basal ganglia, which also express high levels of FOXP2. Since human speech and learned vocalizations in songbirds bear behavioral and neural parallels, songbirds provide a genuine model for investigating the basic principles of speech and its pathologies. In zebra finch Area X, a basal ganglia structure necessary for song learning, FoxP2 expression increases during the time when song learning occurs. Here, we used lentivirus-mediated RNA interference (RNAi) to reduce FoxP2 levels in Area X during song development. Knockdown of FoxP2 resulted in an incomplete and inaccurate imitation of tutor song. Inaccurate vocal imitation was already evident early during song ontogeny and persisted into adulthood. The acoustic structure and the duration of adult song syllables were abnormally variable, similar to word production in children with DVD. Our findings provide the first example of a functional gene analysis in songbirds and suggest that normal auditory-guided vocal motor learning requires FoxP2. PMID:18052609

  9. Knockdown of HNRNPA1 inhibits lung adenocarcinoma cell proliferation through cell cycle arrest at G0/G1 phase.

    PubMed

    Liu, Xianxun; Zhou, Yan; Lou, Yuqing; Zhong, Hua

    2016-02-01

    Heterogeneous nuclear ribonucleoprotein A1 (HNRNPA1), a member of heterogeneous nuclear ribonucleoprotein family in actively growing mammalian cells, is involved in a variety of RNA-related processes. HNRNPA1 can enhance the degradation of inhibitory subunit of nuclear factor κ B alpha (IκBα) and lengthen the telomeres. Recently, it is reported that HNRNPA1 is aberrantly expressed in varied tumors. In this study we found HNRNPA1 protein overexpressed in lung cancer tissues. To explore the exact role of HNRNPA1 in lung cancers, we carried out a loss of function analysis of HNRNPA1 in A549 lung cancer cells by RNA interference (RNAi). The results demonstrated that knockdown of HNRNPA1 inhibited cell viability and colony formation of lung cancer cells and arrested cell cycle in G0/G1 phase. Our study suggested that HNRNPA1 might play an important role in lung adenocarcinoma cells and provided a foundation for further study into the potential of HNRNPA1 for lung cancer therapy. PMID:26581508

  10. Potential and development of inhaled RNAi therapeutics for the treatment of pulmonary tuberculosis.

    PubMed

    Man, Dede K W; Chow, Michael Y T; Casettari, Luca; Gonzalez-Juarrero, Mercedes; Lam, Jenny K W

    2016-07-01

    Tuberculosis (TB), caused by the infection of Mycobacterium tuberculosis (Mtb), continues to pose a serious threat to public health, and the situation is worsening with the rapid emergence of multidrug resistant (MDR) TB. Current TB regimens require long duration of treatment, and their toxic side effects often lead to poor adherence and low success rates. There is an urgent need for shorter and more effective treatment for TB. In recent years, RNA interference (RNAi) has become a powerful tool for studying gene function by silencing the target genes. The survival of Mtb in host macrophages involves the attenuation of the antimicrobial responses mounted by the host cells. RNAi technology has helped to improve our understanding of how these bacilli interferes with the bactericidal effect and host immunity during TB infection. It has been suggested that the host-directed intervention by modulation of host pathways can be employed as a novel and effective therapy against TB. This therapeutic approach could be achieved by RNAi, which holds enormous potential beyond a laboratory to the clinic. RNAi therapy targeting TB is being investigated for enhancing host antibacterial capacity or improving drug efficacy on drug resistance strains while minimizing the associated adverse effects. One of the key challenges of RNAi therapeutics arises from the delivery of the RNAi molecules into the target cells, and inhalation could serve as a direct administration route for the treatment of pulmonary TB in a non-invasive manner. However, there are still major obstacles that need to be overcome. This review focuses on the RNAi candidates that are currently explored for the treatment of TB and discusses the major barriers of pulmonary RNAi delivery. From this, we hope to stimulate further studies of local RNAi therapeutics for pulmonary TB treatment. PMID:27108702

  11. Self-Assembled Cell Microarray (SAMcell) for High-Throughput RNAi Screening.

    PubMed

    Zhang, Hanshuo; Li, Juan

    2016-01-01

    RNAi has now become a valuable research tool for cell-based high-throughput screening. However, traditional RNAi high-throughput methods are based on multi-well plates, relying on expensive instruments and complicated operations. In this chapter, we describe a method termed self-assembled cell microarray (SAMcell), which integrates micro-fabrication, reverse transfection, and RNAi technologies and allows for cell behavior investigations to be performed directly on the cell chip. This method has been successfully employed to perform large-scale functional screening assays to identify gene modulators of cell migration, cell proliferation, and cellular apoptosis. PMID:27581287

  12. TOPICAL REVIEW: RNAi nanomedicines: challenges and opportunities within the immune system

    NASA Astrophysics Data System (ADS)

    Weinstein, Shiri; Peer, Dan

    2010-06-01

    RNAi, as a novel therapeutic modality, has an enormous potential to bring the era of personalized medicine one step further from notion into reality. However, delivery of RNAi effector molecules into their target tissues and cells remain extremely challenging. Major attempts have been made in recent years to develop sophisticated nanocarriers that could overcome these hurdles. This review will present the recent progress with the challenges and opportunities in this emerging field, focusing mostly on the in vivo applications with special emphasis on the strategies for RNAi delivery into immune cells.

  13. Adseverin knockdown inhibits osteoclastogenesis in RAW264.7 cells

    PubMed Central

    QI, WENTING; GAO, YAN; TIAN, JUN; JIANG, HONGWEI

    2014-01-01

    Osteoclastogenesis is a complex process that is highly dependent on the dynamic regulation of the actin cytoskeleton. Adseverin (Ads), a member of the gelsolin superfamily of actin-binding proteins, regulates actin remodeling by severing and capping actin filaments. The objective of the present study was to characterize the role of Ads during osteoclastogenesis by assessing Ads expression and using a knockdown strategy. Immunoblot analyses were used to examine Ads expression during osteoclastogenesis. A stable Ads knockdown macrophage cell line was generated using a retroviral shRNA construct. Osteoclast differentiation was morphologically examined via cell staining with osteoclast specific markers and light microscopy. The results showed that Ads expression was significantly increased in response to receptor activator of nuclear factor-κB ligand during osteoclastogenesis, and Ads was highly expressed in mature osteoclasts. Ads-knockdown macrophages showed major osteoclastogenesis defects, most likely caused by a pre-osteoclast fusion defect. These results indicate that Ads deficiency in monocytes inhibits osteoclastogenesis. Thus, in future studies it could be noteworthy to investigate the function of Ads in bone marrow monocytes during osteoclastogenesis. PMID:25339151

  14. Neuromedin U receptor 2 knockdown in the paraventricular nucleus modifies behavioral responses to obesogenic high-fat food and leads to increased body weight.

    PubMed

    Benzon, C R; Johnson, S B; McCue, D L; Li, D; Green, T A; Hommel, J D

    2014-01-31

    Neuromedin U (NMU) is a highly conserved neuropeptide which regulates food intake and body weight. Transgenic mice lacking NMU are hyperphagic and obese, making NMU a novel target for understanding and treating obesity. Neuromedin U receptor 2 (NMUR2) is a high-affinity receptor for NMU found in discrete regions of the central nervous system, in particular the paraventricular nucleus of the hypothalamus (PVN), where it may be responsible for mediating the anorectic effects of NMU. We hypothesized that selective knock down of NMUR2 in the PVN of rats would increase their sensitivity to the reinforcing properties of food resulting in increased intake and preference for high-fat obesogenic food. To this end, we used viral-mediated RNAi to selectively knock down NMUR2 gene expression in the PVN. In rats fed a standard chow, NMUR2 knockdown produced no significant effect on food intake or body weight. However, when the same rats were fed a high-fat diet (45% fat), they consumed significantly more food, gained more body weight, and had increased feed efficiency relative to controls. Furthermore, NMUR2 knockdown rats demonstrated significantly greater binge-type food consumption of the high-fat diet and showed a greater preference for higher-fat food. These results demonstrate that NMUR2 signaling in the PVN regulates consumption and preference for high-fat foods without disrupting feeding behavior associated with non-obesogenic standard chow. PMID:24269937

  15. Knock-down of argonaute 2 (AGO2) induces apoptosis in myeloid leukaemia cells and inhibits siRNA-mediated silencing of transfected oncogenes in HEK-293 cells.

    PubMed

    Naoghare, Pravin K; Tak, Yu Kyung; Kim, Min Jung; Han, Eunyoung; Song, Joon Myong

    2011-10-01

    Understanding the role of oncomirs allows new insights into the development of modern therapeutic approaches for the repression of multiple oncomirs in cancer cells. At present, no suitable approach is available to repress the development of multiple oncomirs in cancer cells. Herein, we report that argonaute 2 (AGO2) could be a unique molecule to regulate the development of multiple oncomirs in cancer cells. Knock-down of AGO2 by custom-made AGO2 siRNA resulted in the induction of apoptosis in myeloid leukaemia cells (HL-60). Further investigations revealed that knock-down of AGO2 by custom-made AGO2 siRNA in HEK-293 cells resulted in silencing of the expression of target genes vascular endothelial growth factor A and histone deacetylase 2, which are known to be involved in the development of myeloid leukaemia. From these results, it can be predicted that AGO2 could regulate siRNA-mediated RNAi pathways in cancer cells. Furthermore, we investigated the possible implication of AGO2 in drug-induced apoptosis. Investigations revealed that treatment with the newly synthesized drug analogue SH-03[{(7S,7aR,13aS)-9,10-dimethoxy-3,3-dimethyl-7,7a,13,13atetrahydro-3H-chromeno[3,4-b]pyrano[2,3-h]chromen-7-ol}] could induce AGO2-mediated apoptosis in myeloid leukaemia cells via intrinsic apoptotic pathways independent of Dicer. PMID:21535412

  16. Synthetic heterochromatin bypasses RNAi and centromeric repeats to establish functional centromeres.

    PubMed

    Kagansky, Alexander; Folco, Hernan Diego; Almeida, Ricardo; Pidoux, Alison L; Boukaba, Abdelhalim; Simmer, Femke; Urano, Takeshi; Hamilton, Georgina L; Allshire, Robin C

    2009-06-26

    In the central domain of fission yeast centromeres, the kinetochore is assembled on CENP-A(Cnp1) nucleosomes. Normally, small interfering RNAs generated from flanking outer repeat transcripts direct histone H3 lysine 9 methyltransferase Clr4 to homologous loci to form heterochromatin. Outer repeats, RNA interference (RNAi), and centromeric heterochromatin are required to establish CENP-A(Cnp1) chromatin. We demonstrated that tethering Clr4 via DNA-binding sites at euchromatic loci induces heterochromatin assembly, with or without active RNAi. This synthetic heterochromatin completely substitutes for outer repeats on plasmid-based minichromosomes, promoting de novo CENP-A(Cnp1) and kinetochore assembly, to allow their mitotic segregation, even with RNAi inactive. Thus, the role of outer repeats in centromere establishment is simply the provision of RNAi substrates to direct heterochromatin formation; H3K9 methylation-dependent heterochromatin is alone sufficient to form functional centromeres. PMID:19556509

  17. Engineered mammalian RNAi can elicit antiviral protection that negates the requirement for the interferon response

    PubMed Central

    Bouhaddou, Mehdi; Sachs, David; tenOever, Benjamin Robert

    2015-01-01

    SUMMARY While the intrinsic antiviral cell defenses of many kingdoms utilize pathogen-specific small RNAs, the antiviral response of chordates is primarily protein-based and not uniquely tailored to the incoming microbe. In an effort to explain this evolutionary bifurcation, we determined whether antiviral RNA interference (RNAi) was sufficient to replace the protein-based type I interferon (IFN-I) system of mammals. To this end, we recreated an RNAi-like response in mammals and determined its effectiveness to combat influenza A virus in vivo in the presence and absence of the canonical IFN-I system. Mammalian antiviral RNAi, elicited by either host- or virus-derived small RNAs, effectively attenuated virus and prevented disease independently of the innate immune response. These data find that chordates could have utilized RNAi as their primary antiviral cell defense and suggest that the IFN-I system emerged as a result of natural selection imposed by ancient pathogens. PMID:26549455

  18. Soaking RNAi in Bombyx mori BmN4-SID1 cells arrests cell cycle progression.

    PubMed

    Mon, Hiroaki; Li, Zhiqing; Kobayashi, Isao; Tomita, Shuichiro; Lee, JaeMan; Sezutsu, Hideki; Tamura, Toshiki; Kusakabe, Takahiro

    2013-01-01

    RNA interference (RNAi) is an evolutionarily conserved mechanism for sequence-specific gene silencing. Previously, the BmN4-SID1 cell expressing Caenorhabditis ele gans SID-1 was established, in which soaking RNAi could induce effective gene silencing. To establish its utility, 6 cell cycle progression related cDNAs, CDK1, MYC, MYB, RNRS, CDT1, and GEMININ, were isolated from the silkworm, Bombyx mori L. (Lepidoptera: Bombycidae), and their expressions were further silenced by soaking RNAi in the BmN4-SID1 cells. The cell cycle progression analysis using flow cytometer demonstrated that the small amount of double stranded RNA was enough to arrest cell cycle progression at the specific cell phases. These data suggest that RNAi in the BmN4-SID1 cells can be used as a powerful tool for loss-of-function analysis of B. mori genes. PMID:24773378

  19. Branch-PCR Constructed Stable shRNA Transcription Nanoparticles Have Long-Lasting RNAi Effect.

    PubMed

    Liu, Jianbing; Wang, Runyu; Ma, Dejun; Li, Yanyan; Wei, Chao; Xi, Zhen

    2016-06-01

    RNA interference (RNAi) is a cellular process for gene silencing. Because of poor serum stability, transferring dsRNA directly into the target cells is a challenge. We report a facile and universal strategy to construct short hairpin RNA (shRNA) transcription nanoparticles with multiple shRNA transcription templates by PCR with flexible branched primers (branch-PCR). Compared with conventional linear shRNA transcription templates, these shRNA transcription nanoparticles show excellent stability against digestion by exonuclease III. Importantly, we found that our highly stable shRNA transcription nanoparticles can also be transcribed and thus induce efficient and long-lasting RNAi with picomolar activity in living mammalian cells. These chemically well-defined branch-PCR-generated stable shRNA transcription nanoparticles might facilitate RNAi delivery with a long-lasting RNAi effects. PMID:26972444

  20. RNAi screens in mice identify physiological regulators of oncogenic growth

    PubMed Central

    Beronja, Slobodan; Janki, Peter; Heller, Evan; Lien, Wen-Hui; Keyes, Brice; Oshimori, Naoki; Fuchs, Elaine

    2013-01-01

    Summary Tissue growth is the multifaceted outcome of a cell’s intrinsic capabilities and its interactions with the surrounding environment. Decoding these complexities is essential for understanding human development and tumorigenesis. Here, we tackle this problem by carrying out the first genome-wide RNAi-mediated screens in mice. Focusing on skin development and oncogenic (HrasG12V-induced) hyperplasia, our screens uncover novel as well as anticipated regulators of embryonic epidermal growth. Among top oncogenic screen hits are Mllt6 and the Wnt effector β-catenin; they maintain HrasG12V-dependent hyperproliferation. We also expose β-catenin as an unanticipated antagonist of normal epidermal growth, functioning through Wnt-independent intercellular adhesion. Finally, we document physiological relevance to mouse and human cancers, thereby establishing the feasibility of in vivo mammalian genome-wide investigations to dissect tissue development and tumorigenesis. By documenting some oncogenic growth regulators, we pave the way for future investigations of other hits and raise promise for unearthing new targets for cancer therapies. PMID:23945586

  1. Vitellogenin RNAi halts ovarian growth and diverts reproductive proteins and lipids in young grasshoppers.

    PubMed

    Tokar, Derek R; Veleta, Katherine A; Canzano, Joseph; Hahn, Daniel A; Hatle, John D

    2014-11-01

    Reduced reproduction extends lifespan of females in many animals. To test the effects of reproduction on storage of macronutrients, we block reproductive output in the lubber grasshopper by injecting RNAi against the precursor to egg-yolk protein, vitellogenin, in early adulthood. Controls were injected with either buffer or RNAi against the major storage protein in the hemolymph, hexamerin-90. Vitellogenin RNAi greatly reduced both levels of mRNA for vitellogenin and ovarian growth, in comparison to both controls. Fat body mass was increased upon vitellogenin RNAi, but concentrations of the three hexameric storage proteins from the hemolymph were not. Surprisingly, hemolymph vitellogenin levels were increased upon vitellogenin RNAi. Total reproductive protein (hemolymph vitellogenin plus ovarian vitellin) was unchanged by vitellogenin RNAi, as reproductive protein was diverted to the hemolymph. Similarly, the increased lipid storage upon vitellogenin RNAi was largely attributable to the reduction in lipid in the ovary, due to decreased ovarian growth. A BLAST search revealed that the 515 bp sequence of vitellogenin used for RNAi had three 11 bp regions identical to the vitellogenin receptor of the cockroach Leucophaea maderae. This suggests that our treatment, in addition to reducing levels of vitellogenin transcript, may have also blocked transport of vitellogenin from the hemolymph to the ovary. This would be consistent with halted ovarian growth simultaneous with high levels of vitellogenin in the hemolymph. Nonetheless, the accumulation of vitellogenin, instead of hexameric storage proteins, is inconsistent with a simple model of the trade-off between reproduction and storage. This was observed in young females; future studies will address whether investment of proteins may shift to the soma as individuals age. Overall, our results suggest that blockage of reproduction in young grasshoppers redirects lipids to storage and reproductive proteins to the hemolymph

  2. The Insect Ecdysone Receptor is a Good Potential Target for RNAi-based Pest Control

    PubMed Central

    Yu, Rong; Xu, Xinping; Liang, Yongkang; Tian, Honggang; Pan, Zhanqing; Jin, Shouheng; Wang, Na; Zhang, Wenqing

    2014-01-01

    RNA interference (RNAi) has great potential for use in insect pest control. However, some significant challenges must be overcome before RNAi-based pest control can become a reality. One challenge is the proper selection of a good target gene for RNAi. Here, we report that the insect ecdysone receptor (EcR) is a good potential target for RNAi-based pest control in the brown planthopper Nilaparvata lugens, a serious insect pest of rice plants. We demonstrated that the use of a 360 bp fragment (NlEcR-c) that is common between NlEcR-A and NlEcR-B for feeding RNAi experiments significantly decreased the relative mRNA expression levels of NlEcR compared with those in the dsGFP control. Feeding RNAi also resulted in a significant reduction in the number of offspring per pair of N. lugens. Consequently, a transgenic rice line expressing NlEcR dsRNA was constructed by Agrobacterium- mediated transformation. The results of qRT-PCR showed that the total copy number of the target gene in all transgenic rice lines was 2. Northern blot analysis showed that the small RNA of the hairpin dsNlEcR-c was successfully expressed in the transgenic rice lines. After newly hatched nymphs of N. lugens fed on the transgenic rice lines, effective RNAi was observed. The NlEcR expression levels in all lines examined were decreased significantly compared with the control. In all lines, the survival rate of the nymphs was nearly 90%, and the average number of offspring per pair in the treated groups was significantly less than that observed in the control, with a decrease of 44.18-66.27%. These findings support an RNAi-based pest control strategy and are also important for the management of rice insect pests. PMID:25516715

  3. The insect ecdysone receptor is a good potential target for RNAi-based pest control.

    PubMed

    Yu, Rong; Xu, Xinping; Liang, Yongkang; Tian, Honggang; Pan, Zhanqing; Jin, Shouheng; Wang, Na; Zhang, Wenqing

    2014-01-01

    RNA interference (RNAi) has great potential for use in insect pest control. However, some significant challenges must be overcome before RNAi-based pest control can become a reality. One challenge is the proper selection of a good target gene for RNAi. Here, we report that the insect ecdysone receptor (EcR) is a good potential target for RNAi-based pest control in the brown planthopper Nilaparvata lugens, a serious insect pest of rice plants. We demonstrated that the use of a 360 bp fragment (NlEcR-c) that is common between NlEcR-A and NlEcR-B for feeding RNAi experiments significantly decreased the relative mRNA expression levels of NlEcR compared with those in the dsGFP control. Feeding RNAi also resulted in a significant reduction in the number of offspring per pair of N. lugens. Consequently, a transgenic rice line expressing NlEcR dsRNA was constructed by Agrobacterium- mediated transformation. The results of qRT-PCR showed that the total copy number of the target gene in all transgenic rice lines was 2. Northern blot analysis showed that the small RNA of the hairpin dsNlEcR-c was successfully expressed in the transgenic rice lines. After newly hatched nymphs of N. lugens fed on the transgenic rice lines, effective RNAi was observed. The NlEcR expression levels in all lines examined were decreased significantly compared with the control. In all lines, the survival rate of the nymphs was nearly 90%, and the average number of offspring per pair in the treated groups was significantly less than that observed in the control, with a decrease of 44.18-66.27%. These findings support an RNAi-based pest control strategy and are also important for the management of rice insect pests. PMID:25516715

  4. Genome-Wide RNAi Screens in C. elegans to Identify Genes Influencing Lifespan and Innate Immunity.

    PubMed

    Sinha, Amit; Rae, Robbie

    2016-01-01

    RNA interference is a rapid, inexpensive, and highly effective tool used to inhibit gene function. In C. elegans, whole genome screens have been used to identify genes involved with numerous traits including aging and innate immunity. RNAi in C. elegans can be carried out via feeding, soaking, or injection. Here we outline protocols used to maintain, grow, and carry out RNAi via feeding in C. elegans and determine whether the inhibited genes are essential for lifespan or innate immunity. PMID:27581293

  5. Online Phenotype Discovery in High-Content RNAi Screens using Gap Statistics

    NASA Astrophysics Data System (ADS)

    Yin, Zheng; Zhou, Xiaobo; Bakal, Chris; Li, Fuhai; Sun, Youxian; Perrimon, Norbert; Wong, Stephen T. C.

    2007-11-01

    Discovering and identifying novel phenotypes from images inputting online is a major challenge in high-content RNA interference (RNAi) screens. Discovered phenotypes should be visually distinct from existing ones and make biological sense. An online phenotype discovery method featuring adaptive phenotype modeling and iterative cluster merging using gap statistics is proposed. The method works well on discovering new phenotypes adaptively when applied to both of synthetic data sets and RNAi high content screen (HCS) images with ground truth labels.

  6. siRNA knockdown validation 101: Incorporating negative controls in antibody research

    PubMed Central

    Olds, Will; Li, Jason

    2016-01-01

    More antibody validation protocols would identify non-specific reagents – a major source of irreproducible research – with the inclusion of negative controls. This article presents an overview of one such method: siRNA knockdown. The authors outline a general protocol, the knockdown mechanism, and tips for evaluating knockdown experiments. PMID:26998240

  7. The status of RNAi-based transgenic research in plant nematology

    PubMed Central

    Dutta, Tushar K.; Banakar, Prakash; Rao, Uma

    2015-01-01

    With the understanding of nematode-plant interactions at the molecular level, new avenues for engineering resistance have opened up, with RNA interference being one of them. Induction of RNAi by delivering double-stranded RNA (dsRNA) has been very successful in the model non-parasitic nematode, Caenorhabditis elegans, while in plant nematodes, dsRNA delivery has been accomplished by soaking nematodes with dsRNA solution mixed with synthetic neurostimulants. The success of in vitro RNAi of target genes has inspired the use of in planta delivery of dsRNA to feeding nematodes. The most convincing success of host-delivered RNAi has been achieved against root-knot nematodes. Plant-mediated RNAi has been shown to lead to the specific down-regulation of target genes in invading nematodes, which had a profound effect on nematode development. RNAi-based transgenics are advantageous as they do not produce any functional foreign proteins and target organisms in a sequence-specific manner. Although the development of RNAi-based transgenics against plant nematodes is still in the preliminary stage, they offer novel management strategy for the future. PMID:25628609

  8. Lipid Nanoparticles as Carriers for RNAi against Viral Infections: Current Status and Future Perspectives

    PubMed Central

    Rodríguez-Gascón, Alicia; Solinís, María Ángeles; del Pozo-Rodríguez, Ana

    2014-01-01

    The efforts made to develop RNAi-based therapies have led to productive research in the field of infections in humans, such as hepatitis C virus (HCV), hepatitis B virus (HBV), human immunodeficiency virus (HIV), human cytomegalovirus (HCMV), herpetic keratitis, human papillomavirus, or influenza virus. Naked RNAi molecules are rapidly digested by nucleases in the serum, and due to their negative surface charge, entry into the cell cytoplasm is also hampered, which makes necessary the use of delivery systems to exploit the full potential of RNAi therapeutics. Lipid nanoparticles (LNP) represent one of the most widely used delivery systems for in vivo application of RNAi due to their relative safety and simplicity of production, joint with the enhanced payload and protection of encapsulated RNAs. Moreover, LNP may be functionalized to reach target cells, and they may be used to combine RNAi molecules with conventional drug substances to reduce resistance or improve efficiency. This review features the current application of LNP in RNAi mediated therapy against viral infections and aims to explore possible future lines of action in this field. PMID:25184135

  9. RNAi revised - target mRNA-dependent enhancement of gene silencing

    PubMed Central

    Dornseifer, Simon; Willkomm, Sarah; Far, Rosel Kretschmer-Kazemi; Liebschwager, Janine; Beltsiou, Foteini; Frank, Kirsten; Laufer, Sandra D.; Martinetz, Thomas; Sczakiel, Georg; Claussen, Jens Christian; Restle, Tobias

    2015-01-01

    The discovery of RNA interference (RNAi) gave rise to the development of new nucleic acid-based technologies as powerful investigational tools and potential therapeutics. Mechanistic key details of RNAi in humans need to be deciphered yet, before such approaches take root in biomedicine and molecular therapy. We developed and validated an in silico-based model of siRNA-mediated RNAi in human cells in order to link in vitro-derived pre-steady state kinetic data with a quantitative and time-resolved understanding of RNAi on the cellular level. The observation that product release by Argonaute 2 is accelerated in the presence of an excess of target RNA in vitro inspired us to suggest an associative mechanism for the RNA slicer reaction where incoming target mRNAs actively promote dissociation of cleaved mRNA fragments. This novel associative model is compatible with high multiple turnover rates of RNAi-based gene silencing in living cells and accounts for target mRNA concentration-dependent enhancement of the RNAi machinery. PMID:26578554

  10. RNAi revised--target mRNA-dependent enhancement of gene silencing.

    PubMed

    Dornseifer, Simon; Willkomm, Sarah; Far, Rosel Kretschmer-Kazemi; Liebschwager, Janine; Beltsiou, Foteini; Frank, Kirsten; Laufer, Sandra D; Martinetz, Thomas; Sczakiel, Georg; Claussen, Jens Christian; Restle, Tobias

    2015-12-15

    The discovery of RNA interference (RNAi) gave rise to the development of new nucleic acid-based technologies as powerful investigational tools and potential therapeutics. Mechanistic key details of RNAi in humans need to be deciphered yet, before such approaches take root in biomedicine and molecular therapy. We developed and validated an in silico-based model of siRNA-mediated RNAi in human cells in order to link in vitro-derived pre-steady state kinetic data with a quantitative and time-resolved understanding of RNAi on the cellular level. The observation that product release by Argonaute 2 is accelerated in the presence of an excess of target RNA in vitro inspired us to suggest an associative mechanism for the RNA slicer reaction where incoming target mRNAs actively promote dissociation of cleaved mRNA fragments. This novel associative model is compatible with high multiple turnover rates of RNAi-based gene silencing in living cells and accounts for target mRNA concentration-dependent enhancement of the RNAi machinery. PMID:26578554

  11. A Multi-RNAi Microsponge Platform for Simultaneous Controlled Delivery of Multiple Small Interfering RNAs.

    PubMed

    Roh, Young Hoon; Deng, Jason Z; Dreaden, Erik C; Park, Jae Hyon; Yun, Dong Soo; Shopsowitz, Kevin E; Hammond, Paula T

    2016-03-01

    Packaging multiple small interfering RNA (siRNA) molecules into nanostructures at precisely defined ratios is a powerful delivery strategy for effective RNA interference (RNAi) therapy. We present a novel RNA nanotechnology based approach to produce multiple components of polymerized siRNA molecules that are simultaneously self-assembled and densely packaged into composite sponge-like porous microstructures (Multi-RNAi-MSs) by rolling circle transcription. The Multi-RNAi-MSs were designed to contain a combination of multiple polymeric siRNA molecules with precisely controlled stoichiometry within a singular microstructure by manipulating the types and ratios of the circular DNA templates. The Multi-RNAi-MSs were converted into nanosized complexes by polyelectrolyte condensation to manipulate their physicochemical properties (size, shape, and surface charge) for favorable delivery, while maintaining the multifunctional properties of the siRNAs for combined therapeutic effects. These Multi-RNAi-MS systems have great potential in RNAi-mediated biomedical applications, for example, for the treatment of cancer, genetic disorders, and viral infections. PMID:26695874

  12. Manipulating the in vivo immune response by targeted gene knockdown

    PubMed Central

    Lieberman, Judy

    2015-01-01

    Aptamers, nucleic acids selected for high affinity binding to proteins, can be used to activate or antagonize immune mediators or receptors in a location and cell-type specific manner and to enhance antigen presentation. They can also be linked to other molecules (other aptamers, siRNAs or miRNAs, proteins, toxins) to produce multifunctional compounds for targeted immune modulation in vivo. Aptamer-siRNA chimeras (AsiCs) that induce efficient cell-specific knockdown in immune cells in vitro and in vivo can be used as an immunological research tool or potentially as an immunomodulating therapeutic. PMID:26149459

  13. Manipulating the in vivo immune response by targeted gene knockdown.

    PubMed

    Lieberman, Judy

    2015-08-01

    Aptamers, nucleic acids selected for high affinity binding to proteins, can be used to activate or antagonize immune mediators or receptors in a location and cell-type specific manner and to enhance antigen presentation. They can also be linked to other molecules (other aptamers, siRNAs or miRNAs, proteins, toxins) to produce multifunctional compounds for targeted immune modulation in vivo. Aptamer-siRNA chimeras (AsiCs) that induce efficient cell-specific knockdown in immune cells in vitro and in vivo can be used as an immunological research tool or potentially as an immunomodulating therapeutic. PMID:26149459

  14. Feasibility, limitation and possible solutions of RNAi-based technology for insect pest control.

    PubMed

    Zhang, Hao; Li, Hai-Chao; Miao, Xue-Xia

    2013-02-01

    Numerous studies indicate that target gene silencing by RNA interference (RNAi) could lead to insect death. This phenomenon has been considered as a potential strategy for insect pest control, and it is termed RNAi-mediated crop protection. However, there are many limitations using RNAi-based technology for pest control, with the effectiveness target gene selection and reliable double-strand RNA (dsRNA) delivery being two of the major challenges. With respect to target gene selection, at present, the use of homologous genes and genome-scale high-throughput screening are the main strategies adopted by researchers. Once the target gene is identified, dsRNA can be delivered by micro-injection or by feeding as a dietary component. However, micro-injection, which is the most common method, can only be used in laboratory experiments. Expression of dsRNAs directed against insect genes in transgenic plants and spraying dsRNA reagents have been shown to induce RNAi effects on target insects. Hence, RNAi-mediated crop protection has been considered as a potential new-generation technology for pest control, or as a complementary method of existing pest control strategies; however, further development to improve the efficacy of protection and range of species affected is necessary. In this review, we have summarized current research on RNAi-based technology for pest insect management. Current progress has proven that RNAi technology has the potential to be a tool for designing a new generation of insect control measures. To accelerate its practical application in crop protection, further study on dsRNA uptake mechanisms based on the knowledge of insect physiology and biochemistry is needed. PMID:23955822

  15. RNAi-mediated silencing of HLA A2 suppressed acute rejection against human fibroblast xenografts in the striatum of 6-OHDA lesioned rats.

    PubMed

    Liang, Caixia; Xu, Yunzhi; Zheng, Deyu; Sun, Xiaohong; Xu, Qunyuan; Duan, Deyi

    2016-08-15

    Major histocompatibility complex class l (MHC I) molecules play a role in determining whether transplanted cells will be accepted or rejected, and masking of MHC I on donor cells has been found useful for immunoprotection of neural xenografts. In the present study, primary human embryonic lung fibroblasts (HELF), HELF treated with lentivirus-mediated small interfering RNAs (siRNAs) targeting human leukocyte antigen A2 (HLA A2, MHC I in humans) (siHELF), and rat embryonic lung fibroblasts (RELF) were stereotaxically grafted into the striatum of 6-hydroxydopamine lesioned rats to explore whether knockdown of HLA A2 could reduce host immune responses against xenografts. Before lentiviral infection, the cells were transduced with retroviruses harboring tyrosine hydroxylase cDNA. Knockdown of HLA A2 protein was examined by Western blotting. The immune responses (the number of CD4 and CD8 T-cells in the brain and peripheral blood), glial reaction, and survival of human fibroblasts were quantitatively evaluated by flow cytometry and immunohistochemistry at 4d, 2w, and 6w post-graft. Animal behaviors were assessed by counting apomorphine-induced rotations pre- and post-grafts. It was shown that a lower level of HLA A2 was observed in siHELF grafts than in HELF grafts, and knockdown of HLA A2 decreased rat immune responses, as indicated by less remarkable increases in the number of CD8 and CD4 T-cells in the brain and the ratio of CD4:CD8 T-cells in the peripheral blood in rats grafted with siHELF. Rats grafted with siHELF exhibited a significant improvement in motor asymmetry post-transplantation and a better survival of human fibroblasts at 2w. The increasing number of activated microglia and the decreasing number of astrocytes were found in three groups of rats post-implantation. These data suggested that RNAi-mediated knockdown of HLA A2 could suppress acute rejection against xenogeneic human cell transplants in the rat brain. PMID:27397073

  16. The Transgenic RNAi Project at Harvard Medical School: Resources and Validation.

    PubMed

    Perkins, Lizabeth A; Holderbaum, Laura; Tao, Rong; Hu, Yanhui; Sopko, Richelle; McCall, Kim; Yang-Zhou, Donghui; Flockhart, Ian; Binari, Richard; Shim, Hye-Seok; Miller, Audrey; Housden, Amy; Foos, Marianna; Randkelv, Sakara; Kelley, Colleen; Namgyal, Pema; Villalta, Christians; Liu, Lu-Ping; Jiang, Xia; Huan-Huan, Qiao; Wang, Xia; Fujiyama, Asao; Toyoda, Atsushi; Ayers, Kathleen; Blum, Allison; Czech, Benjamin; Neumuller, Ralph; Yan, Dong; Cavallaro, Amanda; Hibbard, Karen; Hall, Don; Cooley, Lynn; Hannon, Gregory J; Lehmann, Ruth; Parks, Annette; Mohr, Stephanie E; Ueda, Ryu; Kondo, Shu; Ni, Jian-Quan; Perrimon, Norbert

    2015-11-01

    To facilitate large-scale functional studies in Drosophila, the Drosophila Transgenic RNAi Project (TRiP) at Harvard Medical School (HMS) was established along with several goals: developing efficient vectors for RNAi that work in all tissues, generating a genome-scale collection of RNAi stocks with input from the community, distributing the lines as they are generated through existing stock centers, validating as many lines as possible using RT-qPCR and phenotypic analyses, and developing tools and web resources for identifying RNAi lines and retrieving existing information on their quality. With these goals in mind, here we describe in detail the various tools we developed and the status of the collection, which is currently composed of 11,491 lines and covering 71% of Drosophila genes. Data on the characterization of the lines either by RT-qPCR or phenotype is available on a dedicated website, the RNAi Stock Validation and Phenotypes Project (RSVP, http://www.flyrnai.org/RSVP.html), and stocks are available from three stock centers, the Bloomington Drosophila Stock Center (United States), National Institute of Genetics (Japan), and TsingHua Fly Center (China). PMID:26320097

  17. Key enzymes and proteins of crop insects as candidate for RNAi based gene silencing

    PubMed Central

    Kola, Vijaya Sudhakara Rao; Renuka, P.; Madhav, Maganti Sheshu; Mangrauthia, Satendra K.

    2015-01-01

    RNA interference (RNAi) is a mechanism of homology dependent gene silencing present in plants and animals. It operates through 21–24 nucleotides small RNAs which are processed through a set of core enzymatic machinery that involves Dicer and Argonaute proteins. In recent past, the technology has been well appreciated toward the control of plant pathogens and insects through suppression of key genes/proteins of infecting organisms. The genes encoding key enzymes/proteins with the great potential for developing an effective insect control by RNAi approach are actylcholinesterase, cytochrome P450 enzymes, amino peptidase N, allatostatin, allatotropin, tryptophan oxygenase, arginine kinase, vacuolar ATPase, chitin synthase, glutathione-S-transferase, catalase, trehalose phosphate synthase, vitellogenin, hydroxy-3-methylglutaryl coenzyme A reductase, and hormone receptor genes. Through various studies, it is demonstrated that RNAi is a reliable molecular tool which offers great promises in meeting the challenges imposed by crop insects with careful selection of key enzymes/proteins. Utilization of RNAi tool to target some of these key proteins of crop insects through various approaches is described here. The major challenges of RNAi based insect control such as identifying potential targets, delivery methods of silencing trigger, off target effects, and complexity of insect biology are very well illustrated. Further, required efforts to address these challenges are also discussed. PMID:25954206

  18. Mmi1 RNA surveillance machinery directs RNAi complex RITS to specific meiotic genes in fission yeast

    PubMed Central

    Hiriart, Edwige; Vavasseur, Aurélia; Touat-Todeschini, Leila; Yamashita, Akira; Gilquin, Benoit; Lambert, Emeline; Perot, Jonathan; Shichino, Yuichi; Nazaret, Nicolas; Boyault, Cyril; Lachuer, Joel; Perazza, Daniel; Yamamoto, Masayuki; Verdel, André

    2012-01-01

    RNA interference (RNAi) silences gene expression by acting both at the transcriptional and post-transcriptional levels in a broad range of eukaryotes. In the fission yeast Schizosaccharomyces pombe the RNA-Induced Transcriptional Silencing (RITS) RNAi complex mediates heterochromatin formation at non-coding and repetitive DNA. However, the targeting and role of RITS at other genomic regions, including protein-coding genes, remain unknown. Here we show that RITS localizes to specific meiotic genes and mRNAs. Remarkably, RITS is guided to these meiotic targets by the RNA-binding protein Mmi1 and its associated RNA surveillance machinery that together degrade selective meiotic mRNAs during vegetative growth. Upon sexual differentiation, RITS localization to the meiotic genes and mRNAs is lost. Large-scale identification of Mmi1 RNA targets reveals that RITS subunit Chp1 associates with the vast majority of them. In addition, loss of RNAi affects the effective repression of sexual differentiation mediated by the Mmi1 RNA surveillance machinery. These findings uncover a new mechanism for recruiting RNAi to specific meiotic genes and suggest that RNAi participates in the control of sexual differentiation in fission yeast. PMID:22522705

  19. Two sets of RNAi components are required for heterochromatin formation in trans triggered by truncated transgenes.

    PubMed

    Götz, Ulrike; Marker, Simone; Cheaib, Miriam; Andresen, Karsten; Shrestha, Simon; Durai, Dilip A; Nordström, Karl J; Schulz, Marcel H; Simon, Martin

    2016-07-01

    Across kingdoms, RNA interference (RNAi) has been shown to control gene expression at the transcriptional- or the post-transcriptional level. Here, we describe a mechanism which involves both aspects: truncated transgenes, which fail to produce intact mRNA, induce siRNA accumulation and silencing of homologous loci in trans in the ciliate Paramecium We show that silencing is achieved by co-transcriptional silencing, associated with repressive histone marks at the endogenous gene. This is accompanied by secondary siRNA accumulation, strictly limited to the open reading frame of the remote locus. Our data shows that in this mechanism, heterochromatic marks depend on a variety of RNAi components. These include RDR3 and PTIWI14 as well as a second set of components, which are also involved in post-transcriptional silencing: RDR2, PTIWI13, DCR1 and CID2. Our data indicates differential processing of nascent un-spliced and long, spliced transcripts thus suggesting a hitherto-unrecognized functional interaction between post-transcriptional and co-transcriptional RNAi. Both sets of RNAi components are required for efficient trans-acting RNAi at the chromatin level and our data indicates similar mechanisms contributing to genome wide regulation of gene expression by epigenetic mechanisms. PMID:27085807

  20. Two sets of RNAi components are required for heterochromatin formation in trans triggered by truncated transgenes

    PubMed Central

    Götz, Ulrike; Marker, Simone; Cheaib, Miriam; Andresen, Karsten; Shrestha, Simon; Durai, Dilip A.; Nordström, Karl J.; Schulz, Marcel H.; Simon, Martin

    2016-01-01

    Across kingdoms, RNA interference (RNAi) has been shown to control gene expression at the transcriptional- or the post-transcriptional level. Here, we describe a mechanism which involves both aspects: truncated transgenes, which fail to produce intact mRNA, induce siRNA accumulation and silencing of homologous loci in trans in the ciliate Paramecium. We show that silencing is achieved by co-transcriptional silencing, associated with repressive histone marks at the endogenous gene. This is accompanied by secondary siRNA accumulation, strictly limited to the open reading frame of the remote locus. Our data shows that in this mechanism, heterochromatic marks depend on a variety of RNAi components. These include RDR3 and PTIWI14 as well as a second set of components, which are also involved in post-transcriptional silencing: RDR2, PTIWI13, DCR1 and CID2. Our data indicates differential processing of nascent un-spliced and long, spliced transcripts thus suggesting a hitherto-unrecognized functional interaction between post-transcriptional and co-transcriptional RNAi. Both sets of RNAi components are required for efficient trans-acting RNAi at the chromatin level and our data indicates similar mechanisms contributing to genome wide regulation of gene expression by epigenetic mechanisms. PMID:27085807

  1. Recent advances in RNAi-based strategies for therapy and prevention of HIV-1/AIDS.

    PubMed

    Swamy, Manjunath N; Wu, Haoquan; Shankar, Premlata

    2016-08-01

    RNA interference (RNAi) provides a powerful tool to silence specific gene expression and has been widely used to suppress host factors such as CCR5 and/or viral genes involved in HIV-1 replication. Newer nuclease-based gene-editing technologies, such as zinc finger nucleases (ZFN), transcription activator-like effector nucleases (TALEN) and the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 system, also provide powerful tools to ablate specific genes. Because of differences in co-receptor usage and the high mutability of the HIV-1 genome, a combination of host factors and viral genes needs to be suppressed for effective prevention and treatment of HIV-1 infection. Whereas the continued presence of small interfering/short hairpin RNA (si/shRNA) mediators is needed for RNAi to be effective, the continued expression of nucleases in the gene-editing systems is undesirable. Thus, RNAi provides the only practical way for expression of multiple silencers in infected and uninfected cells, which is needed for effective prevention/treatment of infection. There have been several advances in the RNAi field in terms of si/shRNA design, targeted delivery to HIV-1 susceptible cells, and testing for efficacy in preclinical humanized mouse models. Here, we comprehensively review the latest advances in RNAi technology towards prevention and treatment of HIV-1. PMID:27013255

  2. Intelligent Interfaces for Mining Large-Scale RNAi-HCS Image Databases

    PubMed Central

    Lin, Chen; Mak, Wayne; Hong, Pengyu; Sepp, Katharine; Perrimon, Norbert

    2010-01-01

    Recently, High-content screening (HCS) has been combined with RNA interference (RNAi) to become an essential image-based high-throughput method for studying genes and biological networks through RNAi-induced cellular phenotype analyses. However, a genome-wide RNAi-HCS screen typically generates tens of thousands of images, most of which remain uncategorized due to the inadequacies of existing HCS image analysis tools. Until now, it still requires highly trained scientists to browse a prohibitively large RNAi-HCS image database and produce only a handful of qualitative results regarding cellular morphological phenotypes. For this reason we have developed intelligent interfaces to facilitate the application of the HCS technology in biomedical research. Our new interfaces empower biologists with computational power not only to effectively and efficiently explore large-scale RNAi-HCS image databases, but also to apply their knowledge and experience to interactive mining of cellular phenotypes using Content-Based Image Retrieval (CBIR) with Relevance Feedback (RF) techniques. PMID:21278820

  3. Phosphodiesterase-4D Knock-down in the Prefrontal Cortex Alleviates Chronic Unpredictable Stress-Induced Depressive-Like Behaviors and Memory Deficits in Mice

    PubMed Central

    Wang, Zhen-Zhen; Yang, Wei-Xing; Zhang, Yi; Zhao, Nan; Zhang, You-Zhi; Liu, Yan-Qin; Xu, Ying; Wilson, Steven P.; O'Donnell, James M.; Zhang, Han-Ting; Li, Yun-Feng

    2015-01-01

    Phosphodiesterase 4 (PDE4) has four isoforms (PDE4A-D) with at least 25 splice variants. PDE4 subtype nonselective inhibitors produce potent antidepressant-like and cognition-enhancing effects via increased intracellular cyclic AMP (cAMP) signaling in the brain. Our previous data have demonstrated that long-form PDE4Ds appear to be involved in these pharmacological properties of PDE4 inhibitors in the normal animals. However, it is not clear whether long-form PDE4Ds are critical for the behaviors and related cellular signaling/neuronal plasticity/neuroendocrine alterations in the depressed animals. In the present study, animals exposed to the chronic unpredictable stress (CUS), a rodent model of depression, exhibited elevated corticosterone, depressive-like behavior, memory deficits, accompanied with decreased cAMP-PKA-CREB and cAMP-ERK1/2-CREB signaling and neuroplasticity. These alterations induced by CUS were reversed by RNA interference (RNAi)-mediated prefrontal cortex long-form PDE4Ds (especially PDE4D4 and PDE4D5) knock-down, similar to the effects of the PDE4 subtype nonselective inhibitor rolipram. Furthermore, these effects of RNAi were not enhanced by rolipram. These data indicate a predominant role of long-form PDE4Ds in the pharmacotherapies of PDE4 inhibitors for depression and concomitant memory deficits. Long-form PDE4Ds, especially PDE4D4 and PDE4D5, appear to be the promising targets for the development of antidepressants with high therapeutic indices. PMID:26161529

  4. Practical Recommendations for the Use of the GeneSwitch Gal4 System to Knock-Down Genes in Drosophila melanogaster

    PubMed Central

    Scialo, Filippo; Sriram, Ashwin; Stefanatos, Rhoda; Sanz, Alberto

    2016-01-01

    Drosophila melanogaster is a popular research model organism thanks to its’ powerful genetic tools that allow spatial and temporal control of gene expression. The inducible GeneSwitch Gal4 system (GS) system is a modified version of the classic UAS/GAL4 system which allows inducible regulation of gene expression and eliminates background effects. It is widely acknowledged that the GS system is leaky, with low level expression of UAS transgenes in absence of the inducer RU-486 (the progesterone analog that activates the modified GAL4 protein). However, in the course of our experiments, we have observed that the extent of this leak depends on the nature of the transgene being expressed. In the absence of RU-486, when strong drivers are used to express protein coding transgenes, leaky expression is low or negligible, however expression of RNA interference (RNAi) transgenes results in complete depletion of protein levels. The majority of published studies, using the GS system and RNAi transgenes validate knock-down efficiency by comparing target gene mRNA levels between induced and non-induced groups. Here, we demonstrate that this approach is lacking and that both additional control groups and further validation is required at the protein level. Unfortunately, this experimental limitation of the GS system eliminates “the background advantage”, but does offer the possibility of performing more complex experiments (e.g. studying depletion and overexpression of different proteins in the same genetic background). The limitations and new possible applications of the GS system are discussed in detail. PMID:27570965

  5. Practical Recommendations for the Use of the GeneSwitch Gal4 System to Knock-Down Genes in Drosophila melanogaster.

    PubMed

    Scialo, Filippo; Sriram, Ashwin; Stefanatos, Rhoda; Sanz, Alberto

    2016-01-01

    Drosophila melanogaster is a popular research model organism thanks to its' powerful genetic tools that allow spatial and temporal control of gene expression. The inducible GeneSwitch Gal4 system (GS) system is a modified version of the classic UAS/GAL4 system which allows inducible regulation of gene expression and eliminates background effects. It is widely acknowledged that the GS system is leaky, with low level expression of UAS transgenes in absence of the inducer RU-486 (the progesterone analog that activates the modified GAL4 protein). However, in the course of our experiments, we have observed that the extent of this leak depends on the nature of the transgene being expressed. In the absence of RU-486, when strong drivers are used to express protein coding transgenes, leaky expression is low or negligible, however expression of RNA interference (RNAi) transgenes results in complete depletion of protein levels. The majority of published studies, using the GS system and RNAi transgenes validate knock-down efficiency by comparing target gene mRNA levels between induced and non-induced groups. Here, we demonstrate that this approach is lacking and that both additional control groups and further validation is required at the protein level. Unfortunately, this experimental limitation of the GS system eliminates "the background advantage", but does offer the possibility of performing more complex experiments (e.g. studying depletion and overexpression of different proteins in the same genetic background). The limitations and new possible applications of the GS system are discussed in detail. PMID:27570965

  6. Effective AAV-mediated gene therapy in a mouse model of ethylmalonic encephalopathy

    PubMed Central

    Di Meo, Ivano; Auricchio, Alberto; Lamperti, Costanza; Burlina, Alberto; Viscomi, Carlo; Zeviani, Massimo

    2012-01-01

    Ethylmalonic encephalopathy (EE) is an invariably fatal disease, characterized by the accumulation of hydrogen sulfide (H2S), a highly toxic compound. ETHE1, encoding sulfur dioxygenase (SDO), which takes part in the mitochondrial pathway that converts sulfide into harmless sulfate, is mutated in EE. The main source of H2S is the anaerobic bacterial flora of the colon, although in trace amount it is also produced by tissues, where it acts as a ‘gasotransmitter’. Here, we show that AAV2/8-mediated, ETHE1-gene transfer to the liver of a genetically, metabolically and clinically faithful EE mouse model resulted in full restoration of SDO activity, correction of plasma thiosulfate, a biomarker reflecting the accumulation of H2S, and spectacular clinical improvement. Most of treated animals were alive and well >6–8 months after birth, whereas untreated individuals live 26 ± 7 days. Our results provide proof of concept on the efficacy and safety of AAV2/8-mediated livergene therapy for EE, and alike conditions caused by the accumulation of harmful compounds in body fluids and tissues, which can directly be transferred to the clinic. PMID:22903887

  7. AAV-Mediated Cone Rescue in a Naturally Occurring Mouse Model of CNGA3-Achromatopsia

    PubMed Central

    Dai, Xufeng; Lei, Bo; Everhart, Drew; Umino, Yumiko; Li, Jie; Zhang, Keqing; Mao, Song; Boye, Sanford L.; Liu, Li; Chiodo, Vince A.; Liu, Xuan; Shi, Wei; Tao, Ye; Chang, Bo; Hauswirth, William W.

    2012-01-01

    Achromatopsia is a rare autosomal recessive disorder which shows color blindness, severely impaired visual acuity, and extreme sensitivity to bright light. Mutations in the alpha subunits of the cone cyclic nucleotide-gated channels (CNGA3) are responsible for about 1/4 of achromatopsia in the U.S. and Europe. Here, we test whether gene replacement therapy using an AAV5 vector could restore cone-mediated function and arrest cone degeneration in the cpfl5 mouse, a naturally occurring mouse model of achromatopsia with a CNGA3 mutation. We show that gene therapy leads to significant rescue of cone-mediated ERGs, normal visual acuities and contrast sensitivities. Normal expression and outer segment localization of both M- and S-opsins were maintained in treated retinas. The therapeutic effect of treatment lasted for at least 5 months post-injection. This study is the first demonstration of substantial, relatively long-term restoration of cone-mediated light responsiveness and visual behavior in a naturally occurring mouse model of CNGA3 achromatopsia. The results provide the foundation for development of an AAV5-based gene therapy trial for human CNGA3 achromatopsia. PMID:22509403

  8. AAV-Mediated Gene Therapy for Choroideremia: Preclinical Studies in Personalized Models

    PubMed Central

    Vasireddy, Vidyullatha; Kohnke, Monika; Black, Aaron D.; Alexandrov, Krill; Zhou, Shangzhen; Maguire, Albert M.; Chung, Daniel C.; Mac, Helen; Sullivan, Lisa; Gadue, Paul; Bennicelli, Jeannette L.; French, Deborah L.; Bennett, Jean

    2013-01-01

    Choroideremia (CHM) is an X- linked retinal degeneration that is symptomatic in the 1st or 2nd decade of life causing nyctalopia and loss of peripheral vision. The disease progresses through mid-life, when most patients become blind. CHM is a favorable target for gene augmentation therapy, as the disease is due to loss of function of a protein necessary for retinal cell health, Rab Escort Protein 1 (REP1).The CHM cDNA can be packaged in recombinant adeno-associated virus (rAAV), which has an established track record in human gene therapy studies, and, in addition, there are sensitive and quantitative assays to document REP1 activity. An animal model that accurately reflects the human condition is not available. In this study, we tested the ability to restore REP1 function in personalized in vitro models of CHM: lymphoblasts and induced pluripotent stems cells (iPSCs) from human patients. The initial step of evaluating safety of the treatment was carried out by evaluating for acute retinal histopathologic effects in normal-sighted mice and no obvious toxicity was identified. Delivery of the CHM cDNA to affected cells restores REP1 enzymatic activity and also restores proper protein trafficking. The gene transfer is efficient and the preliminary safety data are encouraging. These studies pave the way for a human clinical trial of gene therapy for CHM. PMID:23667438

  9. Mitigation of cerebellar neuropathy in globoid cell leukodystrophy mice by AAV-mediated gene therapy.

    PubMed

    Lin, Dar-Shong; Hsiao, Chung-Der; Lee, Allan Yueh-Luen; Ho, Che-Sheng; Liu, Hsuan-Liang; Wang, Tuen-Jen; Jian, Yuan-Ren; Hsu, Jui-Cheng; Huang, Zon-Darr; Lee, Tsung-Han; Chiang, Ming-Fu

    2015-10-15

    Globoid cell leukodystrophy (GLD) is an autosomal recessive, lysosomal storage disease caused by deficiency of the enzyme galactocerebrosidase (GALC). The absence of GALC activity leads to the accumulation of the toxic substance psychosine and the preferential loss of myelinating cells in the central and peripheral nervous systems. Profound demyelination, astrogliosis and axonopathy are the hallmarks of the pathogenesis of GLD, and cerebellar ataxia is one of the dominant manifestations in adolescents and adults affected with GLD. To date, studies regarding cerebellar degeneration in GLD are limited. In this study, the efficacy of cerebellum-targeted gene therapy on the cerebellar neuropathology in twitcher mice (a murine model of GLD) has been validated. We observed degeneration of Purkinje cells, Bergmann glia, and granule cells in addition to astrocytosis and demyelination in the cerebellum of the twitcher mice. Ultrastructural analysis revealed dark cell degeneration and disintegration of the cellular composition of Purkinje cells in untreated twitcher mice. In addition, the expressions of neurotrophic factors CNTF, GDNF and IGF-I were up-regulated and the expression of BDNF was down-regulated. Intracerebellar-mediated gene therapy efficiently corrected enzymatic deficiency by direct transduction to Purkinje cells and cross-correction in other cell types in the cerebellum, leading to the amelioration of both neuroinflammation and demyelination. The population, dendritic territory, and axonal processes of Purkinje cells remained normal in the cerebellum of treated twitcher mice, where radial fibers of Bergmann glia spanned the molecular layer and collateral branches ensheathed the dendritic processes of Purkinje cells. Moreover, the aberrant expressions of neurotrophic factors were mitigated in the cerebellum of treated twitcher mice, indicating the preservation of cellular function in addition to maintaining the neuronal architecture. The life span of the treated twitcher mice was significantly prolonged and their neurobehavioral performance was improved. Taken together, our findings underscore the complexity of cerebellar neurodegeneration in GLD and highlight the potential effectiveness of gene therapy in mitigating neuropathological deficits in GLD and other neurodegenerative disorders in which Purkinje cells are involved. PMID:26115766

  10. AAV-mediated in vivo functional selection of tissue-protective factors against ischaemia.

    PubMed

    Ruozi, Giulia; Bortolotti, Francesca; Falcione, Antonella; Dal Ferro, Matteo; Ukovich, Laura; Macedo, Antero; Zentilin, Lorena; Filigheddu, Nicoletta; Gortan Cappellari, Gianluca; Baldini, Giovanna; Zweyer, Marina; Barazzoni, Rocco; Graziani, Andrea; Zacchigna, Serena; Giacca, Mauro

    2015-01-01

    Functional screening of expression libraries in vivo would offer the possibility of identifying novel biotherapeutics without a priori knowledge of their biochemical function. Here we describe a procedure for the functional selection of tissue-protective factors based on the in vivo delivery of arrayed cDNA libraries from the mouse secretome using adeno-associated virus (AAV) vectors. Application of this technique, which we call FunSel, in the context of acute ischaemia, revealed that the peptide ghrelin protects skeletal muscle and heart from ischaemic damage. When delivered to the heart using an AAV9 vector, ghrelin markedly reduces infarct size and preserves cardiac function over time. This protective activity associates with the capacity of ghrelin to sustain autophagy and remove dysfunctional mitochondria after myocardial infarction. Our findings describe an innovative tool to identify biological therapeutics and reveal a novel role of ghrelin as an inducer of myoprotective autophagy. PMID:26066847

  11. Evaluation of AAV-mediated Gene Therapy for Central Nervous System Disease in Canine Mucopolysaccharidosis VII

    PubMed Central

    Gurda, Brittney L; De Guilhem De Lataillade, Adrien; Bell, Peter; Zhu, Yanqing; Yu, Hongwei; Wang, Ping; Bagel, Jessica; Vite, Charles H; Sikora, Tracey; Hinderer, Christian; Calcedo, Roberto; Yox, Alexander D; Steet, Richard A; Ruane, Therese; O'Donnell, Patricia; Gao, Guangping; Wilson, James M; Casal, Margret; Ponder, Katherine P; Haskins, Mark E

    2016-01-01

    Mucopolysaccharidosis VII (MPS VII) is a lysosomal storage disease arising from mutations in β-d-glucuronidase (GUSB), which results in glycosaminoglycan (GAG) accumulation and a variety of clinical manifestations including neurological disease. Herein, MPS VII dogs were injected intravenously (i.v.) and/or intrathecally (i.t.) via the cisterna magna with AAV9 or AAVrh10 vectors carrying the canine GUSB cDNA. Although i.v. injection alone at 3 days of age resulted in normal cerebrospinal fluid (CSF) GUSB activity, brain tissue homogenates had only ~1 to 6% normal GUSB activity and continued to have elevated GAG storage. In contrast, i.t. injection at 3 weeks of age resulted in CSF GUSB activity 44-fold normal while brain tissue homogenates had >100% normal GUSB activity and reduced GAGs compared with untreated dogs. Markers for secondary storage and inflammation were eliminated in i.t.-treated dogs and reduced in i.v.-treated dogs compared with untreated dogs. Given that i.t.-treated dogs expressed higher levels of GUSB in the CNS tissues compared to those treated i.v., we conclude that i.t. injection of AAV9 or AAVrh10 vectors is more effective than i.v. injection alone in the large animal model of MPS VII. PMID:26447927

  12. AAV-mediated Gene Therapy Halts Retinal Degeneration in PDE6β-deficient Dogs.

    PubMed

    Pichard, Virginie; Provost, Nathalie; Mendes-Madeira, Alexandra; Libeau, Lyse; Hulin, Philippe; Tshilenge, Kizito-Tshitoko; Biget, Marine; Ameline, Baptiste; Deschamps, Jack-Yves; Weber, Michel; Le Meur, Guylène; Colle, Marie-Anne; Moullier, Philippe; Rolling, Fabienne

    2016-05-01

    We previously reported that subretinal injection of AAV2/5 RK.cpde6β allowed long-term preservation of photoreceptor function and vision in the rod-cone dysplasia type 1 (rcd1) dog, a large animal model of naturally occurring PDE6β deficiency. The present study builds on these earlier findings to provide a detailed assessment of the long-term effects of gene therapy on the spatiotemporal pattern of retinal degeneration in rcd1 dogs treated at 20 days of age. We analyzed the density distribution of the retinal layers and of particular photoreceptor cells in 3.5-year-old treated and untreated rcd1 dogs. Whereas no rods were observed outside the bleb or in untreated eyes, gene transfer halted rod degeneration in all vector-exposed regions. Moreover, while gene therapy resulted in the preservation of cones, glial cells and both the inner nuclear and ganglion cell layers, no cells remained in vector-unexposed retinas, except in the visual streak. Finally, the retinal structure of treated 3.5-year-old rcd1 dogs was identical to that of unaffected 4-month-old rcd1 dogs, indicating near complete preservation. Our findings indicate that gene therapy arrests the degenerative process even if intervention is initiated after the onset of photoreceptor degeneration, and point to significant potential of this therapeutic approach in future clinical trials. PMID:26857842

  13. Inner Limiting Membrane Barriers to AAV-mediated Retinal Transduction From the Vitreous

    PubMed Central

    Dalkara, Deniz; Kolstad, Kathleen D; Caporale, Natalia; Visel, Meike; Klimczak, Ryan R; Schaffer, David V; Flannery, John G

    2009-01-01

    Adeno-associated viral gene therapy has shown great promise in treating retinal disorders, with three promising clinical trials in progress. Numerous adeno-associated virus (AAV) serotypes can infect various cells of the retina when administered subretinally, but the retinal detachment accompanying this injection induces changes that negatively impact the microenvironment and survival of retinal neurons. Intravitreal administration could circumvent this problem, but only AAV2 can infect retinal cells from the vitreous, and transduction is limited to the inner retina. We therefore sought to investigate and reduce barriers to transduction from the vitreous. We fluorescently labeled several AAV serotype capsids and followed their retinal distribution after intravitreal injection. AAV2, 8, and 9 accumulate at the vitreoretinal junction. AAV1 and 5 show no accumulation, indicating a lack of appropriate receptors at the inner limiting membrane (ILM). Importantly, mild digestion of the ILM with a nonspecific protease enabled substantially enhanced transduction of multiple retinal cell types from the vitreous, with AAV5 mediating particularly remarkable expression in all retinal layers. This protease treatment has no effect on retinal function as shown by electroretinogram (ERG) and visual cortex cell population responses. These findings may help avoid limitations, risks, and damage associated with subretinal injections currently necessary for clinical gene therapy. PMID:19672248

  14. Evaluation of AAV-mediated Gene Therapy for Central Nervous System Disease in Canine Mucopolysaccharidosis VII.

    PubMed

    Gurda, Brittney L; De Guilhem De Lataillade, Adrien; Bell, Peter; Zhu, Yanqing; Yu, Hongwei; Wang, Ping; Bagel, Jessica; Vite, Charles H; Sikora, Tracey; Hinderer, Christian; Calcedo, Roberto; Yox, Alexander D; Steet, Richard A; Ruane, Therese; O'Donnell, Patricia; Gao, Guangping; Wilson, James M; Casal, Margret; Ponder, Katherine P; Haskins, Mark E

    2016-02-01

    Mucopolysaccharidosis VII (MPS VII) is a lysosomal storage disease arising from mutations in β-d-glucuronidase (GUSB), which results in glycosaminoglycan (GAG) accumulation and a variety of clinical manifestations including neurological disease. Herein, MPS VII dogs were injected intravenously (i.v.) and/or intrathecally (i.t.) via the cisterna magna with AAV9 or AAVrh10 vectors carrying the canine GUSB cDNA. Although i.v. injection alone at 3 days of age resulted in normal cerebrospinal fluid (CSF) GUSB activity, brain tissue homogenates had only ~1 to 6% normal GUSB activity and continued to have elevated GAG storage. In contrast, i.t. injection at 3 weeks of age resulted in CSF GUSB activity 44-fold normal while brain tissue homogenates had >100% normal GUSB activity and reduced GAGs compared with untreated dogs. Markers for secondary storage and inflammation were eliminated in i.t.-treated dogs and reduced in i.v.-treated dogs compared with untreated dogs. Given that i.t.-treated dogs expressed higher levels of GUSB in the CNS tissues compared to those treated i.v., we conclude that i.t. injection of AAV9 or AAVrh10 vectors is more effective than i.v. injection alone in the large animal model of MPS VII. PMID:26447927

  15. AAV-mediated in vivo functional selection of tissue-protective factors against ischaemia

    PubMed Central

    Ruozi, Giulia; Bortolotti, Francesca; Falcione, Antonella; Dal Ferro, Matteo; Ukovich, Laura; Macedo, Antero; Zentilin, Lorena; Filigheddu, Nicoletta; Cappellari, Gianluca Gortan; Baldini, Giovanna; Zweyer, Marina; Barazzoni, Rocco; Graziani, Andrea; Zacchigna, Serena; Giacca, Mauro

    2015-01-01

    Functional screening of expression libraries in vivo would offer the possibility of identifying novel biotherapeutics without a priori knowledge of their biochemical function. Here we describe a procedure for the functional selection of tissue-protective factors based on the in vivo delivery of arrayed cDNA libraries from the mouse secretome using adeno-associated virus (AAV) vectors. Application of this technique, which we call FunSel, in the context of acute ischaemia, revealed that the peptide ghrelin protects skeletal muscle and heart from ischaemic damage. When delivered to the heart using an AAV9 vector, ghrelin markedly reduces infarct size and preserves cardiac function over time. This protective activity associates with the capacity of ghrelin to sustain autophagy and remove dysfunctional mitochondria after myocardial infarction. Our findings describe an innovative tool to identify biological therapeutics and reveal a novel role of ghrelin as an inducer of myoprotective autophagy. PMID:26066847

  16. A Genome-Wide Map of AAV-Mediated Human Gene Targeting

    PubMed Central

    Deyle, David R.; Hansen, R. Scott; Cornea, Anda M.; Li, Li B.; Burt, Amber A.; Alexander, Ian E.; Sandstrom, Richard S.; Stamatoyannopoulos, John A.; Wei, Chia-Lin; Russell, David W.

    2014-01-01

    To determine which genomic features promote homologous recombination, we created a genome-wide map of gene targeting sites. An adeno-associated virus vector was used to target identical loci introduced as transcriptionally active retroviral vector proviruses. A comparison of ~2,000 targeted and untargeted sites showed that targeting occurred throughout the human genome and was not influenced by the presence of nearby CpG islands, sequence repeats, or DNase I hypersensitive sites. Targeted sites were preferentially found within transcription units, especially when the target loci were transcribed in the opposite orientation to their surrounding chromosomal genes. The impact of DNA replication was determined by mapping replication forks, which revealed a preference for recombination at target loci transcribed towards an incoming fork. Our results constitute the first genome-wide screen of gene targeting in mammalian cells, and they demonstrate a strong recombinogenic effect of colliding polymerases. PMID:25282150

  17. A new Houdini act: multiple routes for HIV-1 escape from RNAi-mediated inhibition.

    PubMed

    Berkhout, Ben

    2009-03-01

    Evaluation of: Leonard JN, Shah PS, Burnett JC, Schaffer DV: HIV evades RNA interference directed at TAR by an indirect compensatory mechanism. Cell Host Microbe 4, 484-494 (2008). RNAi can be used to induce the silencing of messenger RNAs in a sequence-specific manner. Several therapeutic RNAi applications are actively being pursued, including the targeting of the RNA genome of human pathogenic viruses such as HIV-1. Viruses are able to escape from RNAi attack by mutation of the targeted sequence. In this report, Leonard and co-workers present evidence of a more indirect viral escape route by selection of up-mutations in the promoter that boosts viral gene expression. This indirect route may serve as a general viral evasion mechanism. PMID:19257841

  18. Biosafety research for non-target organism risk assessment of RNAi-based GE plants

    PubMed Central

    Roberts, Andrew F.; Devos, Yann; Lemgo, Godwin N. Y.; Zhou, Xuguo

    2015-01-01

    RNA interference, or RNAi, refers to a set of biological processes that make use of conserved cellular machinery to silence genes. Although there are several variations in the source and mechanism, they are all triggered by double stranded RNA (dsRNA) which is processed by a protein complex into small, single stranded RNA, referred to as small interfering RNAs (siRNA) with complementarity to sequences in genes targeted for silencing. The use of the RNAi mechanism to develop new traits in plants has fueled a discussion about the environmental safety of the technology for these applications, and this was the subject of a symposium session at the 13th ISBGMO in Cape Town, South Africa. This paper continues that discussion by proposing research areas that may be beneficial for future environmental risk assessments of RNAi-based genetically modified plants, with a particular focus on non-target organism assessment. PMID:26594220

  19. Biosafety research for non-target organism risk assessment of RNAi-based GE plants.

    PubMed

    Roberts, Andrew F; Devos, Yann; Lemgo, Godwin N Y; Zhou, Xuguo

    2015-01-01

    RNA interference, or RNAi, refers to a set of biological processes that make use of conserved cellular machinery to silence genes. Although there are several variations in the source and mechanism, they are all triggered by double stranded RNA (dsRNA) which is processed by a protein complex into small, single stranded RNA, referred to as small interfering RNAs (siRNA) with complementarity to sequences in genes targeted for silencing. The use of the RNAi mechanism to develop new traits in plants has fueled a discussion about the environmental safety of the technology for these applications, and this was the subject of a symposium session at the 13th ISBGMO in Cape Town, South Africa. This paper continues that discussion by proposing research areas that may be beneficial for future environmental risk assessments of RNAi-based genetically modified plants, with a particular focus on non-target organism assessment. PMID:26594220

  20. Vitellogenin-RNAi and ovariectomy each increase lifespan, increase protein storage, and decrease feeding, but are not additive in grasshoppers.

    PubMed

    Tetlak, Alicia G; Burnett, Jacob B; Hahn, Daniel A; Hatle, John D

    2015-12-01

    Reduced reproduction has been shown to increase lifespan in many animals, yet the mechanisms behind this trade-off are unclear. We addressed this question by combining two distinct, direct means of life-extension via reduced reproduction, to test whether they were additive. In the lubber grasshopper, Romalea microptera, ovariectomized (OVX) individuals had a ~20% increase in lifespan and a doubling of storage relative to controls (Sham operated). Similarly, young female grasshoppers treated with RNAi against vitellogenin (the precursor to egg yolk protein) had increased fat body mass and halted ovarian growth. In this study, we compared VgRNAi to two control groups that do not reduce reproduction, namely buffer injection (Buffer) and injection with RNAi against a hexameric storage protein (Hex90RNAi). Each injection treatment was tested with and without ovariectomy. Hence, we tested feeding, storage, and lifespans in six groups: OVX and Buffer, OVX and Hex90RNAi, OVX and VgRNAi, Sham and Buffer, Sham and Hex90RNAi, and Sham and VgRNAi. Ovariectomized grasshoppers and VgRNAi grasshoppers each had similar reductions in feeding (~40%), increases in protein storage in the hemolymph (150-300%), and extensions in lifespan (13-21%). Ovariectomized grasshoppers had higher vitellogenin protein levels than did VgRNAi grasshoppers. Last but not least, when ovariectomy and VgRNAi were applied together, there was no greater effect on feeding, protein storage, or longevity. Hence, feeding regulation, and protein storage in insects, may be conserved components of life-extension via reduced reproduction. PMID:26298568

  1. Persistence and transgenerational effect of plant-mediated RNAi in aphids

    PubMed Central

    Coleman, A. D.; Wouters, R. H. M.; Mugford, S. T.; Hogenhout, S. A.

    2015-01-01

    Plant-mediated RNA interference (RNAi) has been successfully used as a tool to study gene function in aphids. The persistence and transgenerational effects of plant-mediated RNAi in the green peach aphid (GPA) Myzus persicae were investigated, with a focus on three genes with different functions in the aphid. Rack1 is a key component of various cellular processes inside aphids, while candidate effector genes MpC002 and MpPIntO2 (Mp2) modulate aphid–plant interactions. The gene sequences and functions did not affect RNAi-mediated down-regulation and persistence levels in the aphids. Maximal reduction of gene expression was ~70% and this was achieved at between 4 d and 8 d of exposure of the aphids to double-stranded RNA (dsRNA)-producing transgenic Arabidopsis thaliana. Moreover, gene expression levels returned to wild-type levels within ~6 d after removal of the aphids from the transgenic plants, indicating that a continuous supply of dsRNA is required to maintain the RNAi effect. Target genes were also down-regulated in nymphs born from mothers exposed to dsRNA-producing transgenic plants, and the RNAi effect lasted twice as long (12–14 d) in these nymphs. Investigations of the impact of RNAi over three generations of aphids revealed that aphids reared on dsMpC002 transgenic plants experienced a 60% decline in aphid reproduction levels compared with a 40% decline of aphids reared on dsRack1 and dsMpPIntO2 plants. In a field setting, a reduction of the aphid reproduction by 40–60% would dramatically decrease aphid population growth, contributing to a substantial reduction in agricultural losses. PMID:25403918

  2. Persistence and transgenerational effect of plant-mediated RNAi in aphids.

    PubMed

    Coleman, A D; Wouters, R H M; Mugford, S T; Hogenhout, S A

    2015-02-01

    Plant-mediated RNA interference (RNAi) has been successfully used as a tool to study gene function in aphids. The persistence and transgenerational effects of plant-mediated RNAi in the green peach aphid (GPA) Myzus persicae were investigated, with a focus on three genes with different functions in the aphid. Rack1 is a key component of various cellular processes inside aphids, while candidate effector genes MpC002 and MpPIntO2 (Mp2) modulate aphid-plant interactions. The gene sequences and functions did not affect RNAi-mediated down-regulation and persistence levels in the aphids. Maximal reduction of gene expression was ~70% and this was achieved at between 4 d and 8 d of exposure of the aphids to double-stranded RNA (dsRNA)-producing transgenic Arabidopsis thaliana. Moreover, gene expression levels returned to wild-type levels within ~6 d after removal of the aphids from the transgenic plants, indicating that a continuous supply of dsRNA is required to maintain the RNAi effect. Target genes were also down-regulated in nymphs born from mothers exposed to dsRNA-producing transgenic plants, and the RNAi effect lasted twice as long (12-14 d) in these nymphs. Investigations of the impact of RNAi over three generations of aphids revealed that aphids reared on dsMpC002 transgenic plants experienced a 60% decline in aphid reproduction levels compared with a 40% decline of aphids reared on dsRack1 and dsMpPIntO2 plants. In a field setting, a reduction of the aphid reproduction by 40-60% would dramatically decrease aphid population growth, contributing to a substantial reduction in agricultural losses. PMID:25403918

  3. RNAiFold2T: Constraint Programming design of thermo-IRES switches

    PubMed Central

    Garcia-Martin, Juan Antonio; Dotu, Ivan; Fernandez-Chamorro, Javier; Lozano, Gloria; Ramajo, Jorge; Martinez-Salas, Encarnacion; Clote, Peter

    2016-01-01

    Motivation: RNA thermometers (RNATs) are cis-regulatory elements that change secondary structure upon temperature shift. Often involved in the regulation of heat shock, cold shock and virulence genes, RNATs constitute an interesting potential resource in synthetic biology, where engineered RNATs could prove to be useful tools in biosensors and conditional gene regulation. Results: Solving the 2-temperature inverse folding problem is critical for RNAT engineering. Here we introduce RNAiFold2T, the first Constraint Programming (CP) and Large Neighborhood Search (LNS) algorithms to solve this problem. Benchmarking tests of RNAiFold2T against existent programs (adaptive walk and genetic algorithm) inverse folding show that our software generates two orders of magnitude more solutions, thus allowing ample exploration of the space of solutions. Subsequently, solutions can be prioritized by computing various measures, including probability of target structure in the ensemble, melting temperature, etc. Using this strategy, we rationally designed two thermosensor internal ribosome entry site (thermo-IRES) elements, whose normalized cap-independent translation efficiency is approximately 50% greater at 42 °C than 30 °C, when tested in reticulocyte lysates. Translation efficiency is lower than that of the wild-type IRES element, which on the other hand is fully resistant to temperature shift-up. This appears to be the first purely computational design of functional RNA thermoswitches, and certainly the first purely computational design of functional thermo-IRES elements. Availability: RNAiFold2T is publicly available as part of the new release RNAiFold3.0 at https://github.com/clotelab/RNAiFold and http://bioinformatics.bc.edu/clotelab/RNAiFold, which latter has a web server as well. The software is written in C ++ and uses OR-Tools CP search engine. Contact: clote@bc.edu Supplementary information: Supplementary data are available at Bioinformatics online. PMID

  4. SKI knockdown inhibits human melanoma tumor growth in vivo.

    PubMed

    Chen, Dahu; Lin, Qiushi; Box, Neil; Roop, Dennis; Ishii, Shunsuke; Matsuzaki, Koichi; Fan, Tao; Hornyak, Thomas J; Reed, Jon A; Stavnezer, Ed; Timchenko, Nikolai A; Medrano, Estela E

    2009-12-01

    The SKI protein represses the TGF-beta tumor suppressor pathway by associating with the Smad transcription factors. SKI is upregulated in human malignant melanoma tumors in a disease-progression manner and its overexpression promotes proliferation and migration of melanoma cells in vitro. The mechanisms by which SKI antagonizes TGF-beta signaling in vivo have not been fully elucidated. Here we show that human melanoma cells in which endogenous SKI expression was knocked down by RNAi produced minimal orthotopic tumor xenograft nodules that displayed low mitotic rate and prominent apoptosis. These minute tumors exhibited critical signatures of active TGF-beta signaling including high levels of nuclear Smad3 and p21(Waf-1), which are not found in the parental melanomas. To understand how SKI promotes tumor growth we used gain- and loss-of-function approaches and found that simultaneously to blocking the TGF-beta-growth inhibitory pathway, SKI promotes the switch of Smad3 from tumor suppression to oncogenesis by favoring phosphorylations of the Smad3 linker region in melanoma cells but not in normal human melanocytes. In this context, SKI is required for preventing TGF-beta-mediated downregulation of the oncogenic protein c-MYC, and for inducing the plasminogen activator inhibitor-1, a mediator of tumor growth and angiogenesis. Together, the results indicate that SKI exploits multiple regulatory levels of the TGF-beta pathway and its deficiency restores TGF-beta tumor suppressor and apoptotic activities in spite of the likely presence of oncogenic mutations in melanoma tumors. PMID:19845874

  5. Tumor Selective Silencing Using an RNAi-Conjugated Polymeric Nanopharmaceutical.

    PubMed

    Svenson, Sonke; Case, Roy I; Cole, Roderick O; Hwang, Jungyeon; Kabir, Sujan R; Lazarus, Douglas; Lim Soo, Patrick; Ng, Pei-Sze; Peters, Christian; Shum, Pochi; Sweryda-Krawiec, Beata; Tripathi, Snehlata; van der Poll, Derek; Eliasof, Scott

    2016-03-01

    Small interfering RNA (siRNA) therapeutics have potential advantages over traditional small molecule drugs such as high specificity and the ability to inhibit otherwise "undruggable" targets. However, siRNAs have short plasma half-lives in vivo, can induce a cytokine response, and show poor cellular uptake. Formulating siRNA into nanoparticles offers two advantages: enhanced siRNA stability against nuclease degradation beyond what chemical modification alone can provide; and improved site-specific delivery that takes advantage of the enhanced permeability and retention (EPR) effect. Existing delivery systems generally suffer from poor delivery to tumors. Here we describe the formation and biological activity of polymeric nanopharmaceuticals (PNPs) based on biocompatible and biodegradable poly(lactic-co-glycolic acid) (PLGA) conjugated to siRNA via an intracellular cleavable disulfide linker (PLGA-siRNA). Additionally, these PNPs contain (1) PLGA conjugated to polyethylene glycol (PEG) for enhanced pharmacokinetics of the nanocarrier; (2) a cation for complexation of siRNA and charge compensation to avoid high negative zeta potential; and (3) neutral poly(vinyl alcohol) (PVA) to stabilize the PNPs and support the PEG shell to prevent particle aggregation and protein adsorption. The biological data demonstrate that these PNPs achieve prolonged circulation, tumor accumulation that is uniform throughout the tumor, and prolonged tumor-specific knockdown. PNPs employed in this study had no effect on body weight, blood cell count, serum chemistry, or cytokine response at doses >10 times the effective dose. PNPs, therefore, constitute a promising solution for achieving durable siRNA delivery and gene silencing in tumors. PMID:26835715

  6. Choosing the Right Tool for the Job: RNAi, TALEN or CRISPR

    PubMed Central

    Boettcher, Michael; McManus, Michael T.

    2015-01-01

    The most widely used approach for defining a genes’ function is to reduce or completely disrupt its normal expression. For over a decade, RNAi has ruled the lab, offering a magic bullet to disrupt gene expression in many organisms. However, new biotechnological tools - specifically CRISPR-based technologies - have become available and are squeezing out RNAi dominance in mammalian cell studies. These seemingly competing technologies leave research investigators with the question: ‘Which technology should I use in my experiment?’ This review offers a practical resource to compare and contrast these technologies, guiding the investigator when and where to use this fantastic array of powerful tools. PMID:26000843

  7. Knockdown of the cellular protein LRPPRC attenuates HIV-1 infection.

    PubMed

    Schweitzer, Cameron J; Matthews, John M; Madson, Christian J; Donnellan, Meghan R; Cerny, Ronald L; Belshan, Michael

    2012-01-01

    HIV-1 exploits numerous host cellular pathways for productive infection. To identify novel factors involved in HIV-1 replication, HIV-1 integrase and matrix protein complexes were captured at 4 hours post infection for proteomic analysis using an affinity purification system. Leucine-rich PPR-motif containing (LRPPRC) protein, a cellular protein involved in mitochondrial function, cell metabolism, and cell-cycle progression was identified as one of the candidate HIV-1 factors. Co-immunoprecipitation RT-PCR experiments confirmed that LRPPRC associated with HIV-1 nucleic acids during the early steps of virus infection. To establish if LRPPRC was critical for HIV-1 infection, three independent LRPPRC knockdown cell lines were constructed (2.7, 3.6, and 4.1). Subcellular fractionation of these cell lines revealed differential knockdown of LRPPRC in subcellular compartments. LRPPRC was knocked down in the insoluble/cytoskeletal fractions of all three cell lines, but the 3.6 and 4.1 cells also showed a reduction in nuclear LRPPRC. Additionally, several cellular factors were downregulated and/or disrupted by loss of LRPPRC. HIV-1 infection was reduced in all three cell lines, but virus production and RNA encapsidation were unaffected, suggesting that LRPPRC was critical for the afferent stage of virus replication. Two of the three cell lines (3.6, 4.1) were refractory for murine leukemia virus infection, a virus dependent on cellular proliferation for productive infection. Consistent with this, these two cell lines exhibited reduced cellular growth with no loss of cellular viability or change in cell cycle phenotype. The early steps of virus infection were also differentially affected among the cell lines. A reduced level of preintegration complex formation was observed in all three cell lines, but viral DNA nuclear import was reduced only in the 3.6 and 4.1 cells. Combined, these data identify LRPPRC as a HIV-1 factor that is involved in HIV-1 replication through more

  8. Suppression of RNAi by dsRNA-degrading RNaseIII enzymes of viruses in animals and plants.

    PubMed

    Weinheimer, Isabel; Jiu, Yaming; Rajamäki, Minna-Liisa; Matilainen, Olli; Kallijärvi, Jukka; Cuellar, Wilmer J; Lu, Rui; Saarma, Mart; Holmberg, Carina I; Jäntti, Jussi; Valkonen, Jari P T

    2015-03-01

    Certain RNA and DNA viruses that infect plants, insects, fish or poikilothermic animals encode Class 1 RNaseIII endoribonuclease-like proteins. dsRNA-specific endoribonuclease activity of the RNaseIII of rock bream iridovirus infecting fish and Sweet potato chlorotic stunt crinivirus (SPCSV) infecting plants has been shown. Suppression of the host antiviral RNA interference (RNAi) pathway has been documented with the RNaseIII of SPCSV and Heliothis virescens ascovirus infecting insects. Suppression of RNAi by the viral RNaseIIIs in non-host organisms of different kingdoms is not known. Here we expressed PPR3, the RNaseIII of Pike-perch iridovirus, in the non-hosts Nicotiana benthamiana (plant) and Caenorhabditis elegans (nematode) and found that it cleaves double-stranded small interfering RNA (ds-siRNA) molecules that are pivotal in the host RNA interference (RNAi) pathway and thereby suppresses RNAi in non-host tissues. In N. benthamiana, PPR3 enhanced accumulation of Tobacco rattle tobravirus RNA1 replicon lacking the 16K RNAi suppressor. Furthermore, PPR3 suppressed single-stranded RNA (ssRNA)--mediated RNAi and rescued replication of Flock House virus RNA1 replicon lacking the B2 RNAi suppressor in C. elegans. Suppression of RNAi was debilitated with the catalytically compromised mutant PPR3-Ala. However, the RNaseIII (CSR3) produced by SPCSV, which cleaves ds-siRNA and counteracts antiviral RNAi in plants, failed to suppress ssRNA-mediated RNAi in C. elegans. In leaves of N. benthamiana, PPR3 suppressed RNAi induced by ssRNA and dsRNA and reversed silencing; CSR3, however, suppressed only RNAi induced by ssRNA and was unable to reverse silencing. Neither PPR3 nor CSR3 suppressed antisense-mediated RNAi in Drosophila melanogaster. These results show that the RNaseIII enzymes of RNA and DNA viruses suppress RNAi, which requires catalytic activities of RNaseIII. In contrast to other viral silencing suppression proteins, the RNaseIII enzymes are homologous in

  9. Suppression of RNAi by dsRNA-Degrading RNaseIII Enzymes of Viruses in Animals and Plants

    PubMed Central

    Matilainen, Olli; Kallijärvi, Jukka; Cuellar, Wilmer J.; Lu, Rui; Saarma, Mart; Holmberg, Carina I.; Jäntti, Jussi; Valkonen, Jari P. T.

    2015-01-01

    Certain RNA and DNA viruses that infect plants, insects, fish or poikilothermic animals encode Class 1 RNaseIII endoribonuclease-like proteins. dsRNA-specific endoribonuclease activity of the RNaseIII of rock bream iridovirus infecting fish and Sweet potato chlorotic stunt crinivirus (SPCSV) infecting plants has been shown. Suppression of the host antiviral RNA interference (RNAi) pathway has been documented with the RNaseIII of SPCSV and Heliothis virescens ascovirus infecting insects. Suppression of RNAi by the viral RNaseIIIs in non-host organisms of different kingdoms is not known. Here we expressed PPR3, the RNaseIII of Pike-perch iridovirus, in the non-hosts Nicotiana benthamiana (plant) and Caenorhabditis elegans (nematode) and found that it cleaves double-stranded small interfering RNA (ds-siRNA) molecules that are pivotal in the host RNA interference (RNAi) pathway and thereby suppresses RNAi in non-host tissues. In N. benthamiana, PPR3 enhanced accumulation of Tobacco rattle tobravirus RNA1 replicon lacking the 16K RNAi suppressor. Furthermore, PPR3 suppressed single-stranded RNA (ssRNA)—mediated RNAi and rescued replication of Flock House virus RNA1 replicon lacking the B2 RNAi suppressor in C. elegans. Suppression of RNAi was debilitated with the catalytically compromised mutant PPR3-Ala. However, the RNaseIII (CSR3) produced by SPCSV, which cleaves ds-siRNA and counteracts antiviral RNAi in plants, failed to suppress ssRNA-mediated RNAi in C. elegans. In leaves of N. benthamiana, PPR3 suppressed RNAi induced by ssRNA and dsRNA and reversed silencing; CSR3, however, suppressed only RNAi induced by ssRNA and was unable to reverse silencing. Neither PPR3 nor CSR3 suppressed antisense-mediated RNAi in Drosophila melanogaster. These results show that the RNaseIII enzymes of RNA and DNA viruses suppress RNAi, which requires catalytic activities of RNaseIII. In contrast to other viral silencing suppression proteins, the RNaseIII enzymes are homologous in

  10. Vertically integrated translational studies of PDX1 as a therapeutic target for pancreatic cancer via a novel bifunctional RNAi platform.

    PubMed

    Wu, J; Liu, S; Yu, J; Zhou, G; Rao, D; Jay, C M; Kumar, P; Sanchez, R; Templeton, N; Senzer, N; Maples, P; Nemunaitis, J; Brunicardi, F C

    2014-02-01

    RNA interference (RNAi) represents a powerful, new tool for scientific investigation as well as a promising new form of targeted gene therapy, with applications currently in clinical trials. Bifunctional short hairpin RNA (shRNA) are synthetic RNAi molecules, engineered to utilize multiple endogenous RNAi pathways to specifically silence target genes. Pancreatic and duodenal homeobox 1 (PDX1) is a key regulator of pancreatic development, β-cell differentiation, normal β-cell function and pancreatic cancer. Our aim is to review the process of identifying PDX1 as a specific, potential RNAi target in pancreatic cancer, as well as the underlying mechanisms and various forms of RNAi, with subsequent testing and development of PDX1-targeted bifunctional shRNA therapy. PMID:24457987

  11. RNAi silencing of P/Q-type calcium channels in Purkinje neurons of adult mouse leads to episodic ataxia type 2.

    PubMed

    Salvi, Julie; Bertaso, Federica; Mausset-Bonnefont, Anne-Laure; Metz, Alexandra; Lemmers, Céline; Ango, Fabrice; Fagni, Laurent; Lory, Philippe; Mezghrani, Alexandre

    2014-08-01

    Episodic ataxia type-2 (EA2) is a dominantly inherited human neurological disorder caused by loss of function mutations in the CACNA1A gene, which encodes the CaV2.1 subunit of P/Q-type voltage-gated calcium channels. It remains however unknown whether the deficit of cerebellar CaV2.1 in adult is in direct link with the disease. To address this issue, we have used lentiviral based-vector RNA interference (RNAi) to knock-down CaV2.1 expression in the cerebellum of adult mice. We show that suppression of the P/Q-type channels in Purkinje neurons induced motor abnormalities, such as imbalance and ataxic gait. Interestingly, moderate channel suppression caused no basal ataxia, while β-adrenergic activation and exercise mimicked stress induced motor disorders. Moreover, stress-induced ataxia was stable, non-progressive and totally abolished by acetazolamide, a carbonic anhydrase inhibitor used to treat EA2. Altogether, these data reveal that P/Q-type channel suppression in adult mice supports the episodic status of EA2 disease. PMID:24768804

  12. Uncoupling protein-2 knockdown mediates the cytotoxic effects of cisplatin.

    PubMed

    Santandreu, Francisca M; Roca, Pilar; Oliver, Jordi

    2010-08-15

    Cisplatin is among the most important chemotherapeutic agents ever developed. However, more than a generation after its clinical introduction, its exact mechanism of action on tumor cells is not fully defined. The aim of this study was to investigate the role of oxidative stress as a mediator of cisplatin action on colon cancer cells, studying the influence of mitochondrial physiology and composition on its effectiveness. The chemosensitivity shown by cancer cells to mechanistically dissimilar antitumor drugs is shown to be associated with their capacity to induce early alterations in mitochondrial and redox metabolism. Specifically, cisplatin exerted a marked pro-oxidative action on mitochondria by inhibiting resting respiration and stimulating the immediate generation of ROS in isolated mitochondria. Antioxidants and mitochondrial uncouplers counteracted cisplatin-induced cytotoxicity in tumor cells, reflecting that oxidative stress and the inhibition of mitochondrial uncoupling are relevant to its antiproliferative activity. Additionally, inhibition of uncoupling protein-2 (UCP2) caused cytotoxicity in colon cancer cells via ROS of mitochondrial origin. In conclusion, we show for the first time that UCP2 knockdown participates in the mechanism of action of cisplatin, thus providing evidence that targeting UCP2 may offer clinical benefit in the treatment of cancer. PMID:20595066

  13. Hunchback knockdown induces supernumerary segment formation in Bombyx.

    PubMed

    Nakao, Hajime

    2016-05-15

    Insect segment number within species appears to be fixed irrespective of germ types: long vs. short/intermediate. The present study showed induction of supernumerary segment formation by the knockdown of Bombyx hunchback (Bm-hb), presumably by terminal segment addition, a short/intermediate-like-segmentation mode that is not observed in normal Bombyx embryogenesis. This suggests that Bm-hb suppresses segmentation. The results obtained also suggest that the gap gene Bm-Kr (Bombyx Krüppel) provides a permissive environment for the progression of segmentation by suppressing the expression Bm-hb, which terminates segmentation. This indicates a novel mechanism by which the gap gene is involved in segmentation. It appears that Bm-Kr and Bm-hb are involved in segment counting and their interplay contributes to the correct number of segments being formed in Bombyx. Similar mechanisms may be operating in insects that employ the non-Drosophilan mode of segmentation such as in short/intermediate-germ insects. PMID:27016505

  14. Knockdown of cathepsin L sensitizes ovarian cancer cells to chemotherapy

    PubMed Central

    ZHANG, HONGMEI; ZHANG, LUOSHENG; WEI, LIXIA; GAO, XINGWANG; TANG, LI; GONG, WEI; MIN, NA; ZHANG, LI; YUAN, YAWEI

    2016-01-01

    Ovarian cancer is a leading gynecological malignancy associated with high mortality. The development of acquired drug resistance is the primary cause of chemotherapy failure in the treatment of ovarian cancer. To examine the mechanism underlying paclitaxel resistance in ovarian cancer and attempt to reverse it, the present study induced a TAX-resistant ovarian cancer cell line, SKOV3/TAX. Cathepsin L (CTSL) has been found to be overexpressed in ovarian cancer. The aim of the present study was to investigate the possible involvement of CTSL in the development of TAX resistance in ovarian cancer. CTSL expression was knocked down in SKOV3 ovarian cancer cells and their phenotypic changes were analyzed. The effects of silenced CTSL on the resistant cell line were investigated by proliferation and apoptosis analysis compared with control SKOV3 cells. CTSL was more highly expressed in SKOV3/TAX cells compared with SKOV3 cells. Paclitaxel treatment downregulated the expression of CTSL in SKOV-3 but not in the paclitaxel-resistant SKOV3/TAX cells. CTSL small hairpin RNA (shRNA) knockdown significantly potentiated apoptosis induced by paclitaxel compared with SKOV3/TAX cells transfected with control shRNA, suggesting that CTSL contributes to paclitaxel resistance in ovarian cancer cells and that CTSL silencing can enhance paclitaxel-mediated cell apoptosis. Thus, CTSL should be explored as a candidate of therapeutic target for modulating paclitaxel sensitivity in ovarian cancer. PMID:27313771

  15. Knockdown of a JmjC domain-containing gene JMJ524 confers altered gibberellin responses by transcriptional regulation of GRAS protein lacking the DELLA domain genes in tomato

    PubMed Central

    Li, Jinhua; Yu, Chuying; Wu, Hua; Luo, Zhidan; Ouyang, Bo; Cui, Long; Zhang, Junhong; Ye, Zhibiao

    2015-01-01

    Plants integrate responses to independent hormonal and environmental signals to survive adversity. In particular, the phytohormone gibberellin (GA) regulates a variety of developmental processes and stress responses. In this study, the Jumonji-C (JmjC) domain-containing gene JMJ524 was characterized in tomato. JMJ524 responded to circadian rhythms and was upregulated by GA treatment. Knockdown of JMJ524 by RNAi caused a GA-insensitive dwarf phenotype with shrunken leaves and shortened internodes. However, in these transgenic plants, higher levels of endogenous GAs were detected. A genome-wide gene expression analysis by RNA-seq indicated that the expression levels of two DELLA-like genes, SlGLD1 (‘GRAS protein Lacking the DELLA domain’) and SlGLD2, were increased in JMJ524-RNAi transgenic plants. Nevertheless, only the overexpression of SlGLD1 in tomato resulted in a GA-insensitive dwarf phenotype, suggesting that SlGLD1 acts as a repressor of GA signalling. This study proposes that JMJ524 is required for stem elongation by altering GA responses, at least partially by regulating SlGLD1. PMID:25680796

  16. Effects of PHENYLALANINE AMMONIA LYASE (PAL) knockdown on cell wall composition, biomass digestibility, and biotic and abiotic stress responses in Brachypodium

    PubMed Central

    Cass, Cynthia L.; Peraldi, Antoine; Dowd, Patrick F.; Mottiar, Yaseen; Santoro, Nicholas; Karlen, Steven D.; Bukhman, Yury V.; Foster, Cliff E.; Thrower, Nick; Bruno, Laura C.; Moskvin, Oleg V.; Johnson, Eric T.; Willhoit, Megan E.; Phutane, Megha; Ralph, John; Mansfield, Shawn D.; Nicholson, Paul; Sedbrook, John C.

    2015-01-01

    The phenylpropanoid pathway in plants synthesizes a variety of structural and defence compounds, and is an important target in efforts to reduce cell wall lignin for improved biomass conversion to biofuels. Little is known concerning the trade-offs in grasses when perturbing the function of the first gene family in the pathway, PHENYLALANINE AMMONIA LYASE (PAL). Therefore, PAL isoforms in the model grass Brachypodium distachyon were targeted, by RNA interference (RNAi), and large reductions (up to 85%) in stem tissue transcript abundance for two of the eight putative BdPAL genes were identified. The cell walls of stems of BdPAL-knockdown plants had reductions of 43% in lignin and 57% in cell wall-bound ferulate, and a nearly 2-fold increase in the amounts of polysaccharide-derived carbohydrates released by thermochemical and hydrolytic enzymic partial digestion. PAL-knockdown plants exhibited delayed development and reduced root growth, along with increased susceptibilities to the fungal pathogens Fusarium culmorum and Magnaporthe oryzae. Surprisingly, these plants generally had wild-type (WT) resistances to caterpillar herbivory, drought, and ultraviolet light. RNA sequencing analyses revealed that the expression of genes associated with stress responses including ethylene biosynthesis and signalling were significantly altered in PAL knocked-down plants under non-challenging conditions. These data reveal that, although an attenuation of the phenylpropanoid pathway increases carbohydrate availability for biofuel, it can adversely affect plant growth and disease resistance to fungal pathogens. The data identify notable differences between the stress responses of these monocot pal mutants versus Arabidopsis (a dicot) pal mutants and provide insights into the challenges that may arise when deploying phenylpropanoid pathway-altered bioenergy crops. PMID:26093023

  17. Effects of PHENYLALANINE AMMONIA LYASE (PAL) knockdown on cell wall composition, biomass digestibility, and biotic and abiotic stress responses in Brachypodium.

    PubMed

    Cass, Cynthia L; Peraldi, Antoine; Dowd, Patrick F; Mottiar, Yaseen; Santoro, Nicholas; Karlen, Steven D; Bukhman, Yury V; Foster, Cliff E; Thrower, Nick; Bruno, Laura C; Moskvin, Oleg V; Johnson, Eric T; Willhoit, Megan E; Phutane, Megha; Ralph, John; Mansfield, Shawn D; Nicholson, Paul; Sedbrook, John C

    2015-07-01

    The phenylpropanoid pathway in plants synthesizes a variety of structural and defence compounds, and is an important target in efforts to reduce cell wall lignin for improved biomass conversion to biofuels. Little is known concerning the trade-offs in grasses when perturbing the function of the first gene family in the pathway, PHENYLALANINE AMMONIA LYASE (PAL). Therefore, PAL isoforms in the model grass Brachypodium distachyon were targeted, by RNA interference (RNAi), and large reductions (up to 85%) in stem tissue transcript abundance for two of the eight putative BdPAL genes were identified. The cell walls of stems of BdPAL-knockdown plants had reductions of 43% in lignin and 57% in cell wall-bound ferulate, and a nearly 2-fold increase in the amounts of polysaccharide-derived carbohydrates released by thermochemical and hydrolytic enzymic partial digestion. PAL-knockdown plants exhibited delayed development and reduced root growth, along with increased susceptibilities to the fungal pathogens Fusarium culmorum and Magnaporthe oryzae. Surprisingly, these plants generally had wild-type (WT) resistances to caterpillar herbivory, drought, and ultraviolet light. RNA sequencing analyses revealed that the expression of genes associated with stress responses including ethylene biosynthesis and signalling were significantly altered in PAL knocked-down plants under non-challenging conditions. These data reveal that, although an attenuation of the phenylpropanoid pathway increases carbohydrate availability for biofuel, it can adversely affect plant growth and disease resistance to fungal pathogens. The data identify notable differences between the stress responses of these monocot pal mutants versus Arabidopsis (a dicot) pal mutants and provide insights into the challenges that may arise when deploying phenylpropanoid pathway-altered bioenergy crops. PMID:26093023

  18. Multiple shRNA expressions in a single plasmid vector improve RNAi against the XPA gene

    SciTech Connect

    Nagao, Akihiro; Zhao, Xia; Takegami, Tsutomu; Nakagawa, Hideaki; Matsui, Shinobu; Matsunaga, Tsukasa; Ishigaki, Yasuhito

    2008-05-30

    To improve the efficiency of stable knockdown with short hairpin RNA (shRNA), we inserted multiple shRNA expression sequences into a single plasmid vector. In this study, the DNA repair factor XPA was selected as a target gene since it is not essential for cell viability and it is easy to check the functional knockdown of this gene. The efficiency of knockdown was compared among single and triple expression vectors. The single shRNA-expressing vector caused limited knockdown of the target protein in stable transfectants, however, the multiple expression vectors apparently increased the frequency of knockdown transfectants. There were correlations between the knockdown level and marker expression in multiple-expressing transfectants, whereas poorer correlations were observed in single vector transfectants. Multiple-transfectants exhibited reduced efficiency of repair of UV-induced DNA damage and an increased sensitivity to ultraviolet light-irradiation. We propose that multiple shRNA expression vectors might be a useful strategy for establishing knockdown cells.

  19. VSG switching in Trypanosoma brucei: antigenic variation analysed using RNAi in the absence of immune selection

    PubMed Central

    Aitcheson, Niall; Talbot, Suzanne; Shapiro, Jesse; Hughes, Katie; Adkin, Carl; Butt, Thomas; Sheader, Karen; Rudenko, Gloria

    2006-01-01

    Summary Trypanosoma brucei relies on antigenic variation of its Variant Surface Glycoprotein (VSG) coat for survival. We show that VSG switching can be efficiently studied in vitro using VSG RNAi in place of an immune system to select for switch variants. Contrary to models predicting an instant switch after inhibition of VSG synthesis, switching was not induced by VSG RNAi and occurred at a rate of 10−4 per division. We find a highly reproducible hierarchy of VSG activation which appears to be capable of resetting, whereby more than half of the switch events over 12 experiments were to one of two VSGs. We characterised switched clones according to switch mechanism using marker genes in the active VSG expression site (ES). Transcriptional switches between ESs were the preferred switching mechanism, whereby at least 10 of the 17 ESs identified in T. brucei 427 can be functionally active in vitro. We could specifically select for switches mediated by DNA rearrangements by inducing VSG RNAi in the presence of drug selection for the active ES. Most of the preferentially activated VSGs could be activated by multiple mechanisms. This VSG RNAi based procedure provides a rapid and powerful means for analysing VSG switching in African trypanosomes entirely in vitro. PMID:16135228

  20. RNA Interference (RNAi) Induced Gene Silencing: A Promising Approach of Hi-Tech Plant Breeding

    PubMed Central

    Younis, Adnan; Siddique, Muhammad Irfan; Kim, Chang-Kil; Lim, Ki-Byung

    2014-01-01

    RNA interference (RNAi) is a promising gene regulatory approach in functional genomics that has significant impact on crop improvement which permits down-regulation in gene expression with greater precise manner without affecting the expression of other genes. RNAi mechanism is expedited by small molecules of interfering RNA to suppress a gene of interest effectively. RNAi has also been exploited in plants for resistance against pathogens, insect/pest, nematodes, and virus that cause significant economic losses. Keeping beside the significance in the genome integrity maintenance as well as growth and development, RNAi induced gene syntheses are vital in plant stress management. Modifying the genes by the interference of small RNAs is one of the ways through which plants react to the environmental stresses. Hence, investigating the role of small RNAs in regulating gene expression assists the researchers to explore the potentiality of small RNAs in abiotic and biotic stress management. This novel approach opens new avenues for crop improvement by developing disease resistant, abiotic or biotic stress tolerant, and high yielding elite varieties. PMID:25332689

  1. [Mutation frequencies in HIV-1 subtype-A genome in regions containing efficient RNAi targets].

    PubMed

    Kravatsky, Y V; Chechetkin, V R; Fedoseeva, D M; Gorbacheva, M A; Kretova, O V; Tchurikov, N A

    2016-01-01

    The development of gene-therapy technology using RNAi for AIDS/HIV-1 treatment is a prospective alternative to traditional anti-retroviral therapy. RNAi targets could be selected in HIV-1 transcripts and in CCR5 mRNA. Previously, we experimentally selected a number of efficient siRNAs that target HIV-1 RNAs. The viral genome mutates frequently, and RNAi strength is very sensitive, even for a single mismatches. That is why it is important to study nucleotide sequences of targets in clinical isolates of HIV-1. In the present study, we analyzed mutations in 6 of about 300-bp regions containing RNAi targets from HIV-1 subtype A isolates in Russia. Estimates of the mean frequencies of mutations in the targets were obtained and the frequencies of mutations in the different codon positions were compared. The frequencies of mutations in the vicinity of the targets and directly within the targets were also compared and have been shown to be approximately the same. The frequencies of indels in the chosen regions have been assessed. Their frequencies have proved to be two to three orders of magnitude less compared to that for mutations. PMID:27414786

  2. RNAiFold: a web server for RNA inverse folding and molecular design.

    PubMed

    Garcia-Martin, Juan Antonio; Clote, Peter; Dotu, Ivan

    2013-07-01

    Synthetic biology and nanotechnology are poised to make revolutionary contributions to the 21st century. In this article, we describe a new web server to support in silico RNA molecular design. Given an input target RNA secondary structure, together with optional constraints, such as requiring GC-content to lie within a certain range, requiring the number of strong (GC), weak (AU) and wobble (GU) base pairs to lie in a certain range, the RNAiFold web server determines one or more RNA sequences, whose minimum free-energy secondary structure is the target structure. RNAiFold provides access to two servers: RNA-CPdesign, which applies constraint programming, and RNA-LNSdesign, which applies the large neighborhood search heuristic; hence, it is suitable for larger input structures. Both servers can also solve the RNA inverse hybridization problem, i.e. given a representation of the desired hybridization structure, RNAiFold returns two sequences, whose minimum free-energy hybridization is the input target structure. The web server is publicly accessible at http://bioinformatics.bc.edu/clotelab/RNAiFold, which provides access to two specialized servers: RNA-CPdesign and RNA-LNSdesign. Source code for the underlying algorithms, implemented in COMET and supported on linux, can be downloaded at the server website. PMID:23700314

  3. Transcriptome analysis and systemic RNAi response in the African sweetpotato weevil (Cylas puncticollis, Coleoptera, Brentidae).

    PubMed

    Prentice, Katterinne; Pertry, Ine; Christiaens, Olivier; Bauters, Lander; Bailey, Ana; Niblett, Chuck; Ghislain, Marc; Gheysen, Godelieve; Smagghe, Guy

    2015-01-01

    The African sweetpotato weevil (SPW) Cylas puncticollis Boheman is one of the most important constraints of sweetpotato production in Sub-Saharan Africa and yet is largely an uncharacterized insect pest. Here, we report on the transcriptome analysis of SPW generated using an Illumina platform. More than 213 million sequencing reads were obtained and assembled into 89,599 contigs. This assembly was followed by a gene ontology annotation. Subsequently, a transcriptome search showed that the necessary RNAi components relevant to the three major RNAi pathways, were found to be expressed in SPW. To address the functionality of the RNAi mechanism in this species, dsRNA was injected into second instar larvae targeting laccase2, a gene which encodes an enzyme involved in the sclerotization of insect exoskeleton. The body of treated insects showed inhibition of sclerotization, leading eventually to death. Quantitative Real Time PCR (qPCR) confirmed this phenotype to be the result of gene silencing. Together, our results provide valuable sequence data on this important insect pest and demonstrate that a functional RNAi pathway with a strong and systemic effect is present in SPW and can further be explored as a new strategy for controlling this important pest. PMID:25590333

  4. Phenotypic screen for RNAi effects in the codling moth Cydia pomonella.

    Technology Transfer Automated Retrieval System (TEKTRAN)

    RNAi-based technologies have the potential to augment, or replace existing pest management strategies. However, some insect taxa are less susceptible to the induction of the post-transcriptional gene silencing effect than others, such as the Lepidoptera. Here we describe experiments to investigate t...

  5. Transcript analysis of sedentary female reniform nematodes identifies potential targets ofr RNAi-mediated resistance

    Technology Transfer Automated Retrieval System (TEKTRAN)

    RNA-interference (RNAi) has become an attractive avenue of research in the development of crop resistance to sedentary plant-parasitic nematodes. A prerequisite for this type of research is the availability of high quality gene sequence data for the nematode in question. The reniform nematode (Rot...

  6. RNAi and emerging psyllid genomes - increasing the number of genetic targets

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Genomics has changed the strategies used to manage insects and diseases. The ability to effect a change in proteins and transcripts through RNA-interference (RNAi) has produced a rush towards the development of the most state-of-the-art pest suppression strategy available. To rapidly advance these e...

  7. iScreen: Image-Based High-Content RNAi Screening Analysis Tools.

    PubMed

    Zhong, Rui; Dong, Xiaonan; Levine, Beth; Xie, Yang; Xiao, Guanghua

    2015-09-01

    High-throughput RNA interference (RNAi) screening has opened up a path to investigating functional genomics in a genome-wide pattern. However, such studies are often restricted to assays that have a single readout format. Recently, advanced image technologies have been coupled with high-throughput RNAi screening to develop high-content screening, in which one or more cell image(s), instead of a single readout, were generated from each well. This image-based high-content screening technology has led to genome-wide functional annotation in a wider spectrum of biological research studies, as well as in drug and target discovery, so that complex cellular phenotypes can be measured in a multiparametric format. Despite these advances, data analysis and visualization tools are still largely lacking for these types of experiments. Therefore, we developed iScreen (image-Based High-content RNAi Screening Analysis Tool), an R package for the statistical modeling and visualization of image-based high-content RNAi screening. Two case studies were used to demonstrate the capability and efficiency of the iScreen package. iScreen is available for download on CRAN (http://cran.cnr.berkeley.edu/web/packages/iScreen/index.html). The user manual is also available as a supplementary document. PMID:25548139

  8. Human Papillomavirus: Current and Future RNAi Therapeutic Strategies for Cervical Cancer

    PubMed Central

    Jung, Hun Soon; Rajasekaran, Nirmal; Ju, Woong; Shin, Young Kee

    2015-01-01

    Human papillomaviruses (HPVs) are small DNA viruses; some oncogenic ones can cause different types of cancer, in particular cervical cancer. HPV-associated carcinogenesis provides a classical model system for RNA interference (RNAi) based cancer therapies, because the viral oncogenes E6 and E7 that cause cervical cancer are expressed only in cancerous cells. Previous studies on the development of therapeutic RNAi facilitated the advancement of therapeutic siRNAs and demonstrated its versatility by siRNA-mediated depletion of single or multiple cellular/viral targets. Sequence-specific gene silencing using RNAi shows promise as a novel therapeutic approach for the treatment of a variety of diseases that currently lack effective treatments. However, siRNA-based targeting requires further validation of its efficacy in vitro and in vivo, for its potential off-target effects, and of the design of conventional therapies to be used in combination with siRNAs and their drug delivery vehicles. In this review we discuss what is currently known about HPV-associated carcinogenesis and the potential for combining siRNA with other treatment strategies for the development of future therapies. Finally, we present our assessment of the most promising path to the development of RNAi therapeutic strategies for clinical settings. PMID:26239469

  9. RNAi-based insecticidal crops: potential effects on non-target species

    Technology Transfer Automated Retrieval System (TEKTRAN)

    RNAi is a sequence specific mechanism that silences protein production when particular mRNAs are bound and enzymatically cleaved. Genetically modified crops that silence critical gene function in insect pests have been developed, and are a likely future direction for commercial pest management. Pote...

  10. Transcriptome Analysis and Systemic RNAi Response in the African Sweetpotato Weevil (Cylas puncticollis, Coleoptera, Brentidae)

    PubMed Central

    Prentice, Katterinne; Pertry, Ine; Christiaens, Olivier; Bauters, Lander; Bailey, Ana; Niblett, Chuck; Ghislain, Marc; Gheysen, Godelieve; Smagghe, Guy

    2015-01-01

    The African sweetpotato weevil (SPW) Cylas puncticollis Boheman is one of the most important constraints of sweetpotato production in Sub-Saharan Africa and yet is largely an uncharacterized insect pest. Here, we report on the transcriptome analysis of SPW generated using an Illumina platform. More than 213 million sequencing reads were obtained and assembled into 89,599 contigs. This assembly was followed by a gene ontology annotation. Subsequently, a transcriptome search showed that the necessary RNAi components relevant to the three major RNAi pathways, were found to be expressed in SPW. To address the functionality of the RNAi mechanism in this species, dsRNA was injected into second instar larvae targeting laccase2, a gene which encodes an enzyme involved in the sclerotization of insect exoskeleton. The body of treated insects showed inhibition of sclerotization, leading eventually to death. Quantitative Real Time PCR (qPCR) confirmed this phenotype to be the result of gene silencing. Together, our results provide valuable sequence data on this important insect pest and demonstrate that a functional RNAi pathway with a strong and systemic effect is present in SPW and can further be explored as a new strategy for controlling this important pest. PMID:25590333

  11. RNAi as a management tool for the western corn rootworm, Diabrotica virgifera virgifera.

    PubMed

    Fishilevich, Elane; Vélez, Ana M; Storer, Nicholas P; Li, Huarong; Bowling, Andrew J; Rangasamy, Murugesan; Worden, Sarah E; Narva, Kenneth E; Siegfried, Blair D

    2016-09-01

    The western corn rootworm (WCR), Diabrotica virgifera virgifera, is the most important pest of corn in the US Corn Belt. Economic estimates indicate that costs of control and yield loss associated with WCR damage exceed $US 1 billion annually. Historically, corn rootworm management has been extremely difficult because of its ability to evolve resistance to both chemical insecticides and cultural control practices. Since 2003, the only novel commercialized developments in rootworm management have been transgenic plants expressing Bt insecticidal proteins. Four transgenic insecticidal proteins are currently registered for rootworm management, and field resistance to proteins from the Cry3 family highlights the importance of developing traits with new modes of action. One of the newest approaches for controlling rootworm pests involves RNA interference (RNAi). This review describes the current understanding of the RNAi mechanisms in WCR and the use of this technology for WCR management. Further, the review addresses ecological risk assessment of RNAi and insect resistance management of RNAi for corn rootworm. © 2016 Society of Chemical Industry. PMID:27218412

  12. Clone mapper: an online suite of tools for RNAi experiments in Caenorhabditis elegans.

    PubMed

    Thakur, Nishant; Pujol, Nathalie; Tichit, Laurent; Ewbank, Jonathan J

    2014-11-01

    RNA interference (RNAi), mediated by the introduction of a specific double-stranded RNA, is a powerful method to investigate gene function. It is widely used in the Caenorhabditis elegans research community. An expanding number of laboratories conduct genome-wide RNAi screens, using standard libraries of bacterial clones each designed to produce a specific double-stranded RNA. Proper interpretation of results from RNAi experiments requires a series of analytical steps, from the verification of the identity of bacterial clones, to the identification of the clones' potential targets. Despite the popularity of the technique, no user-friendly set of tools allowing these steps to be carried out accurately, automatically, and at a large scale, is currently available. We report here the design and production of Clone Mapper, an online suite of tools specifically adapted to the analysis pipeline typical for RNAi experiments with C. elegans. We show that Clone Mapper overcomes the limitations of existing techniques and provide examples illustrating its potential for the identification of biologically relevant genes. The Clone Mapper tools are freely available via http://www.ciml.univ-mrs.fr/EWBANK_jonathan/software.html. PMID:25187039

  13. RNAi-directed post transcriptional gene silencing of an Arabidopsis Myb transgene in tobacco

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The AtMyb90 gene encodes the 'production of anthocyanin pigment 2' (PAP2) transcription factor of Arabidopsis thaliana and is able to induce a visible hyper-pigmented phenotype when expressed in tobacco. Based upon this phenotype, we have used the AtMyb90 gene as a reporter gene to examine RNAi-dire...

  14. Ribonucleic acid interference (RNAi) technology for control of Asian citrus psyllid

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Ribonucleic acid interference, RNAi, applications and function are described for the non-scientist to bring a better understanding of how this emerging technology is providing environmentally friendly, non-transgenic, insect pest control to the citrus industry. Two part Video presentation....

  15. A novel platform to enable inhaled naked RNAi medicine for lung cancer.

    PubMed

    Fujita, Yu; Takeshita, Fumitaka; Mizutani, Takayuki; Ohgi, Tadaaki; Kuwano, Kazuyoshi; Ochiya, Takahiro

    2013-01-01

    Small interfering RNA (siRNA)-based therapeutics have been used in humans and offer distinct advantages over traditional therapies. However, previous investigations have shown that there are several technical obstacles that need to be overcome before routine clinical applications are used. Currently, we are launching a novel class of RNAi therapeutic agents (PnkRNA™, nkRNA) that show high resistance to degradation and are less immunogenic, less cytotoxic, and capable of efficient intracellular delivery. Here, we develop a novel platform to promote naked RNAi approaches administered through inhalation without sophisticated delivery technology in mice. Furthermore, a naked and unmodified novel RNAi agent, such as ribophorin II (RPN2-PnkRNA), which has been selected as a therapeutic target for lung cancer, resulted in efficient inhibition of tumor growth without any significant toxicity. Thus, this new technology using aerosol delivery could represent a safe, potentially RNAi-based strategy for clinical applications in lung cancer treatment without delivery vehicles. PMID:24270189

  16. Technological development of structural DNA/RNA-based RNAi systems and their applications.

    PubMed

    Jeong, Eun Hye; Kim, Hyejin; Jang, Bora; Cho, Hyesoo; Ryu, Jaehee; Kim, Boyeon; Park, Youngkuk; Kim, Jieun; Lee, Jong Bum; Lee, Hyukjin

    2016-09-01

    RNA interference (RNAi)-based gene therapy has drawn tremendous attention due to its highly specific gene regulation by selective degradation of any target mRNA. There have been multiple reports regarding the development of various cationic materials for efficient siRNA delivery, however, many studies still suffer from the conventional delivery problems such as suboptimal transfection performance, a lack of tissue specificity, and potential cytotoxicity. Despite the huge therapeutic potential of siRNAs, conventional gene carriers have failed to guarantee successful gene silencing in vivo, thus not warranting clinical trials. The relatively short double-stranded structure of siRNAs has resulted in uncompromising delivery formulations, as well as low transfection efficiency, compared with the conventional nucleic acid drugs such as plasmid DNAs. Recent developments in structural siRNA and RNAi nanotechnology have enabled more refined and reliable in vivo gene silencing with multiple advantages over naked siRNAs. This review focuses on recent progress in the development of structural DNA/RNA-based RNAi systems and their potential therapeutic applications. In addition, an extensive list of prior reports on various RNAi systems is provided and categorized by their distinctive molecular characters. PMID:26494399

  17. Targeting Atp6v1c1 Prevents Inflammation and Bone Erosion Caused by Periodontitis and Reveals Its Critical Function in Osteoimmunology

    PubMed Central

    Wang, Lin; Lu, Yun; Li, Qian; Zhu, Zheng; Shao, Jian-Zhong; Chen, Wei

    2015-01-01

    Periodontal disease (Periodontitis) is a serious disease that affects a majority of adult Americans and is associated with other systemic diseases, including diabetes, rheumatoid arthritis, and other inflammatory diseases. While great efforts have been devoted toward understanding the pathogenesis of periodontitis, there remains a pressing need for developing potent therapeutic strategies for targeting this pervasive and destructive disease. In this study, we utilized novel adeno-associated virus (AAV)-mediated Atp6v1c1 knockdown gene therapy to treat bone erosion and inflammatory caused by periodontitis in mouse model. Atp6v1c1 is a subunit of the V-ATPase complex and regulator of the assembly of the V0 and V1 domains of the V-ATPase complex. We demonstrated previously that Atp6v1c1 has an essential function in osteoclast mediated bone resorption. We hypothesized that Atp6v1c1 may be an ideal target to prevent the bone erosion and inflammation caused by periodontitis. To test the hypothesis, we employed AAV RNAi knockdown of Atp6v1c1 gene expression to prevent bone erosion and gingival inflammation simultaneously. We found that lesion-specific injection of AAV-shRNA-Atp6v1c1 into the periodontal disease lesions protected against bone erosion (>85%) and gingival inflammation caused by P. gingivalis W50 infection. AAV-mediated Atp6v1c1 knockdown dramatically reduced osteoclast numbers and inhibited the infiltration of dendritic cells and macrophages in the bacteria-induced inflammatory lesions in periodontitis. Silencing of Atp6v1c1 expression also prevented the expressions of osteoclast-related genes and pro-inflammatory cytokine genes. Our data suggests that AAV-shRNA-Atp6v1c1 treatment can significantly attenuate the bone erosion and inflammation caused by periodontitis, indicating the dual function of AAV-shRNA-Atp6v1c1 as an inhibitor of bone erosion mediated by osteoclasts, and as an inhibitor of inflammation through down-regulation of pro

  18. Mutations in the antiviral RNAi defense pathway modify Brome mosaic virus RNA recombinant profiles.

    PubMed

    Dzianott, Aleksandra; Sztuba-Solińska, Joanna; Bujarski, Jozef J

    2012-01-01

    RNA interference (RNAi) mechanism targets viral RNA for degradation. To test whether RNAi gene products contributed to viral RNA recombination, a series of Arabidopsis thaliana RNAi-defective mutants were infected with Brome mosaic virus (BMV) RNAs that have been engineered to support crossovers within the RNA3 segment. Single-cross RNA3-RNA1, RNA3-RNA2, and RNA3-RNA3 recombinants accumulated in both the wild-type (wt) and all knock-out lines at comparable frequencies. However, a reduced accumulation of novel 3' mosaic RNA3 recombinants was observed in ago1, dcl2, dcl4, and rdr6 lines but not in wt Col-0 or the dcl3 line. A BMV replicase mutant accumulated a low level of RNA3-RNA1 single-cross recombinants in Col-0 plants while, in a dcl2 dcl4 double mutant, the formation of both RNA3-RNA1 and mosaic recombinants was at a low level. A control infection in the cpr5-2 mutant, a more susceptible BMV Arabidopsis host, generated similar-to-Col-0 profiles of both single-cross and mosaic recombinants, indicating that recombinant profiles were, to some extent, independent of a viral replication rate. Also, the relative growth experiments revealed similar selection pressure for recombinants among the host lines. Thus, the altered recombinant RNA profiles have originated at the level of recombinant formation rather than because of altered selection. In conclusion, the viral replicase and the host RNAi gene products contribute in distinct ways to BMV RNA recombination. Our studies reveal that the antiviral RNAi mechanisms are utilized by plant RNA viruses to increase their variability, reminiscent of phenomena previously demonstrated in fungi. PMID:21936664

  19. ATP citrate lyase knockdown impacts cancer stem cells in vitro

    PubMed Central

    Hanai, J-i; Doro, N; Seth, P; Sukhatme, V P

    2013-01-01

    ATP citrate lyase (ACL) knockdown (KD) causes tumor suppression and induces differentiation. We have previously reported that ACL KD reverses epithelial–mesenchymal transition (EMT) in lung cancer cells. Because EMT is often associated with processes that induce stemness, we hypothesized that ACL KD impacts cancer stem cells. By assessing tumorsphere formation and expression of stem cell markers, we showed this to be the case in A549 cells, which harbor a Ras mutation, and in two other non-small-cell lung cancer cell lines, H1975 and H1650, driven by activating EGFR mutations. Inducible ACL KD had the same effect as stable ACL KD. Similar effects were noted in another well-characterized Ras-induced mammary model system (HMLER). Moreover, treatment with hydroxycitrate phenocopied the effects of ACL KD, suggesting that the enzymatic activity of ACL was critical. Indeed, acetate treatment reversed the ACL KD phenotype. Having previously established that ACL KD impacts signaling through the phosphatidylinositol 3-kinase (PI3K) pathway, not the Ras-mitogen-activated protein kinase (MAPK) pathway, and that EMT can be reversed by PI3K inhibitors, we were surprised to find that stemness in these systems was maintained through Ras-MAPK signaling, and not via PI3K signaling. Snail is a downstream transcription factor impacted by Ras-MAPK signaling and known to promote EMT and stemness. We found that snail expression was reduced by ACL KD. In tumorigenic HMLER cells, ACL overexpression increased snail expression and stemness, both of which were reduced by ACL KD. Furthermore, ACL could not initiate either tumorigenesis or stemness by itself. ACL and snail proteins interacted and ACL expression regulated the transcriptional activity of snail. Finally, ACL KD counteracted stem cell characteristics induced in diverse cell systems driven by activation of pathways outside of Ras-MAPK signaling. Our findings unveil a novel aspect of ACL function, namely its impact on cancer

  20. ATP citrate lyase knockdown impacts cancer stem cells in vitro.

    PubMed

    Hanai, J-I; Doro, N; Seth, P; Sukhatme, V P

    2013-01-01

    ATP citrate lyase (ACL) knockdown (KD) causes tumor suppression and induces differentiation. We have previously reported that ACL KD reverses epithelial-mesenchymal transition (EMT) in lung cancer cells. Because EMT is often associated with processes that induce stemness, we hypothesized that ACL KD impacts cancer stem cells. By assessing tumorsphere formation and expression of stem cell markers, we showed this to be the case in A549 cells, which harbor a Ras mutation, and in two other non-small-cell lung cancer cell lines, H1975 and H1650, driven by activating EGFR mutations. Inducible ACL KD had the same effect as stable ACL KD. Similar effects were noted in another well-characterized Ras-induced mammary model system (HMLER). Moreover, treatment with hydroxycitrate phenocopied the effects of ACL KD, suggesting that the enzymatic activity of ACL was critical. Indeed, acetate treatment reversed the ACL KD phenotype. Having previously established that ACL KD impacts signaling through the phosphatidylinositol 3-kinase (PI3K) pathway, not the Ras-mitogen-activated protein kinase (MAPK) pathway, and that EMT can be reversed by PI3K inhibitors, we were surprised to find that stemness in these systems was maintained through Ras-MAPK signaling, and not via PI3K signaling. Snail is a downstream transcription factor impacted by Ras-MAPK signaling and known to promote EMT and stemness. We found that snail expression was reduced by ACL KD. In tumorigenic HMLER cells, ACL overexpression increased snail expression and stemness, both of which were reduced by ACL KD. Furthermore, ACL could not initiate either tumorigenesis or stemness by itself. ACL and snail proteins interacted and ACL expression regulated the transcriptional activity of snail. Finally, ACL KD counteracted stem cell characteristics induced in diverse cell systems driven by activation of pathways outside of Ras-MAPK signaling. Our findings unveil a novel aspect of ACL function, namely its impact on cancer

  1. Knockdown of phosphoethanolamine transmethylation enzymes decreases viability of Haemonchus contortus.

    PubMed

    Witola, William H; Cooks-Fagbodun, Sheritta; Ordonez, Adriana Reyes; Matthews, Kwame; Abugri, Daniel A; McHugh, Mark

    2016-06-15

    The phosphobase methylation pathway, in which phosphoethanolamine N-methyltransferases (PMTs) successively catalyze the methylation of phosphoethanolamine to phosphocholine, is essential in the free-living nematode Caenorhabditis elegans. Two PMT-encoding genes (HcPMT1 and HcPMT2) cloned from Haemonchus contortus have been shown, by in vitro assays, to possess enzymatic characteristics similar to those of C. elegans PMTs, but their physiological significance in H. contortus is yet to be elucidated. Therefore, in this study, we endeavored to determine the importance of HcPMT1 and HcPMT2 in the survival of H. contortus by adapting the use of phosphorodiamidate morpholino oligomers (PPMO) antisense approach to block the translation of HcPMT1 and HcPMT2 in the worms. We found that PPMOs targeting HcPMT1 and HcPMT2 down-regulated the expression of HcPMT1 and HcPMT2 proteins in adult H. contortus. Analysis of the effect of HcPMT1 and HcPMT2 knockdown showed that it significantly decreased worm motility and viability, thus validating HcPMT1 and HcPMT2 as essential enzymes for survival of H. contortus. Studies of gene function in H. contortus have been constrained by limited forward and reverse genetic technologies for use in H. contortus. Thus, our success in adaptation of use of PPMO antisense approach in H. contortus provides an important reverse genetic technological advance for studying this parasitic nematode of veterinary significance. PMID:27198768

  2. Targeted Knockdown of RNA-Binding Protein TIAR for Promoting Self-Renewal and Attenuating Differentiation of Mouse Embryonic Stem Cells.

    PubMed

    Geng, Zhe; Li, Ping; Tan, Li; Song, Houyan

    2015-01-01

    RNA-binding protein TIAR has been suggested to mediate the translational silencing of ARE-containing mRNAs. To analyze the functions of TIAR, we established RNAi and genetic rescue assays. We evaluated the expression of neuroectoderm markers Pax6 and nestin, mesoderm markers brachyury and Flk1, and hypoblast and definitive endoderm markers Sox17 and Gata6 during EB differentiation and found that knockdown TIAR expression restrained the differentiation of E14 cells. We assessed gene expression levels of Flk-1 and VE-cadherin and observed attenuated differentiation of E14 cells into endothelial cells upon downregulation of TIAR gene expression. As such, we hypothesized an essential role of TIAR related to EB differentiation. As TIAR inhibits the translation of c-myc, we proposed that downregulation of TIAR results in restrained differentiation of E14 cells, due in part to the function of c-myc. We found that TIAR inhibited c-myc expression at the translational level in E14 cells; accordingly, a reduction of TIAR expression promoted self-renewal of pluripotent cells and attenuated differentiation. Additionally, we established that TIAR inhibited TIA-1 expression at the translational level in E14 cells. Taken together, we have contributed to the understanding of the regulatory relationships between TIAR and both c-myc and TIA-1. PMID:25918534

  3. Inducible Knock-Down of the Mineralocorticoid Receptor in Mice Disturbs Regulation of the Renin-Angiotensin-Aldosterone System and Attenuates Heart Failure Induced by Pressure Overload

    PubMed Central

    Montes-Cobos, Elena; Li, Xiao; Fischer, Henrike J.; Sasse, André; Kügler, Sebastian; Didié, Michael; Toischer, Karl; Fassnacht, Martin; Dressel, Ralf; Reichardt, Holger M.

    2015-01-01

    Mineralocorticoid receptor (MR) inactivation in mice results in early postnatal lethality. Therefore we generated mice in which MR expression can be silenced during adulthood by administration of doxycycline (Dox). Using a lentiviral approach, we obtained two lines of transgenic mice harboring a construct that allows for regulatable MR inactivation by RNAi and concomitant expression of eGFP. MR mRNA levels in heart and kidney of inducible MR knock-down mice were unaltered in the absence of Dox, confirming the tightness of the system. In contrast, two weeks after Dox administration MR expression was significantly diminished in a variety of tissues. In the kidney, this resulted in lower mRNA levels of selected target genes, which was accompanied by strongly increased serum aldosterone and plasma renin levels as well as by elevated sodium excretion. In the healthy heart, gene expression and the amount of collagen were unchanged despite MR levels being significantly reduced. After transverse aortic constriction, however, cardiac hypertrophy and progressive heart failure were attenuated by MR silencing, fibrosis was unaffected and mRNA levels of a subset of genes reduced. Taken together, we believe that this mouse model is a useful tool to investigate the role of the MR in pathophysiological processes. PMID:26605921

  4. Absence of transitive and systemic pathways allows cell-specific and isoform-specific RNAi in Drosophila

    PubMed Central

    ROIGNANT, JEAN-YVES; CARRÉ, CLÉMENT; MUGAT, BRUNO; SZYMCZAK, DIMITRI; LEPESANT, JEAN-ANTOINE; ANTONIEWSKI, CHRISTOPHE

    2003-01-01

    RNA interference (RNAi) designates the multistep process by which double-stranded RNA induces the silencing of homologous endogenous genes. Some aspects of RNAi appear to be conserved throughout evolution, including the processing of trigger dsRNAs into small 21–23-bp siRNAs and their use to guide the degradation of complementary mRNAs. Two remarkable features of RNAi were uncovered in plants and Caenorhabditid elegans. First, RNA-dependent RNA polymerase activities allow the synthesis of siRNA complementary to sequences upstream of or downstream from the initial trigger region in the target mRNA, leading to a transitive RNAi with sequences that had not been initially targeted. Secondly, systemic RNAi may cause the targeting of gene silencing in one tissue to spread to other tissues. Using transgenes expressing dsRNA, we investigated whether transitive and systemic RNAi occur in Drosophila. DsRNA-producing transgenes targeted RNAi to specific regions of alternative mRNA species of one gene without transitive effect directed to sequences downstream from or upstream of the initial trigger region. Moreover, specific expression of a dsRNA, using either cell-specific GAL4 drivers or random clonal activation of a GAL4 driver, mediated a cell-autonomous RNAi. Together, our results provide evidence that transitive and systemic aspects of RNAi are not conserved in Drosophila and demonstrate that dsRNA-producing transgenes allow powerful reverse genetic approaches to be conducted in this model organism, by knocking down gene functions at the resolution of a single-cell type and of a single isoform. PMID:12592004

  5. Gene Network Polymorphism Illuminates Loss and Retention of Novel RNAi Silencing Components in the Cryptococcus Pathogenic Species Complex

    PubMed Central

    Clancey, Shelly Applen; Wang, Xuying; Heitman, Joseph

    2016-01-01

    RNAi is a ubiquitous pathway that serves central functions throughout eukaryotes, including maintenance of genome stability and repression of transposon expression and movement. However, a number of organisms have lost their RNAi pathways, including the model yeast Saccharomyces cerevisiae, the maize pathogen Ustilago maydis, the human pathogen Cryptococcus deuterogattii, and some human parasite pathogens, suggesting there may be adaptive benefits associated with both retention and loss of RNAi. By comparing the RNAi-deficient genome of the Pacific Northwest Outbreak C. deuterogattii strain R265 with the RNAi-proficient genomes of the Cryptococcus pathogenic species complex, we identified a set of conserved genes that were lost in R265 and all other C. deuterogattii isolates examined. Genetic and molecular analyses reveal several of these lost genes play roles in RNAi pathways. Four novel components were examined further. Znf3 (a zinc finger protein) and Qip1 (a homolog of N. crassa Qip) were found to be essential for RNAi, while Cpr2 (a constitutive pheromone receptor) and Fzc28 (a transcription factor) are involved in sex-induced but not mitosis-induced silencing. Our results demonstrate that the mitotic and sex-induced RNAi pathways rely on the same core components, but sex-induced silencing may be a more specific, highly induced variant that involves additional specialized or regulatory components. Our studies further illustrate how gene network polymorphisms involving known components of key cellular pathways can inform identification of novel elements and suggest that RNAi loss may have been a core event in the speciation of C. deuterogattii and possibly contributed to its pathogenic trajectory. PMID:26943821

  6. Identification and Knockdown of the Olfactory Receptor (OrCo) in Gypsy Moth, Lymantria dispar.

    PubMed

    Lin, Wei; Yu, Yanxue; Zhou, Ping; Zhang, Junhua; Dou, Liduo; Hao, Qin; Chen, Hongjun; Zhu, Shuifang

    2015-01-01

    The gypsy moth, Lymantria dispar, is an important economic pest that causes large-scale damage to forests worldwide. Because of its important role in initiating and controlling insect behavior, olfaction-and olfaction-based pest management-has drawn increasing attention from entomologists. In this study, we identified the gene that encodes the olfactory receptor co-receptor (OrCo). Through amino acid sequence alignment, we found that LdisOrCo shares high identity with other OrCo proteins from different insect orders. Next, we performed RNA-interference (RNAi) to assess the role of OrCo in olfaction. Electroantennographic assays showed that after RNAi, the average value of males' response to sex pheromones was 0.636 mV, significantly lower than that of the positive control (average = 1.472 mV). Females showed no response to sex pheromones before or after RNAi. Finally, quantitative PCR showed a strong decrease in the expression of OrCo after RNAi, by ~74% in males and by 23% in females relative to the positive controls. These results indicate that OrCo is not only critical to odor recognition, but it may also represent a new target for development of semiochemicals that can influence insect behavior. PMID:26078719

  7. Identification and Knockdown of the Olfactory Receptor (OrCo) in Gypsy Moth, Lymantria dispar

    PubMed Central

    Lin, Wei; Yu, Yanxue; Zhou, Ping; Zhang, Junhua; Dou, Liduo; Hao, Qin; Chen, Hongjun; Zhu, Shuifang

    2015-01-01

    The gypsy moth, Lymantria dispar, is an important economic pest that causes large-scale damage to forests worldwide. Because of its important role in initiating and controlling insect behavior, olfaction—and olfaction-based pest management—has drawn increasing attention from entomologists. In this study, we identified the gene that encodes the olfactory receptor co-receptor (OrCo). Through amino acid sequence alignment, we found that LdisOrCo shares high identity with other OrCo proteins from different insect orders. Next, we performed RNA-interference (RNAi) to assess the role of OrCo in olfaction. Electroantennographic assays showed that after RNAi, the average value of males' response to sex pheromones was 0.636 mV, significantly lower than that of the positive control (average = 1.472 mV). Females showed no response to sex pheromones before or after RNAi. Finally, quantitative PCR showed a strong decrease in the expression of OrCo after RNAi, by ~74% in males and by 23% in females relative to the positive controls. These results indicate that OrCo is not only critical to odor recognition, but it may also represent a new target for development of semiochemicals that can influence insect behavior. PMID:26078719

  8. Determination of the role of DDX3 a factor involved in mammalian RNAi pathway using an shRNA-expression library.

    PubMed

    Kasim, Vivi; Wu, Shourong; Taira, Kazunari; Miyagishi, Makoto

    2013-01-01

    RNA interference (RNAi) is an endogenous RNA-destruction phenomenon induced by certain double-stranded RNAs (dsRNAs). In RNAi, dsRNAs are processed into small interfering RNAs (siRNAs) which in turn trigger the cleavage of the target mRNA. Here, using a short hairpin RNA-expression library, we identified a DEAD-box helicase 3, DDX3, as an essential factor involved in RNAi pathway and revealed that DDX3 is colocalized with Ago2, an essential factor in RNAi pathway that cleaves target mRNA. Results of experiments with a dominant negative mutant of DDX3 further confirmed that this factor affects the RNAi activity. Together, DDX3 functions to assure mammalian RNAi pathway. Together, our results indicate that DDX3 is a new key molecule to understand the molecular mechanism underlying RNAi pathway in mammals. PMID:23527197

  9. Accelerating Cancer Modeling with RNAi and Nongermline Genetically Engineered Mouse Models

    PubMed Central

    Livshits, Geulah; Lowe, Scott W.

    2014-01-01

    For more than two decades, genetically engineered mouse models have been key to our mechanistic understanding of tumorigenesis and cancer progression. Recently, the massive quantity of data emerging from cancer genomics studies has demanded a corresponding increase in the efficiency and throughput of in vivo models for functional testing of putative cancer genes. Already a mainstay of cancer research, recent innovations in RNA interference (RNAi) technology have extended its utility for studying gene function and genetic interactions, enabling tissue-specific, inducible and reversible gene silencing in vivo. Concurrent advances in embryonic stem cell (ESC) culture and genome engineering have accelerated several steps of genetically engineered mouse model production and have facilitated the incorporation of RNAi technology into these models. Here, we review the current state of these technologies and examine how their integration has the potential to dramatically enhance the throughput and capabilities of animal models for cancer. PMID:24184755

  10. RNA Pol II subunit Rpb7 promotes centromeric transcription and RNAi-directed chromatin silencing

    PubMed Central

    Djupedal, Ingela; Portoso, Manuela; Spåhr, Henrik; Bonilla, Carolina; Gustafsson, Claes M.; Allshire, Robin C.; Ekwall, Karl

    2005-01-01

    Fission yeast centromeric repeats are transcribed into small interfering RNA (siRNA) precursors (pre-siRNAs), which are processed by Dicer to direct heterochromatin formation. Recently, Rpb1 and Rpb2 subunits of RNA polymerase II (RNA Pol II) were shown to mediate RNA interference (RNAi)-directed chromatin modification but did not affect pre-siRNA levels. Here we show that another Pol II subunit, Rpb7 has a specific role in pre-siRNA transcription. We define a centromeric pre-siRNA promoter from which initiation is exquisitely sensitive to the rpb7-G150D mutation. In contrast to other Pol II subunits, Rpb7 promotes pre-siRNA transcription required for RNAi-directed chromatin silencing. PMID:16204182

  11. What RNAi screens in model organisms revealed about microbicidal response in mammals?

    PubMed Central

    Abnave, Prasad; Conti, Filippo; Torre, Cedric; Ghigo, Eric

    2015-01-01

    The strategies evolved by pathogens to infect hosts and the mechanisms used by the host to eliminate intruders are highly complex. Because several biological pathways and processes are conserved across model organisms, these organisms have been used for many years to elucidate and understand the mechanisms of the host-pathogen relationship and particularly to unravel the molecular processes enacted by the host to kill pathogens. The emergence of RNA interference (RNAi) and the ability to apply it toward studies in model organisms have allowed a breakthrough in the elucidation of host-pathogen interactions. The aim of this mini-review is to highlight and describe recent breakthroughs in the field of host-pathogen interactions using RNAi screens of model organisms. We will focus specifically on the model organisms Drosophila melanogaster, Caenorhabditis elegans, and Danio rerio. Moreover, a recent study examining the immune system of planarian will be discussed. PMID:25629007

  12. RNAi targeting multiple cell adhesion molecules reduces immune cell recruitment and vascular inflammation after myocardial infarction.

    PubMed

    Sager, Hendrik B; Dutta, Partha; Dahlman, James E; Hulsmans, Maarten; Courties, Gabriel; Sun, Yuan; Heidt, Timo; Vinegoni, Claudio; Borodovsky, Anna; Fitzgerald, Kevin; Wojtkiewicz, Gregory R; Iwamoto, Yoshiko; Tricot, Benoit; Khan, Omar F; Kauffman, Kevin J; Xing, Yiping; Shaw, Taylor E; Libby, Peter; Langer, Robert; Weissleder, Ralph; Swirski, Filip K; Anderson, Daniel G; Nahrendorf, Matthias

    2016-06-01

    Myocardial infarction (MI) leads to a systemic surge of vascular inflammation in mice and humans, resulting in secondary ischemic complications and high mortality. We show that, in ApoE(-/-) mice with coronary ligation, increased sympathetic tone up-regulates not only hematopoietic leukocyte production but also plaque endothelial expression of adhesion molecules. To counteract the resulting arterial leukocyte recruitment, we developed nanoparticle-based RNA interference (RNAi) that effectively silences five key adhesion molecules. Simultaneously encapsulating small interfering RNA (siRNA)-targeting intercellular cell adhesion molecules 1 and 2 (Icam1 and Icam2), vascular cell adhesion molecule 1 (Vcam1), and E- and P-selectins (Sele and Selp) into polymeric endothelial-avid nanoparticles reduced post-MI neutrophil and monocyte recruitment into atherosclerotic lesions and decreased matrix-degrading plaque protease activity. Five-gene combination RNAi also curtailed leukocyte recruitment to ischemic myocardium. Therefore, targeted multigene silencing may prevent complications after acute MI. PMID:27280687

  13. RNAi related mechanisms affect both transcriptional and posttranscriptional transgene silencing in Drosophila.

    PubMed

    Pal-Bhadra, Manika; Bhadra, Utpal; Birchler, James A

    2002-02-01

    Two types of transgene silencing were found for the Alcohol dehydrogenase (Adh) transcription unit. Transcriptional gene silencing (TGS) is Polycomb dependent and occurs when Adh is driven by the white eye color gene promoter. Full-length Adh transgenes are silenced posttranscriptionally at high copy number or by a pulsed increase over a threshold. The posttranscriptional gene silencing (PTGS) exhibits molecular hallmarks typical of RNA interference (RNAi), including the production of 21--25 bp length sense and antisense RNAs homologous to the silenced RNA. Mutations in piwi, which belongs to a gene family with members required for RNAi, block PTGS and one aspect of TGS, indicating a connection between the two types of silencing. PMID:11864605

  14. Three-Dimensional Spheroid Cell Culture Model for Target Identification Utilizing High-Throughput RNAi Screens.

    PubMed

    Iles, LaKesla R; Bartholomeusz, Geoffrey A

    2016-01-01

    The intrinsic limitations of 2D monolayer cell culture models have prompted the development of 3D cell culture model systems for in vitro studies. Multicellular tumor spheroid (MCTS) models closely simulate the pathophysiological milieu of solid tumors and are providing new insights into tumor biology as well as differentiation, tissue organization, and homeostasis. They are straightforward to apply in high-throughput screens and there is a great need for the development of reliable and robust 3D spheroid-based assays for high-throughput RNAi screening for target identification and cell signaling studies highlighting their potential in cancer research and treatment. In this chapter we describe a stringent standard operating procedure for the use of MCTS for high-throughput RNAi screens. PMID:27581289

  15. Synthesis and properties of vitamin E analog-conjugated neomycin for delivery of RNAi drugs to liver cells.

    PubMed

    Iwata, Rintaro; Nakayama, Futoshi; Hirochi, Sakie; Sato, Kazuki; Piao, Wenying; Nishina, Kazutaka; Yokota, Takanori; Wada, Takeshi

    2015-02-15

    RNA interference (RNAi) is a promising tool to regulate gene expression by external double stranded RNAs (dsRNAs) such as siRNAs. As an efficient method to deliver siRNAs to liver cells, we propose a novel strategy using vitamin E (VE)-conjugated neomycin derivatives. With the aim of delivering RNAi-based drugs to liver cells, several tripod-type and prodrug-type neomycin derivatives were synthesized, all of which were thermodynamically stabilized RNA duplexes. The prodrug-type derivative 7 and the tripod-type derivative 10 were delivered to liver cancer cells and successfully induced RNAi activity. These results indicated the potential use of natural aminoglycosides as carriers of RNAi drugs. PMID:25597008

  16. funRNA: a fungi-centered genomics platform for genes encoding key components of RNAi

    PubMed Central

    2014-01-01

    Background RNA interference (RNAi) is involved in genome defense as well as diverse cellular, developmental, and physiological processes. Key components of RNAi are Argonaute, Dicer, and RNA-dependent RNA polymerase (RdRP), which have been functionally characterized mainly in model organisms. The key components are believed to exist throughout eukaryotes; however, there is no systematic platform for archiving and dissecting these important gene families. In addition, few fungi have been studied to date, limiting our understanding of RNAi in fungi. Here we present funRNA http://funrna.riceblast.snu.ac.kr/, a fungal kingdom-wide comparative genomics platform for putative genes encoding Argonaute, Dicer, and RdRP. Description To identify and archive genes encoding the abovementioned key components, protein domain profiles were determined from reference sequences obtained from UniProtKB/SwissProt. The domain profiles were searched using fungal, metazoan, and plant genomes, as well as bacterial and archaeal genomes. 1,163, 442, and 678 genes encoding Argonaute, Dicer, and RdRP, respectively, were predicted. Based on the identification results, active site variation of Argonaute, diversification of Dicer, and sequence analysis of RdRP were discussed in a fungus-oriented manner. funRNA provides results from diverse bioinformatics programs and job submission forms for BLAST, BLASTMatrix, and ClustalW. Furthermore, sequence collections created in funRNA are synced with several gene family analysis portals and databases, offering further analysis opportunities. Conclusions funRNA provides identification results from a broad taxonomic range and diverse analysis functions, and could be used in diverse comparative and evolutionary studies. It could serve as a versatile genomics workbench for key components of RNAi. PMID:25522231

  17. An Essential Signal Peptide Peptidase Identified in an RNAi Screen of Serine Peptidases of Trypanosoma brucei

    PubMed Central

    Moss, Catherine X.; Brown, Elaine; Hamilton, Alana; Van der Veken, Pieter; Augustyns, Koen; Mottram, Jeremy C.

    2015-01-01

    The serine peptidases of Trypanosoma brucei have been viewed as potential drug targets. In particular, the S9 prolyl oligopeptidase subfamily is thought to be a good avenue for drug discovery. This is based on the finding that some S9 peptidases are secreted and active in the mammalian bloodstream, and that they are a class of enzyme against which drugs have successfully been developed. We collated a list of all serine peptidases in T. brucei, identifying 20 serine peptidase genes, of which nine are S9 peptidases. We screened all 20 serine peptidases by RNAi to determine which, if any, are essential for bloodstream form T. brucei survival. All S9 serine peptidases were dispensable for parasite survival in vitro, even when pairs of similar genes, coding for oligopeptidase B or prolyl oligopeptidase, were targeted simultaneously. We also found no effect on parasite survival in an animal host when the S9 peptidases oligopeptidase B, prolyl oligopeptidase or dipeptidyl peptidase 8 were targeted. The only serine peptidase to emerge from the RNAi screen as essential was a putative type-I signal peptide peptidase (SPP1). This gene was essential for parasite survival both in vitro and in vivo. The growth defect conferred by RNAi depletion of SPP1 was rescued by expression of a functional peptidase from an RNAi resistant SPP1 gene. However, expression of catalytically inactive SPP1 was unable to rescue cells from the SPP1 depleted phenotype, demonstrating that SPP1 serine peptidase activity is necessary for T. brucei survival. PMID:25816352

  18. RNAi-mediated resistance to Cassava brown streak Uganda virus in transgenic cassava.

    PubMed

    Yadav, Jitender S; Ogwok, Emmanuel; Wagaba, Henry; Patil, Basavaprabhu L; Bagewadi, Basavaraj; Alicai, Titus; Gaitan-Solis, Eliana; Taylor, Nigel J; Fauquet, Claude M

    2011-09-01

    Cassava brown streak disease (CBSD), caused by Cassava brown streak Uganda virus (CBSUV) and Cassava brown streak virus (CBSV), is of new epidemic importance to cassava (Manihot esculenta Crantz) production in East Africa, and an emerging threat to the crop in Central and West Africa. This study demonstrates that at least one of these two ipomoviruses, CBSUV, can be efficiently controlled using RNA interference (RNAi) technology in cassava. An RNAi construct targeting the near full-length coat protein (FL-CP) of CBSUV was expressed constitutively as a hairpin construct in cassava. Transgenic cassava lines expressing small interfering RNAs (siRNAs) against this sequence showed 100% resistance to CBSUV across replicated graft inoculation experiments. Reverse transcriptase-polymerase chain reaction (RT-PCR) analysis showed the presence of CBSUV in leaves and some tuberous roots from challenged controls, but not in the same tissues from transgenic plants. This is the first demonstration of RNAi-mediated resistance to the ipomovirus CBSUV in cassava. PMID:21726367

  19. Silencing mutant SOD1 using RNAi protects against neurodegeneration and extends survival in an ALS model.

    PubMed

    Ralph, G Scott; Radcliffe, Pippa A; Day, Denise M; Carthy, Janine M; Leroux, Marie A; Lee, Debbie C P; Wong, Liang-Fong; Bilsland, Lynsey G; Greensmith, Linda; Kingsman, Susan M; Mitrophanous, Kyriacos A; Mazarakis, Nicholas D; Azzouz, Mimoun

    2005-04-01

    Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease resulting in the selective death of motor neurons in the brain and spinal cord. Some familial cases of ALS are caused by dominant mutations in the gene encoding superoxide dismutase (SOD1). The emergence of interfering RNA (RNAi) for specific gene silencing could be therapeutically beneficial for the treatment of such dominantly inherited diseases. We generated a lentiviral vector to mediate expression of RNAi molecules specifically targeting the human SOD1 gene (SOD1). Injection of this vector into various muscle groups of mice engineered to overexpress a mutated form of human SOD1 (SOD1(G93A)) resulted in an efficient and specific reduction of SOD1 expression and improved survival of vulnerable motor neurons in the brainstem and spinal cord. Furthermore, SOD1 silencing mediated an improved motor performance in these animals, resulting in a considerable delay in the onset of ALS symptoms by more than 100% and an extension in survival by nearly 80% of their normal life span. These data are the first to show a substantial extension of survival in an animal model of a fatal, dominantly inherited neurodegenerative condition using RNAi and provide the highest therapeutic efficacy observed in this field to date. PMID:15768029

  20. Heterochromatin and RNAi are required to establish CENP-A chromatin at centromeres.

    PubMed

    Folco, Hernan Diego; Pidoux, Alison L; Urano, Takeshi; Allshire, Robin C

    2008-01-01

    Heterochromatin is defined by distinct posttranslational modifications on histones, such as methylation of histone H3 at lysine 9 (H3K9), which allows heterochromatin protein 1 (HP1)-related chromodomain proteins to bind. Heterochromatin is frequently found near CENP-A chromatin, which is the key determinant of kinetochore assembly. We have discovered that the RNA interference (RNAi)-directed heterochromatin flanking the central kinetochore domain at fission yeast centromeres is required to promote CENP-A(Cnp1) and kinetochore assembly over the central domain. The H3K9 methyltransferase Clr4 (Suv39); the ribonuclease Dicer, which cleaves heterochromatic double-stranded RNA to small interfering RNA (siRNA); Chp1, a component of the RNAi effector complex (RNA-induced initiation of transcriptional gene silencing; RITS); and Swi6 (HP1) are required to establish CENP-A(Cnp1) chromatin on naïve templates. Once assembled, CENP-A(Cnp1) chromatin is propagated by epigenetic means in the absence of heterochromatin. Thus, another, potentially conserved, role for centromeric RNAi-directed heterochromatin has been identified. PMID:18174443

  1. Genome-Wide RNAi Screening to Dissect the TGF-β Signal Transduction Pathway.

    PubMed

    Chen, Xiaochu; Xu, Lan

    2016-01-01

    The transforming growth factor-β (TGF-β) family of cytokines figures prominently in regulation of embryonic development and adult tissue homeostasis from Drosophila to mammals. Genetic defects affecting TGF-β signaling underlie developmental disorders and diseases such as cancer in human. Therefore, delineating the molecular mechanism by which TGF-β regulates cell biology is critical for understanding normal biology and disease mechanisms. Forward genetic screens in model organisms and biochemical approaches in mammalian tissue culture were instrumental in initial characterization of the TGF-β signal transduction pathway. With complete sequence information of the genomes and the advent of RNA interference (RNAi) technology, genome-wide RNAi screening emerged as a powerful functional genomics approach to systematically delineate molecular components of signal transduction pathways. Here, we describe a protocol for image-based whole-genome RNAi screening aimed at identifying molecules required for TGF-β signaling into the nucleus. Using this protocol we examined >90 % of annotated Drosophila open reading frames (ORF) individually and successfully uncovered several novel factors serving critical roles in the TGF-β pathway. Thus cell-based high-throughput functional genomics can uncover new mechanistic insights on signaling pathways beyond what the classical genetics had revealed. PMID:26520138

  2. Phytochrome RNAi enhances major fibre quality and agronomic traits of the cotton Gossypium hirsutum L

    NASA Astrophysics Data System (ADS)

    Abdurakhmonov, Ibrokhim Y.; Buriev, Zabardast T.; Saha, Sukumar; Jenkins, Johnie N.; Abdukarimov, Abdusattor; Pepper, Alan E.

    2014-01-01

    Simultaneous improvement of fibre quality, early-flowering, early-maturity and productivity in Upland cotton (G. hirsutum) is a challenging task for conventional breeding. The influence of red/far-red light ratio on the fibre length prompted us to examine the phenotypic effects of RNA interference (RNAi) of the cotton PHYA1 gene. Here we show a suppression of up to ~70% for the PHYA1 transcript, and compensatory overexpression of up to ~20-fold in the remaining phytochromes in somatically regenerated PHYA1 RNAi cotton plants. Two independent transformants of three generations exhibited vigorous root and vegetative growth, early-flowering, significantly improved upper half mean fibre length and an improvement in other major fibre characteristics. Small decreases in lint traits were observed but seed cotton yield was increased an average 10-17% compared with controls. RNAi-associated phenotypes were heritable and transferable via sexual hybridization. These results should aid in the development of early-maturing and productive Upland cultivars with superior fibre quality.

  3. Harnessing RNAi-based nanomedicines for therapeutic gene silencing in B-cell malignancies

    PubMed Central

    Weinstein, Shiri; Toker, Itai A.; Emmanuel, Rafi; Ramishetti, Srinivas; Hazan-Halevy, Inbal; Rosenblum, Daniel; Goldsmith, Meir; Abraham, Avigdor; Benjamini, Ohad; Bairey, Osnat; Raanani, Pia; Nagler, Arnon; Lieberman, Judy

    2016-01-01

    Despite progress in systemic small interfering RNA (siRNA) delivery to the liver and to solid tumors, systemic siRNA delivery to leukocytes remains challenging. The ability to silence gene expression in leukocytes has great potential for identifying drug targets and for RNAi-based therapy for leukocyte diseases. However, both normal and malignant leukocytes are among the most difficult targets for siRNA delivery as they are resistant to conventional transfection reagents and are dispersed in the body. We used mantle cell lymphoma (MCL) as a prototypic blood cancer for validating a novel siRNA delivery strategy. MCL is an aggressive B-cell lymphoma that overexpresses cyclin D1 with relatively poor prognosis. Down-regulation of cyclin D1 using RNA interference (RNAi) is a potential therapeutic approach to this malignancy. Here, we designed lipid-based nanoparticles (LNPs) coated with anti-CD38 monoclonal antibodies that are specifically taken up by human MCL cells in the bone marrow of xenografted mice. When loaded with siRNAs against cyclin D1, CD38-targeted LNPs induced gene silencing in MCL cells and prolonged survival of tumor-bearing mice with no observed adverse effects. These results highlight the therapeutic potential of cyclin D1 therapy in MCL and present a novel RNAi delivery system that opens new therapeutic opportunities for treating MCL and other B-cell malignancies. PMID:26699502

  4. RNAi-based inhibition of porcine reproductive and respiratory syndrome virus replication in transgenic pigs.

    PubMed

    Li, Li; Li, Qiuyan; Bao, Yonghua; Li, Jinxiu; Chen, Zhisheng; Yu, Xiuling; Zhao, Yaofeng; Tian, Kegong; Li, Ning

    2014-02-10

    Porcine reproductive and respiratory syndrome (PRRS) is an economically devastating viral disease causing heavy losses to the swine industry worldwide. Many studies have shown that transient delivery of small interfering RNA (siRNA) or adenovirus-mediated RNA interfere (RNAi) could potentially inhibit porcine reproductive and respiratory syndrome virus (PRRSV) replication in vivo and in vitro. Here, we applied RNAi to produce transgenic (TG) pigs that constitutively expressed PRRSV-specific siRNA derived from small hairpin RNA (shRNA). First, we evaluated siRNA expression in the founding and F1 generation pigs and confirmed stable transmission. Then, we detected the expression of IFN-β and protein kinase R (PKR) and found no difference among TG, non-transgenic (NTG), and wild-type pigs. Lastly, the F1 generation pigs, including TG and NTG piglets, were challenged with 3×10⁴·⁵ TCID₅₀ of JXA1, a highly pathogenic porcine reproductive and respiratory syndrome virus (HP-PRRSV). Our results showed that the in vivo siRNA expression substantially reduced the serum HP-PRRSV titers and increased survival time by 3 days when TG pigs were compared with the NTG controls. These data suggested that RNAi-based genetic modification might be used to breed viral-resistant livestock with stable siRNA expression with no complications of siRNA toxicity. PMID:24333125

  5. Compositional equivalency of RNAi-mediated virus-resistant transgenic soybean and its nontransgenic counterpart.

    PubMed

    Zhang, Xiuchun; Zhao, Pingjuan; Wu, Kunxin; Zhang, Yuliang; Peng, Ming; Liu, Zhixin

    2014-05-14

    RNA silencing or RNA interference (RNAi), which is triggered by double-stranded RNA (dsRNA), is an evolutionarily conserved process that is active in a wide variety of eukaryotic organisms. Engineering plants with hairpin construct in which the viral gene is arranged in inverted repeats (IR) renders plants resistant to plant virus infection. However, there is no report on whether biologically important changes occurred by the insertion of IR, which confer transgenic plants virus resistance. In the present study, the compositions of virus-resistant transgenic soybean seeds developed by insertion of three short IRs, each containing the specific, highly conserved sequences derived from one virus, were compared with those of nontransgenic counterparts by applying the principle of substantial equivalence to determine whether significant undesirable biological changes occurred by IR insertion. The results revealed that the nutrient components as well as antinutrient contents of these virus-resistant soybean lines are substantially equivalent to those of the nontransgenic counterparts, and the majority of the measured amounts of nutritional components and antinutrient contents are well within the range of values reported for other commercial soybean lines. The results imply that no biologically important changes occurred by the insertion of IRs in the RNAi-mediated virus-resistant transgenic soybeans. The results can serve as baseline information for developing RNAi-mediated transgenic soybean cultivars or other crops with broader spectrum virus resistance. PMID:24754373

  6. Harnessing RNAi-based nanomedicines for therapeutic gene silencing in B-cell malignancies.

    PubMed

    Weinstein, Shiri; Toker, Itai A; Emmanuel, Rafi; Ramishetti, Srinivas; Hazan-Halevy, Inbal; Rosenblum, Daniel; Goldsmith, Meir; Abraham, Avigdor; Benjamini, Ohad; Bairey, Osnat; Raanani, Pia; Nagler, Arnon; Lieberman, Judy; Peer, Dan

    2016-01-01

    Despite progress in systemic small interfering RNA (siRNA) delivery to the liver and to solid tumors, systemic siRNA delivery to leukocytes remains challenging. The ability to silence gene expression in leukocytes has great potential for identifying drug targets and for RNAi-based therapy for leukocyte diseases. However, both normal and malignant leukocytes are among the most difficult targets for siRNA delivery as they are resistant to conventional transfection reagents and are dispersed in the body. We used mantle cell lymphoma (MCL) as a prototypic blood cancer for validating a novel siRNA delivery strategy. MCL is an aggressive B-cell lymphoma that overexpresses cyclin D1 with relatively poor prognosis. Down-regulation of cyclin D1 using RNA interference (RNAi) is a potential therapeutic approach to this malignancy. Here, we designed lipid-based nanoparticles (LNPs) coated with anti-CD38 monoclonal antibodies that are specifically taken up by human MCL cells in the bone marrow of xenografted mice. When loaded with siRNAs against cyclin D1, CD38-targeted LNPs induced gene silencing in MCL cells and prolonged survival of tumor-bearing mice with no observed adverse effects. These results highlight the therapeutic potential of cyclin D1 therapy in MCL and present a novel RNAi delivery system that opens new therapeutic opportunities for treating MCL and other B-cell malignancies. PMID:26699502

  7. Brain-targeting delivery for RNAi neuroprotection against cerebral ischemia reperfusion injury.

    PubMed

    An, Sai; Kuang, Yuyang; Shen, Teng; Li, Jianfeng; Ma, Haojun; Guo, Yubo; He, Xi; Jiang, Chen

    2013-11-01

    Nanoparticles (NPs) with modification of brain-targeting molecules have been extensively exploited for therapeutic gene delivery across the blood-brain barrier (BBB). As one of the effective RNA interference (RNAi) approaches, short hairpin RNA (shRNA) has been proved to be promising in the field of gene therapy. Apoptosis signal-regulating kinase 1 (Ask1) has been reported to be an important target for gene therapy against cerebral ischemia reperfusion injury. In this study, dendrigraft poly-l-lysine (DGL) was decorated by dermorphin (a μ-opiate receptor agonist) through PEG for efficient brain-targeting, then complexed with anti-Ask1 shRNA plasmid DNA, yielding the DGL-PEG-dermorphin/shRNA NPs. The DGL-PEG-dermorphin/shRNA NPs were characterized and estimated the brain-targeting ability. In vitro, increased cellular uptake and transfection efficiency were explored; in vivo, preferable accumulation and gene transfection in brain were showed in images. The DGL-PEG-dermorphin/shRNA NPs also revealed high efficiency of neuroprotection. As a result of RNAi, corresponding mRNA was distinctly degraded, expression of Ask1 protein was obviously suppressed, apoptotic cell death was apparently decreased and cerebral infarct area was significantly reduced. Above all, DGL-PEG-dermorphin/shRNA NPs were proved to be efficient and safe for brain-targeting RNAi neuroprotection against cerebral ischemia reperfusion injury. PMID:23968852

  8. Tafazzin knockdown interrupts cell cycle progression in cultured neonatal ventricular fibroblasts.

    PubMed

    He, Quan; Wang, Miao; Harris, Nicole; Han, Xianlin

    2013-11-01

    Mutation of the mitochondrial protein tafazzin causes dilated cardiomyopathy in Barth syndrome. Previous studies have shown that tafazzin knockdown promotes hypertrophy of neonatal cardiac myocytes. The current investigation was designed to show whether tafazzin knockdown affects cardiac fibroblast proliferation and collagen secretion, which contribute to fibrosis in dilated cardiomyopathy. In primary cultures of neonatal ventricular fibroblasts (NVFs) transduced with a tafazzin short hairpin RNA adenovirus, tafazzin knockdown increased production of reactive oxygen species and activation of mitogen-activated protein kinases and induced protein and DNA synthesis via cell cycle regulators. It also reduced intracellular ATP, activated AMPK, and caused multinucleation, hypertrophy, and enhanced collagen secretion. We concluded that tafazzin knockdown interrupts the NVF cell cycle and this in turn may contribute to fibrosis and dilated cardiomyopathy in Barth syndrome. PMID:23997105

  9. RNAiFold 2.0: a web server and software to design custom and Rfam-based RNA molecules

    PubMed Central

    Garcia-Martin, Juan Antonio; Dotu, Ivan; Clote, Peter

    2015-01-01

    Several algorithms for RNA inverse folding have been used to design synthetic riboswitches, ribozymes and thermoswitches, whose activity has been experimentally validated. The RNAiFold software is unique among approaches for inverse folding in that (exhaustive) constraint programming is used instead of heuristic methods. For that reason, RNAiFold can generate all sequences that fold into the target structure or determine that there is no solution. RNAiFold 2.0 is a complete overhaul of RNAiFold 1.0, rewritten from the now defunct COMET language to C++. The new code properly extends the capabilities of its predecessor by providing a user-friendly pipeline to design synthetic constructs having the functionality of given Rfam families. In addition, the new software supports amino acid constraints, even for proteins translated in different reading frames from overlapping coding sequences; moreover, structure compatibility/incompatibility constraints have been expanded. With these features, RNAiFold 2.0 allows the user to design single RNA molecules as well as hybridization complexes of two RNA molecules. Availability: the web server, source code and linux binaries are publicly accessible at http://bioinformatics.bc.edu/clotelab/RNAiFold2.0. PMID:26019176

  10. Lentiviral Delivery of RNAi for In Vivo Lineage-Specific Modulation of Gene Expression in Mouse Lung Macrophages

    PubMed Central

    Wilson, Andrew A; Kwok, Letty W; Porter, Emily L; Payne, Julie G; McElroy, Gregory S; Ohle, Sarah J; Greenhill, Sara R; Blahna, Matthew T; Yamamoto, Kazuko; Jean, Jyh C; Mizgerd, Joseph P; Kotton, Darrell N

    2013-01-01

    Although RNA interference (RNAi) has become a ubiquitous laboratory tool since its discovery 12 years ago, in vivo delivery to selected cell types remains a major technical challenge. Here, we report the use of lentiviral vectors for long-term in vivo delivery of RNAi selectively to resident alveolar macrophages (AMs), key immune effector cells in the lung. We demonstrate the therapeutic potential of this approach by RNAi-based downregulation of p65 (RelA), a component of the pro-inflammatory transcriptional regulator, nuclear factor κB (NF-κB) and a key participant in lung disease pathogenesis. In vivo RNAi delivery results in decreased induction of NF-κB and downstream neutrophilic chemokines in transduced AMs as well as attenuated lung neutrophilia following stimulation with lipopolysaccharide (LPS). Through concurrent delivery of a novel lentiviral reporter vector (lenti-NF-κB-luc-GFP) we track in vivo expression of NF-κB target genes in real time, a critical step towards extending RNAi-based therapy to longstanding lung diseases. Application of this system reveals that resident AMs persist in the airspaces of mice following the resolution of LPS-induced inflammation, thus allowing these localized cells to be used as effective vehicles for prolonged RNAi delivery in disease settings. PMID:23403494

  11. A network-based integrative approach to prioritize reliable hits from multiple genome-wide RNAi screens in Drosophila

    PubMed Central

    Wang, Li; Tu, Zhidong; Sun, Fengzhu

    2009-01-01

    Background The recently developed RNA interference (RNAi) technology has created an unprecedented opportunity which allows the function of individual genes in whole organisms or cell lines to be interrogated at genome-wide scale. However, multiple issues, such as off-target effects or low efficacies in knocking down certain genes, have produced RNAi screening results that are often noisy and that potentially yield both high rates of false positives and false negatives. Therefore, integrating RNAi screening results with other information, such as protein-protein interaction (PPI), may help to address these issues. Results By analyzing 24 genome-wide RNAi screens interrogating various biological processes in Drosophila, we found that RNAi positive hits were significantly more connected to each other when analyzed within a protein-protein interaction network, as opposed to random cases, for nearly all screens. Based on this finding, we developed a network-based approach to identify false positives (FPs) and false negatives (FNs) in these screening results. This approach relied on a scoring function, which we termed NePhe, to integrate information obtained from both PPI network and RNAi screening results. Using a novel rank-based test, we compared the performance of different NePhe scoring functions and found that diffusion kernel-based methods generally outperformed others, such as direct neighbor-based methods. Using two genome-wide RNAi screens as examples, we validated our approach extensively from multiple aspects. We prioritized hits in the original screens that were more likely to be reproduced by the validation screen and recovered potential FNs whose involvements in the biological process were suggested by previous knowledge and mutant phenotypes. Finally, we demonstrated that the NePhe scoring system helped to biologically interpret RNAi results at the module level. Conclusion By comprehensively analyzing multiple genome-wide RNAi screens, we conclude that

  12. The Pratylenchus penetrans Transcriptome as a Source for the Development of Alternative Control Strategies: Mining for Putative Genes Involved in Parasitism and Evaluation of in planta RNAi

    PubMed Central

    Vieira, Paulo; Eves-van den Akker, Sebastian; Verma, Ruchi; Wantoch, Sarah; Eisenback, Jonathan D.; Kamo, Kathryn

    2015-01-01

    The root lesion nematode Pratylenchus penetrans is considered one of the most economically important species within the genus. Host range studies have shown that nearly 400 plant species can be parasitized by this species. To obtain insight into the transcriptome of this migratory plant-parasitic nematode, we used Illumina mRNA sequencing analysis of a mixed population, as well as nematode reads detected in infected soybean roots 3 and 7 days after nematode infection. Over 140 million paired end reads were obtained for this species, and de novo assembly resulted in a total of 23,715 transcripts. Homology searches showed significant hit matches to 58% of the total number of transcripts using different protein and EST databases. In general, the transcriptome of P. penetrans follows common features reported for other root lesion nematode species. We also explored the efficacy of RNAi, delivered from the host, as a strategy to control P. penetrans, by targeted knock-down of selected nematode genes. Different comparisons were performed to identify putative nematode genes with a role in parasitism, resulting in the identification of transcripts with similarities to other nematode parasitism genes. Focusing on the predicted nematode secreted proteins found in this transcriptome, we observed specific members to be up-regulated at the early time points of infection. In the present study, we observed an enrichment of predicted secreted proteins along the early time points of parasitism by this species, with a significant number being pioneer candidate genes. A representative set of genes examined using RT-PCR confirms their expression during the host infection. The expression patterns of the different candidate genes raise the possibility that they might be involved in critical steps of P. penetrans parasitism. This analysis sheds light on the transcriptional changes that accompany plant infection by P. penetrans, and will aid in identifying potential gene targets for

  13. Knockdown of microplitis mediator odorant receptor involved in the sensitive detection of two chemicals.

    PubMed

    Li, Ke-Ming; Ren, Li-Yan; Zhang, Yong-Jun; Wu, Kong-Ming; Guo, Yu-Yuan

    2012-03-01

    Odorant receptors are thought to play critical roles in the perception of chemosensory stimuli by insects. The primary method to address the functions of odorant receptors in insects is to use in vitro binding assays between the receptors and potential chemical stimuli. We injected MmedOrco dsRNA into the abdominal cavity of a braconid wasp, Microplitis mediator, and assayed for expression of this gene 72 h after treatment (RNAi). Quantitative real-time PCR demonstrated that the level of mRNA expression in MmedOrco dsRNA-treated M. mediator was significantly reduced (>90%) when compared with water-treated controls. Furthermore, electroantennogram (EAG) responses of M. mediator to two chemical attractants, nonanal and farnesene, were also reduced significantly (~70%) in RNAi-treated M. mediator when compared to controls. RNAi-treated M. mediator also responded by walking/flying at a lower rate to both chemicals when compared with controls in a Y-tube olfactometer bioassay, which provides direct evidence that MmedOrco plays an important role in perception of nonanal and farnesene in M. mediator. PMID:22402893

  14. Ribose 5-Phosphate Isomerase B Knockdown Compromises Trypanosoma brucei Bloodstream Form Infectivity

    PubMed Central

    Loureiro, Inês; Faria, Joana; Clayton, Christine; Macedo-Ribeiro, Sandra; Santarém, Nuno; Roy, Nilanjan; Cordeiro-da-Siva, Anabela; Tavares, Joana

    2015-01-01

    Ribose 5-phosphate isomerase is an enzyme involved in the non-oxidative branch of the pentose phosphate pathway, and catalyzes the inter-conversion of D-ribose 5-phosphate and D-ribulose 5-phosphate. Trypanosomatids, including the agent of African sleeping sickness namely Trypanosoma brucei, have a type B ribose-5-phosphate isomerase. This enzyme is absent from humans, which have a structurally unrelated ribose 5-phosphate isomerase type A, and therefore has been proposed as an attractive drug target waiting further characterization. In this study, Trypanosoma brucei ribose 5-phosphate isomerase B showed in vitro isomerase activity. RNAi against this enzyme reduced parasites' in vitro growth, and more importantly, bloodstream forms infectivity. Mice infected with induced RNAi clones exhibited lower parasitaemia and a prolonged survival compared to control mice. Phenotypic reversion was achieved by complementing induced RNAi clones with an ectopic copy of Trypanosoma cruzi gene. Our results present the first functional characterization of Trypanosoma brucei ribose 5-phosphate isomerase B, and show the relevance of an enzyme belonging to the non-oxidative branch of the pentose phosphate pathway in the context of Trypanosoma brucei infection. PMID:25568941

  15. Knockdown of p53 suppresses Nanog expression in embryonic stem cells

    SciTech Connect

    Abdelalim, Essam Mohamed; Tooyama, Ikuo

    2014-01-10

    Highlights: •We investigate the role of p53 in ESCs in the absence of DNA damage. •p53 knockdown suppresses ESC proliferation. •p53 knockdown downregulates Nanog expression. •p53 is essential for mouse ESC self-renewal. -- Abstract: Mouse embryonic stem cells (ESCs) express high levels of cytoplasmic p53. Exposure of mouse ESCs to DNA damage leads to activation of p53, inducing Nanog suppression. In contrast to earlier studies, we recently reported that chemical inhibition of p53 suppresses ESC proliferation. Here, we confirm that p53 signaling is involved in the maintenance of mouse ESC self-renewal. RNA interference-mediated knockdown of p53 induced downregulation of p21 and defects in ESC proliferation. Furthermore, p53 knockdown resulted in a significant downregulation in Nanog expression at 24 and 48 h post-transfection. p53 knockdown also caused a reduction in Oct4 expression at 48 h post-transfection. Conversely, exposure of ESCs to DNA damage caused a higher reduction of Nanog expression in control siRNA-treated cells than in p53 siRNA-treated cells. These data show that in the absence of DNA damage, p53 is required for the maintenance of mouse ESC self-renewal by regulating Nanog expression.

  16. Selenoprotein X Gene Knockdown Aggravated H2O2-Induced Apoptosis in Liver LO2 Cells.

    PubMed

    Tang, Jiayong; Cao, Lei; Li, Qiang; Wang, Longqiong; Jia, Gang; Liu, Guangmang; Chen, Xiaoling; Cai, Jingyi; Shang, Haiying; Zhao, Hua

    2016-09-01

    To determine the roles of selenoprotein X gene (Selx) in protecting liver cells against oxidative damage, the influences of Selx knockdown on H2O2-induced apoptosis in human normal hepatocyte (LO2) cells were studied. pSilencer 3.1 was used to develop knockdown vector targeting the 3'-UTR of human Selx. The Selx knockdown and control cells were further exposed to H2O2, and cell viability, cell apoptosis rate, and the expression levels of mRNA and protein of apoptosis-related genes were detected. The results showed that vector targeting the 3'-UTR of Selx successfully silenced mRNA or protein expression of SelX in LO2 cells. Selx knockdown resulted in decreased cell viability, increased percentage of early apoptotic cells, decreased Bcl2A1 and Bcl-2 expression, and increased phosphorylation of P38 in LO2 cells. When Selx knockdown LO2 cells were exposed to H2O2, characteristics of H2O2-induced cell dysfunctions were further exacerbated. Taken together, our findings suggested that SelX played important roles in protecting LO2 cells against oxidative damage and reducing H2O2-induced apoptosis in liver cells. PMID:26899321

  17. Mitochondria-targeted antioxidant prevents cardiac dysfunction induced by tafazzin gene knockdown in cardiac myocytes.

    PubMed

    He, Quan; Harris, Nicole; Ren, Jun; Han, Xianlin

    2014-01-01

    Tafazzin, a mitochondrial acyltransferase, plays an important role in cardiolipin side chain remodeling. Previous studies have shown that dysfunction of tafazzin reduces cardiolipin content, impairs mitochondrial function, and causes dilated cardiomyopathy in Barth syndrome. Reactive oxygen species (ROS) have been implicated in the development of cardiomyopathy and are also the obligated byproducts of mitochondria. We hypothesized that tafazzin knockdown increases ROS production from mitochondria, and a mitochondria-targeted antioxidant prevents tafazzin knockdown induced mitochondrial and cardiac dysfunction. We employed cardiac myocytes transduced with an adenovirus containing tafazzin shRNA as a model to investigate the effects of the mitochondrial antioxidant, mito-Tempo. Knocking down tafazzin decreased steady state levels of cardiolipin and increased mitochondrial ROS. Treatment of cardiac myocytes with mito-Tempo normalized tafazzin knockdown enhanced mitochondrial ROS production and cellular ATP decline. Mito-Tempo also significantly abrogated tafazzin knockdown induced cardiac hypertrophy, contractile dysfunction, and cell death. We conclude that mitochondria-targeted antioxidant prevents cardiac dysfunction induced by tafazzin gene knockdown in cardiac myocytes and suggest mito-Tempo as a potential therapeutic for Barth syndrome and other dilated cardiomyopathies resulting from mitochondrial oxidative stress. PMID:25247053

  18. The Neuronal PAS Domain Protein 4 (Npas4) Is Required for New and Reactivated Fear Memories

    PubMed Central

    Nguyen, Tam; DiLeone, Ralph J.; Schafe, Glenn E.

    2011-01-01

    The Neuronal PAS domain protein 4 (Npas4) is a neuronal activity-dependent immediate early gene that has recently been identified as a transcription factor which regulates the transcription of genes that control inhibitory synapse development and synaptic plasticity. The role Npas4 in learning and memory, however, is currently unknown. Here, we systematically examine the role of Npas4 in auditory Pavlovian fear conditioning, an amygdala-dependent form of emotional learning. In our first series of experiments, we show that Npas4 mRNA and protein are regulated in the rat lateral nucleus of the amygdala (LA) in a learning-dependent manner. Further, knockdown of Npas4 protein in the LA via adeno-associated viral (AAV) mediated gene delivery of RNAi was observed to impair fear memory formation, while innate fear and the expression of fear memory were not affected. In our second series of experiments, we show that Npas4 protein is regulated in the LA by retrieval of an auditory fear memory and that knockdown of Npas4 in the LA impairs retention of a reactivated, but not a non-reactivated, fear memory. Collectively, our findings provide the first comprehensive look at the functional role of Npas4 in learning and memory. PMID:21887312

  19. RNAi-mediated Gene Silencing of Mutant Myotilin Improves Myopathy in LGMD1A Mice.

    PubMed

    Liu, Jian; Wallace, Lindsay M; Garwick-Coppens, Sara E; Sloboda, Darcée D; Davis, Carol S; Hakim, Chady H; Hauser, Michael A; Brooks, Susan V; Mendell, Jerry R; Harper, Scott Q

    2014-01-01

    Recent progress suggests gene therapy may one day be an option for treating some forms of limb girdle muscular dystrophy (LGMD). Nevertheless, approaches targeting LGMD have so far focused on gene replacement strategies for recessive forms of the disease. In contrast, no attempts have been made to develop molecular therapies for any of the eight dominantly inherited forms of LGMD. Importantly, the emergence of RNA interference (RNAi) therapeutics in the last decade provided new tools to combat dominantly inherited LGMDs with molecular therapy. In this study, we describe the first RNAi-based, preclinical gene therapy approach for silencing a gene associated with dominant LGMD. To do this, we developed adeno-associated viral vectors (AAV6) carrying designed therapeutic microRNAs targeting mutant myotilin (MYOT), which is the underlying cause of LGMD type 1A (LGMD1A). Our best MYOT-targeted microRNA vector (called miMYOT) significantly reduced mutant myotilin mRNA and soluble protein expression in muscles of LGMD1A mice (the TgT57I model) both 3 and 9 months after delivery, demonstrating short- and long-term silencing effects. This MYOT gene silencing subsequently decreased deposition of MYOT-seeded intramuscular protein aggregates, which is the hallmark feature of LGMD1A. Histological improvements were accompanied by significant functional correction, as miMYOT-treated animals showed increased muscle weight and improved specific force in the gastrocnemius, which is one of the most severely affected muscles in TgT57I mice and patients with dominant myotilin mutations. These promising results in a preclinical model of LGMD1A support the further development of RNAi-based molecular therapy as a prospective treatment for LGMD1A. Furthermore, this study sets a foundation that may be refined and adapted to treat other dominant LGMD and related disorders. PMID:24781192

  20. Chemical & RNAi screening at MSKCC: a collaborative platform to discover & repurpose drugs to fight disease

    PubMed Central

    Bhinder, Bhavneet; Antczak, Christophe; Shum, David; Radu, Constantin; Mahida, Jeni P.; Liu-Sullivan, Nancy; Ibáñez, Glorymar; Raja, Balajee Somalinga; Calder, Paul A.; Djaballah, Hakim

    2014-01-01

    Memorial Sloan-Kettering Cancer Center (MSKCC) has implemented the creation of a full service state-of-the-art High-throughput Screening Core Facility (HTSCF) equipped with modern robotics and custom-built screening data management resources to rapidly store and query chemical and RNAi screening data outputs. The mission of the facility is to provide oncology clinicians and researchers alike with access to cost-effective HTS solutions for both chemical and RNAi screening, with an ultimate goal of novel target identification and drug discovery. HTSCF was established in 2003 to support the institution’s commitment to growth in molecular pharmacology and in the realm of therapeutic agents to fight chronic diseases such as cancer. This endeavor required broad range of expertise in technology development to establish robust and innovative assays, large collections of diverse chemical and RNAi duplexes to probe specific cellular events, sophisticated compound and data handling capabilities, and a profound knowledge in assay development, hit validation, and characterization. Our goal has been to strive for constant innovation, and we strongly believe in shifting the paradigm from traditional drug discovery towards translational research now, making allowance for unmet clinical needs in patients. Our efforts towards repurposing FDA-approved drugs fructified when digoxin, identified through primary HTS, was administered in the clinic for treatment of stage Vb retinoblastoma. In summary, the overall aim of our facility is to identify novel chemical probes, to study cellular processes relevant to investigator’s research interest in chemical biology and functional genomics, and to be instrumental in accelerating the process of drug discovery in academia. PMID:24661215

  1. Combined Gene Expression and RNAi Screening to Identify Alkylation Damage Survival Pathways from Fly to Human

    PubMed Central

    Zanotto-Filho, Alfeu; Dashnamoorthy, Ravi; Loranc, Eva; de Souza, Luis H. T.; Moreira, José C. F.; Suresh, Uthra; Chen, Yidong

    2016-01-01

    Alkylating agents are a key component of cancer chemotherapy. Several cellular mechanisms are known to be important for its survival, particularly DNA repair and xenobiotic detoxification, yet genomic screens indicate that additional cellular components may be involved. Elucidating these components has value in either identifying key processes that can be modulated to improve chemotherapeutic efficacy or may be altered in some cancers to confer chemoresistance. We therefore set out to reevaluate our prior Drosophila RNAi screening data by comparison to gene expression arrays in order to determine if we could identify any novel processes in alkylation damage survival. We noted a consistent conservation of alkylation survival pathways across platforms and species when the analysis was conducted on a pathway/process level rather than at an individual gene level. Better results were obtained when combining gene lists from two datasets (RNAi screen plus microarray) prior to analysis. In addition to previously identified DNA damage responses (p53 signaling and Nucleotide Excision Repair), DNA-mRNA-protein metabolism (transcription/translation) and proteasome machinery, we also noted a highly conserved cross-species requirement for NRF2, glutathione (GSH)-mediated drug detoxification and Endoplasmic Reticulum stress (ER stress)/Unfolded Protein Responses (UPR) in cells exposed to alkylation. The requirement for GSH, NRF2 and UPR in alkylation survival was validated by metabolomics, protein studies and functional cell assays. From this we conclude that RNAi/gene expression fusion is a valid strategy to rapidly identify key processes that may be extendable to other contexts beyond damage survival. PMID:27100653

  2. Reduced stability and intracellular transport of dsRNA contribute to poor RNAi response in lepidopteran insects.

    PubMed

    Shukla, Jayendra Nath; Kalsi, Megha; Sethi, Amit; Narva, Kenneth E; Fishilevich, Elane; Singh, Satnam; Mogilicherla, Kanakachari; Palli, Subba Reddy

    2016-07-01

    RNA interference (RNAi) has become a widely used reverse genetic tool to study gene function in eukaryotic organisms and is being developed as a technology for insect pest management. The efficiency of RNAi varies among organisms. Insects from different orders also display differential efficiency of RNAi, ranging from highly efficient (coleopterans) to very low efficient (lepidopterans). We investigated the reasons for varying RNAi efficiency between lepidopteran and coleopteran cell lines and also between the Colorado potato beetle, Leptinotarsa decemlineata and tobacco budworm, Heliothis virescens. The dsRNA either injected or fed was degraded faster in H. virescens than in L. decemlineata. Both lepidopteran and coleopteran cell lines and tissues efficiently took up the dsRNA. Interestingly, the dsRNA administered to coleopteran cell lines and tissues was taken up and processed to siRNA whereas the dsRNA was taken up by lepidopteran cell lines and tissues but no siRNA was detected in the total RNA isolated from these cell lines and tissues. The data included in this paper showed that the degradation and intracellular transport of dsRNA are the major factors responsible for reduced RNAi efficiency in lepidopteran insects. PMID:27245473

  3. New wind in the sails: improving the agronomic value of crop plants through RNAi-mediated gene silencing.

    PubMed

    Koch, Aline; Kogel, Karl-Heinz

    2014-09-01

    RNA interference (RNAi) has emerged as a powerful genetic tool for scientific research over the past several years. It has been utilized not only in fundamental research for the assessment of gene function, but also in various fields of applied research, such as human and veterinary medicine and agriculture. In plants, RNAi strategies have the potential to allow manipulation of various aspects of food quality and nutritional content. In addition, the demonstration that agricultural pests, such as insects and nematodes, can be killed by exogenously supplied RNAi targeting their essential genes has raised the possibility that plant predation can be controlled by lethal RNAi signals generated in planta. Indeed, recent evidence argues that this strategy, called host-induced gene silencing (HIGS), is effective against sucking insects and nematodes; it also has been shown to compromise the growth and development of pathogenic fungi, as well as bacteria and viruses, on their plant hosts. Here, we review recent studies that reveal the enormous potential RNAi strategies hold not only for improving the nutritive value and safety of the food supply, but also for providing an environmentally friendly mechanism for plant protection. PMID:25040343

  4. The possible impact of persistent virus infection on the function of the RNAi machinery in insects: a hypothesis

    PubMed Central

    Swevers, Luc; Vanden Broeck, Jozef; Smagghe, Guy

    2013-01-01

    RNAi experiments in insects are characterized by great variability in efficiency; for instance beetles and locusts are very amenable to dsRNA-mediated gene silencing, while other insect groups, most notably lepidopterans, are more refractory to RNAi. Several factors can be forwarded that could affect the efficiency of RNAi, such as the composition and function of the intracellular RNAi machinery, the mechanism of dsRNA uptake, the presence of dsRNA- and siRNA-degrading enzymes and non-specific activation of the innate immune response. In this essay, we investigate the evidence whether persistent infection with RNA viruses could be a major factor that affects the response to exogenous dsRNA in insects. The occurrence of RNA viruses in different insect groups will be discussed, as well as several mechanisms by which viruses could interfere with the process of RNAi. Finally, the impact of RNA virus infection on the design of dsRNA-based insect control strategies will be considered. PMID:24204347

  5. Reduced stability and intracellular transport of dsRNA contribute to poor RNAi response in lepidopteran insects

    PubMed Central

    Shukla, Jayendra Nath; Kalsi, Megha; Sethi, Amit; Narva, Kenneth E.; Fishilevich, Elane; Singh, Satnam; Mogilicherla, Kanakachari; Palli, Subba Reddy

    2016-01-01

    ABSTRACT RNA interference (RNAi) has become a widely used reverse genetic tool to study gene function in eukaryotic organisms and is being developed as a technology for insect pest management. The efficiency of RNAi varies among organisms. Insects from different orders also display differential efficiency of RNAi, ranging from highly efficient (coleopterans) to very low efficient (lepidopterans). We investigated the reasons for varying RNAi efficiency between lepidopteran and coleopteran cell lines and also between the Colorado potato beetle, Leptinotarsa decemlineata and tobacco budworm, Heliothis virescens. The dsRNA either injected or fed was degraded faster in H. virescens than in L. decemlineata. Both lepidopteran and coleopteran cell lines and tissues efficiently took up the dsRNA. Interestingly, the dsRNA administered to coleopteran cell lines and tissues was taken up and processed to siRNA whereas the dsRNA was taken up by lepidopteran cell lines and tissues but no siRNA was detected in the total RNA isolated from these cell lines and tissues. The data included in this paper showed that the degradation and intracellular transport of dsRNA are the major factors responsible for reduced RNAi efficiency in lepidopteran insects. PMID:27245473

  6. FTO knockdown in rat ventromedial hypothalamus does not affect energy balance

    PubMed Central

    van Gestel, Margriet A.; Sanders, Loek E.; de Jong, Johannes W.; Luijendijk, Mieneke C. M.; Adan, Roger A. H.

    2014-01-01

    Abstract Single nucleotide polymorphisms (SNPs) clustered in the first intron of the fat mass and obesity‐associated (FTO) gene has been associated with obesity. FTO expression is ubiquitous, with particularly high levels in the hypothalamic area of the brain. To investigate the region‐specific role of FTO, AAV technology was applied to knockdown FTO in the ventromedial hypothalamus (VMH). No effect of FTO knockdown was observed on bodyweight or parameters of energy balance. Animals were exposed twice to an overnight fast, followed by a high‐fat high‐sucrose (HFHS) diet for 1 week. FTO knockdown did not result in a different response to the diets. A region‐specific role for FTO in the VMH in the regulation of energy balance could not be found. PMID:25501432

  7. Knockdown of GALNT1 suppresses malignant phenotype of hepatocellular carcinoma by suppressing EGFR signaling

    PubMed Central

    Huang, Miao-Juei; Hu, Rey-Heng; Chou, Chih-Hsing; Hsu, Chia-Lang; Liu, Ya-Wen; Huang, John; Hung, Ji-Shiang; Lai, I-Rue; Juan, Hsueh-Fen; Yu, Sung-Liang; Wu, Yao-Ming; Huang, Min-Chuan

    2015-01-01

    O-glycosylation is a common protein modification. Aberrant O-glycosylation is associated with many cancers. GALNT1 is a GalNAc-transferase that initiates protein O-glycosylation. We found that GALNT1 is frequently up-regulated in hepatocellular carcinoma (HCC) and is associated with poor patient survival. Overexpression of GALNT1 increased and knockdown decreased HCC cell migration and invasion. Knockdown of GALNT1 inhibited EGF-induced migration and invasion. Knockdown of GALNT1 decreased EGFR activation and increased EGFR degradation, by decreasing EGFR O-glycosylation. This study demonstrates that down-regulation of GALNT1 is sufficient to suppress malignant phenotype of HCC cells by decreasing EGFR signaling. Thus, GALNT1 is a potential target in HCC. PMID:25730904

  8. Sleeping Beauty-mediated knockdown of sheep myostatin by RNA interference.

    PubMed

    Hu, Shengwei; Ni, Wei; Sai, Wujiafu; Zhang, Hui; Cao, Xudong; Qiao, Jun; Sheng, Jinliang; Guo, Fei; Chen, Chuangfu

    2011-10-01

    Myostatin is a negative regulator of skeletal muscle growth. Myostatin dysfunction therefore offers a strategy for promoting animal muscle growth in livestock production. Knockdown of myostatin was achieved by combining RNA interference and the Sleeping Beauty (SB) transposon system in sheep cells. Four targeting sites of sheep myostatin were designed and measured for myostatin silencing in sheep fetal fibroblasts by real-time PCR. The sh3 construct induced significant decrease of myostatin gene expression by 90% (P<0.05). Myostatin silencing induced by SB-mediated sh3 was further tested in stably transfected cells. SB transposition increased the integration frequency of genes into sheep genomes and mediated a more efficient myostatin knockdown than random integration of sh3. We suggest that SB-mediated shRNA provides a novel potential tool for gene knockdown in the donor cells of animal cloning. PMID:21698446

  9. Knockdown of Pokemon protein expression inhibits hepatocellular carcinoma cell proliferation by suppression of AKT activity.

    PubMed

    Zhu, Xiaosan; Dai, Yichen; Chen, Zhangxin; Xie, Junpei; Zeng, Wei; Lin, Yuanyuan

    2013-01-01

    Overexpression of Pokemon, which is an erythroid myeloid ontogenic factor protein, occurs in different cancers, including hepatocellular carcinoma (HCC). Pokemon is also reported to have an oncogenic activity in various human cancers. This study investigated the effect of Pokemon knockdown on the regulation of HCC growth. POK shRNA suppressed the expression of Pokemon protein in HepG2 cells compared to the negative control vector-transfected HCC cells. Pokemon knockdown also reduced HCC cell viability and enhanced cisplatin-induced apoptosis in HCC cells. AKT activation and the expression of various cell cycle-related genes were inhibited following Pokemon knockdown. These data demonstrate that Pokemon may play a role in HCC progression, suggesting that inhibition of Pokemon expression using Pokemon shRNA should be further evaluated as a novel target for the control of HCC. PMID:23924858

  10. Knockdown of Akt Sensitizes Osteosarcoma Cells to Apoptosis Induced by Cisplatin Treatment

    PubMed Central

    Zhang, Guoyou; Li, Ming; Zhu, Xiaodong; Bai, Yushu; Yang, Changwei

    2011-01-01

    Akt plays an important role in the inhibition of apoptosis induced by chemotherapy and other stimuli. We therefore investigated if knockdown of Akt2 promoted drug-induced apoptosis in cultured osteosarcoma cells in vitro. SAOS-2 cells were transfected with Akt2 siRNA. The sensitivity of the transformed cell line to the chemotherapeutic drug cisplatin was assessed. Reduced expression of Akt2 did not directly inhibit the growth rate of the transfected cells; however, it significantly increased their sensitivity to cisplatin. Knockdown of Akt2, together with cisplatin treatment, promoted the expression of p53 up-regulated modulator of apoptosis (PUMA). It is possible that the augmentation of cisplatin cytotoxicity may be mediated by PUMA activation. The results of this study suggest that knockdown of Akt2 expression may have therapeutic applications in enhancing the efficacy of chemotherapy in patients with osteosarcoma. PMID:21686164

  11. Withaferin A inhibits in vivo growth of breast cancer cells accelerated by Notch2 knockdown.

    PubMed

    Kim, Su-Hyeong; Hahm, Eun-Ryeong; Arlotti, Julie A; Samanta, Suman K; Moura, Michelle B; Thorne, Stephen H; Shuai, Yongli; Anderson, Carolyn J; White, Alexander G; Lokshin, Anna; Lee, Joomin; Singh, Shivendra V

    2016-05-01

    The present study offers novel insights into the molecular circuitry of accelerated in vivo tumor growth by Notch2 knockdown in triple-negative breast cancer (TNBC) cells. Therapeutic vulnerability of Notch2-altered growth to a small molecule (withaferin A, WA) is also demonstrated. MDA-MB-231 and SUM159 cells were used for the xenograft studies. A variety of technologies were deployed to elucidate the mechanisms underlying tumor growth augmentation by Notch2 knockdown and its reversal by WA, including Fluorescence Molecular Tomography for measurement of tumor angiogenesis in live mice, Seahorse Flux analyzer for ex vivo measurement of tumor metabolism, proteomics, and Luminex-based cytokine profiling. Stable knockdown of Notch2 resulted in accelerated in vivo tumor growth in both cells reflected by tumor volume and/or latency. For example, the wet tumor weight from mice bearing Notch2 knockdown MDA-MB-231 cells was about 7.1-fold higher compared with control (P < 0.0001). Accelerated tumor growth by Notch2 knockdown was highly sensitive to inhibition by a promising steroidal lactone (WA) derived from a medicinal plant. Molecular underpinnings for tumor growth intensification by Notch2 knockdown included compensatory increase in Notch1 activation, increased cellular proliferation and/or angiogenesis, and increased plasma or tumor levels of growth stimulatory cytokines. WA administration reversed many of these effects providing explanation for its remarkable anti-cancer efficacy. Notch2 functions as a tumor growth suppressor in TNBC and WA offers a novel therapeutic strategy for restoring this function. PMID:27097807

  12. Knockdown of FAK inhibits the invasion and metastasis of Tca‑8113 cells in vitro.

    PubMed

    Xiao, Wenbo; Jiang, Mingxin; Li, Hongdan; Li, Chunshan; Su, Rongjian; Huang, Keqiang

    2013-08-01

    Tongue cancer originating on the surface of the tongue is most commonly squamous cell carcinoma, which has a higher invasive ability and a lower survival rate compared with other forms of tongue cancer. Notably, tongue squamous cell carcinomas metastasize into lymph nodes at early stages. Focal adhesion kinase (FAK) is an important protein tyrosine kinase involved in invasion and metastasis of cancer cells. In the present study, the role of FAK in the invasion and metastasis of tongue cancer was evaluated and the underlying mechanisms involved in this process were explored. FAK knockdown was performed using shRNA in the tongue cancer cell line, Tca‑8113, and the invasion and metastasis potentials were analyzed using wound healing and transwell assays, respectively. Cytoskeletal arrangement was detected by fluorescence using TRITC‑conjugated phalloidin staining. The activity of matrix metalloproteinase (MMP)‑2 and ‑9 was examined by gelatin zymography. Paxillin distribution was observed by immunofluorescence. The levels of E‑cadherin, N‑cadherin, MMP‑2 and ‑9, and c‑Jun N‑terminal kinase (JNK) was detected by western blot analysis. Wound healing and transwell assays demonstrated that FAK knockdown inhibited the invasion and metastasis of Tca‑8113 cells. Further analysis revealed that FAK knockdown caused the rearrangement of the cytoskeleton and decreased the activity of MMP‑2 and ‑9. Immunofluorescence analysis revealed that downregulation of FAK induced the relocalization of paxillin. Paxillin accumulated as dots and patches at the cell membrane in control cells. By contrast, in FAK knockdown cells, paxillin was distributed homogeneously in the cytoplasm. Western blot analysis revealed that FAK knockdown inhibited epithelial-mesenchymal transition (EMT) and decreased levels of MMP‑2 and ‑9, and p‑JNK. Knockdown of FAK inhibits the invasion and metastasis of Tca‑8113 by decreasing MMP‑2 and ‑9 activities and led to the

  13. Use of genome-wide RNAi screens to identify regulators of embryonic stem cell self-renewal and pluripotency

    PubMed Central

    Zheng, Xiaofeng; Hu, Guang

    2014-01-01

    Summary Embryonic stem cells (ESCs) are characterized by two defining features: pluripotency and self-renewal. They hold tremendous promise for both basic research and regenerative medicine. To fully realize their potentials, it is important to understand the molecular mechanisms regulating ESC pluripotency and self-renewal. The development of RNA interference (RNAi) technology has revolutionized functional genetic studies in mammalian cells. In recent years, genome-wide RNAi screens have been adopted to systematically study ESC pluripotency and self-renewal, and have uncovered many previously unknown regulators, including transcription factors, chromatin remodelers, and post-transcriptional modulators. Here, we describe a method for the identification of regulators of ESC pluripotency and self-renewal using RNAi screens, as well as assays for further validation and characterization of the identified candidates. With modifications, this method can also be adapted to study the fate specification events during ESC differentiation. PMID:24743997

  14. Developing an in vivo toxicity assay for RNAi risk assessment in honey bees, Apis mellifera L.

    PubMed

    Vélez, Ana María; Jurzenski, Jessica; Matz, Natalie; Zhou, Xuguo; Wang, Haichuan; Ellis, Marion; Siegfried, Blair D

    2016-02-01

    Maize plants expressing dsRNA for the management of Diabrotica virgifera virgifera are likely to be commercially available by the end of this decade. Honey bees, Apis mellifera, can potentially be exposed to pollen from transformed maize expressing dsRNA. Consequently, evaluation of the biological impacts of RNAi in honey bees is a fundamental component for ecological risk assessment. The insecticidal activity of a known lethal dsRNA target for D. v. virgifera, the vATPase subunit A, was evaluated in larval and adult honey bees. Activity of both D. v. virgifera (Dvv)- and A. mellifera (Am)-specific dsRNA was tested by dietary exposure to dsRNA. Larval development, survival, adult eclosion, adult life span and relative gene expression were evaluated. The results of these tests indicated that Dvv vATPase-A dsRNA has limited effects on larval and adult honey bee survival. Importantly, no effects were observed upon exposure of Am vATPase-A dsRNA suggesting that the lack of response involves factors other than sequence specificity. The results from this study provide guidance for future RNAi risk analyses and for the development of a risk assessment framework that incorporates similar hazard assessments. PMID:26454117

  15. Functional identification of optimized RNAi triggers using a massively parallel Sensor assay

    PubMed Central

    Fellmann, Christof; Zuber, Johannes; McJunkin, Katherine; Chang, Kenneth; Malone, Colin D.; Dickins, Ross A.; Xu, Qikai; Hengartner, Michael O.; Elledge, Stephen J.; Hannon, Gregory J.; Lowe, Scott W.

    2011-01-01

    Summary Short hairpin RNAs (shRNAs) provide powerful experimental tools by enabling stable and regulated gene silencing through programming of endogenous microRNA pathways. Since requirements for efficient shRNA biogenesis and target suppression are largely unknown, many predicted shRNAs fail to efficiently suppress their target. To overcome this barrier, we developed a “Sensor assay” that enables the biological identification of effective shRNAs at large scale. By constructing and evaluating 20,000 RNAi reporters covering every possible target site in 9 mammalian transcripts, we show that our assay reliably identifies potent shRNAs that are surprisingly rare and predominantly missed by existing algorithms. Our unbiased analyses reveal that potent shRNAs share various predicted and previously unknown features associated with specific microRNA processing steps, and suggest a new model for competitive strand selection. Together, our study establishes a powerful tool for large-scale identification of highly potent shRNAs and provides new insights into sequence requirements of effective RNAi. PMID:21353615

  16. In vivo RNAi screen identifies NLK as a negative regulator of mesenchymal activity in glioblastoma

    PubMed Central

    Cho, Hee Jin; Lee, Jin-Ku; Kim, Gi-Soo; Han, Suji; Kim, Woon Jin; Shin, Yong Jae; Joo, Kyeung Min; Paddison, Patrick J.; Ishitani, Tohru; Lee, Jeongwu; Nam, Do-Hyun

    2015-01-01

    Glioblastoma (GBM) is the most lethal brain cancer with profound genomic alterations. While the bona fide tumor suppressor genes such as PTEN, NF1, and TP53 have high frequency of inactivating mutations, there may be the genes with GBM-suppressive roles for which genomic mutation is not a primary cause for inactivation. To identify such genes, we employed in vivo RNAi screening approach using the patient-derived GBM xenograft models. We found that Nemo-Like Kinase (NLK) negatively regulates mesenchymal activities, a characteristic of aggressive GBM, in part via inhibition of WNT/β-catenin signaling. Consistent with this, we found that NLK expression is especially low in a subset of GBMs that harbors high WNT/mesenchymal activities. Restoration of NLK inhibited WNT and mesenchymal activities, decreased clonogenic growth and survival, and impeded tumor growth in vivo. These data unravel a tumor suppressive role of NLK and support the feasibility of combining oncogenomics with in vivo RNAi screen. PMID:26023737

  17. Large Scale RNAi Reveals the Requirement of Nuclear Envelope Breakdown for Nuclear Import of Human Papillomaviruses

    PubMed Central

    Snijder, Berend; Samperio Ventayol, Pilar; Kühbacher, Andreas; Becker, Miriam; Day, Patricia M.; Schiller, John T.; Kann, Michael; Pelkmans, Lucas; Helenius, Ari; Schelhaas, Mario

    2014-01-01

    A two-step, high-throughput RNAi silencing screen was used to identify host cell factors required during human papillomavirus type 16 (HPV16) infection. Analysis of validated hits implicated a cluster of mitotic genes and revealed a previously undetermined mechanism for import of the viral DNA (vDNA) into the nucleus. In interphase cells, viruses were endocytosed, routed to the perinuclear area, and uncoated, but the vDNA failed to be imported into the nucleus. Upon nuclear envelope perforation in interphase cells HPV16 infection occured. During mitosis, the vDNA and L2 associated with host cell chromatin on the metaphase plate. Hence, we propose that HPV16 requires nuclear envelope breakdown during mitosis for access of the vDNA to the nucleoplasm. The results accentuate the value of genes found by RNAi screens for investigation of viral infections. The list of cell functions required during HPV16 infection will, moreover, provide a resource for future virus-host cell interaction studies. PMID:24874089

  18. Engineering cherry rootstocks with resistance to Prunus necrotic ring spot virus through RNAi-mediated silencing.

    PubMed

    Song, Guo-qing; Sink, Kenneth C; Walworth, Aaron E; Cook, Meridith A; Allison, Richard F; Lang, Gregory A

    2013-08-01

    Prunus necrotic ringspot virus (PNRSV) is a major pollen-disseminated ilarvirus that adversely affects many Prunus species. In this study, an RNA interference (RNAi) vector pART27-PNRSV containing an inverted repeat (IR) region of PNRSV was transformed into two hybrid (triploid) cherry rootstocks, 'Gisela 6' (GI 148-1) and 'Gisela 7'(GI 148-8)', which are tolerant and sensitive, respectively, to PNRSV infection. One year after inoculation with PNRSV plus Prune Dwarf Virus, nontransgenic 'Gisela 6' exhibited no symptoms but a significant PNRSV titre, while the transgenic 'Gisela 6' had no symptoms and minimal PNRSV titre. The nontransgenic 'Gisela 7' trees died, while the transgenic 'Gisela 7' trees survived. These results demonstrate the RNAi strategy is useful for developing viral resistance in fruit rootstocks, and such transgenic rootstocks may have potential to enhance production of standard, nongenetically modified fruit varieties while avoiding concerns about transgene flow and exogenous protein production that are inherent for transformed fruiting genotypes. PMID:23521804

  19. Adeno Associated Viral Vector Delivered RNAi for Gene Therapy of SOD1 Amyotrophic Lateral Sclerosis

    PubMed Central

    Stoica, Lorelei; Sena-Esteves, Miguel

    2016-01-01

    Amyotrophic Lateral Sclerosis (ALS) is a fatal neurodegenerative disease caused by progressive loss of upper and lower motor neurons. Mutations in superoxide dismutase 1 (SOD1) are a leading cause of ALS, responsible for up to 20% of familial cases. Although the exact mechanism by which mutant SOD1 causes disease remains unknown, multiple studies have shown that reduction of the mutant species leads to delayed disease onset and extension of lifespan of animal models. This makes SOD1 an ideal target for gene therapy coupling adeno associated virus vector (AAV) gene delivery with RNAi molecules. In this review we summarize the studies done thus far attempting to decrease SOD1 gene expression, using AAV vectors as delivery tools, and RNAi as therapeutic molecules. Current hurdles to be overcome, such as the need for widespread gene delivery through the entire central nervous system (CNS), are discussed. Continued efforts to improve current AAV delivery methods and capsids will accelerate the application of these therapeutics to the clinic. PMID:27531973

  20. Efficiency of gene silencing in Arabidopsis: direct inverted repeats vs. transitive RNAi vectors.

    SciTech Connect

    Filichkin, Sergei A; DiFazio, Steven P; Brunner, Amy M; Davis, John M; Yang, Zamin Koo; Kalluri, Udaya C; Arias, Renee S; Etherington, Elizabeth; Tuskan, Gerald A; Strauss, S

    2007-01-01

    We investigated the efficiency of RNA interference (RNAi) in Arabidopsis using transitive and homologous inverted repeat (hIR) vectors. hIR constructs carry self-complementary intron-spliced fragments of the target gene whereas transitive vectors have the target sequence fragment adjacent to an intron-spliced, inverted repeat of heterologous origin. Both transitive and hIR constructs facilitated specific and heritable silencing in the three genes studied (AP1, ETTIN and TTG1). Both types of vectors produced a phenotypic series that phenocopied reduction of function mutants for the respective target gene. The hIR yielded up to fourfold higher proportions of events with strongly manifested reduction of function phenotypes compared to transitive RNAi. We further investigated the efficiency and potential off-target effects of AP1 silencing by both types of vectors using genome-scale microarrays and quantitative RT-PCR. The depletion of AP1 transcripts coincided with reduction of function phenotypic changes among both hIR and transitive lines and also showed similar expression patterns among differentially regulated genes. We did not detect significant silencing directed against homologous potential off-target genes when constructs were designed with minimal sequence similarity. Both hIR and transitive methods are useful tools in plant biotechnology and genomics. The choice of vector will depend on specific objectives such as cloning throughput, number of events and degree of suppression required.

  1. Control of HCV Replication With iMIRs, a Novel Anti-RNAi Agent

    PubMed Central

    Itami, Saori; Eguchi, Yutaka; Mizutani, Takayuki; Aoki, Eriko; Ohgi, Tadaaki; Kuroda, Masahiko; Ochiya, Takahiro; Kato, Nobuyuki; Suzuki, Hiroshi I; Kawada, Norifumi; Murakami, Yoshiki

    2015-01-01

    MicroRNAs (miRNAs) serve important roles in regulating various physiological activities through RNA interference (RNAi). miR-122 is an important mediator of RNAi that is known to control hepatitis C virus (HCV) replication and is being investigated in clinical trials as a target for anti-HCV therapy. In this study, we developed novel oligonucleotides containing non-nucleotide residues, termed iMIRs, and tested their abilities to inhibit miR-122 function. We compared the inhibitory effects of iMIRs and locked nucleic acids (LNAs) on HCV replication in OR6 cells, which contained full-length HCV (genotype 1b) and a luciferase reporter gene. We found that RNA-type iMIRs with bulge-type, imperfect complementarity with respect to miR-122 were 10-fold more effective than LNAs in inhibiting HCV replication and functioned in a dose-dependent manner. Moreover, iMIR treatment of OR6 cells reduced HCV replication without inducing interferon responses or cellular toxicity. Based on these results, we suggest that iMIRs can inhibit HCV replication more effectively than LNAs and are therefore promising as novel antiviral agents.

  2. Stable association of RNAi machinery is conserved between the cytoplasm and nucleus of human cells

    PubMed Central

    Kalantari, Roya; Hicks, Jessica A.; Li, Liande; Gagnon, Keith T.; Sridhara, Viswanadham; Lemoff, Andrew; Mirzaei, Hamid; Corey, David R.

    2016-01-01

    Argonaute 2 (AGO2), the catalytic engine of RNAi, is typically associated with inhibition of translation in the cytoplasm. AGO2 has also been implicated in nuclear processes including transcription and splicing. There has been little insight into AGO2's nuclear interactions or how they might differ relative to cytoplasm. Here we investigate the interactions of cytoplasmic and nuclear AGO2 using semi-quantitative mass spectrometry. Mass spectrometry often reveals long lists of candidate proteins, complicating efforts to rigorously discriminate true interacting partners from artifacts. We prioritized candidates using orthogonal analytical strategies that compare replicate mass spectra of proteins associated with Flag-tagged and endogenous AGO2. Interactions with TRNC6A, TRNC6B, TNRC6C, and AGO3 are conserved between nuclei and cytoplasm. TAR binding protein interacted stably with cytoplasmic AGO2 but not nuclear AGO2, consistent with strand loading in the cytoplasm. Our data suggest that interactions between functionally important components of RNAi machinery are conserved between the nucleus and cytoplasm but that accessory proteins differ. Orthogonal analysis of mass spectra is a powerful approach to streamlining identification of protein partners. PMID:27198507

  3. Postharvest Analysis of Lowland Transgenic Tomato Fruits Harboring hpRNAi-ACO1 Construct

    PubMed Central

    Behboodian, Bita; Mohd Ali, Zainon; Ismail, Ismanizan; Zainal, Zamri

    2012-01-01

    The plant hormone, ethylene, is an important regulator which involved in regulating fruit ripening and flower senescence. In this study, RNA interference (RNAi) technology was employed to silence the genes involved in ethylene biosynthetic pathway. This was achieved by blocking the expression of specific gene encoding the ACC oxidase. Initially, cDNA corresponding to ACO1 of lowland tomato cultivar (MT1), which has high identity with ACO1 of Solanum lycopersicum in GenBank, was cloned through RT-PCR. Using a partial coding region of ACO1, one hpRNAi transformation vector was constructed and expressed ectopically under the 35S promoter. Results showed that transgenic lines harboring the hpRNA-ACO1 construct had lower ethylene production and a longer shelf life of 32 days as compared to 10 days for wild-type fruits. Changes in cell wall degrading enzyme activities were also investigated in cases where the transgenic fruits exhibited reduced rates of firmness loss, which can be associated with a decrease in pectin methylesterase (PME) and polygalacturonase (PG) activities. However, no significant change was detected in both transgenic and wild-type fruits in terms of β-galactosidase (β-Gal) activity and levels of total soluble solid, titratable acid and ascorbic acid. PMID:22919320

  4. Adeno Associated Viral Vector Delivered RNAi for Gene Therapy of SOD1 Amyotrophic Lateral Sclerosis.

    PubMed

    Stoica, Lorelei; Sena-Esteves, Miguel

    2016-01-01

    Amyotrophic Lateral Sclerosis (ALS) is a fatal neurodegenerative disease caused by progressive loss of upper and lower motor neurons. Mutations in superoxide dismutase 1 (SOD1) are a leading cause of ALS, responsible for up to 20% of familial cases. Although the exact mechanism by which mutant SOD1 causes disease remains unknown, multiple studies have shown that reduction of the mutant species leads to delayed disease onset and extension of lifespan of animal models. This makes SOD1 an ideal target for gene therapy coupling adeno associated virus vector (AAV) gene delivery with RNAi molecules. In this review we summarize the studies done thus far attempting to decrease SOD1 gene expression, using AAV vectors as delivery tools, and RNAi as therapeutic molecules. Current hurdles to be overcome, such as the need for widespread gene delivery through the entire central nervous system (CNS), are discussed. Continued efforts to improve current AAV delivery methods and capsids will accelerate the application of these therapeutics to the clinic. PMID:27531973

  5. Large scale RNAi reveals the requirement of nuclear envelope breakdown for nuclear import of human papillomaviruses.

    PubMed

    Aydin, Inci; Weber, Susanne; Snijder, Berend; Samperio Ventayol, Pilar; Kühbacher, Andreas; Becker, Miriam; Day, Patricia M; Schiller, John T; Kann, Michael; Pelkmans, Lucas; Helenius, Ari; Schelhaas, Mario

    2014-05-01

    A two-step, high-throughput RNAi silencing screen was used to identify host cell factors required during human papillomavirus type 16 (HPV16) infection. Analysis of validated hits implicated a cluster of mitotic genes and revealed a previously undetermined mechanism for import of the viral DNA (vDNA) into the nucleus. In interphase cells, viruses were endocytosed, routed to the perinuclear area, and uncoated, but the vDNA failed to be imported into the nucleus. Upon nuclear envelope perforation in interphase cells HPV16 infection occured. During mitosis, the vDNA and L2 associated with host cell chromatin on the metaphase plate. Hence, we propose that HPV16 requires nuclear envelope breakdown during mitosis for access of the vDNA to the nucleoplasm. The results accentuate the value of genes found by RNAi screens for investigation of viral infections. The list of cell functions required during HPV16 infection will, moreover, provide a resource for future virus-host cell interaction studies. PMID:24874089

  6. RNAi pathways in Mucor: A tale of proteins, small RNAs and functional diversity.

    PubMed

    Torres-Martínez, Santiago; Ruiz-Vázquez, Rosa M

    2016-05-01

    The existence of an RNA-mediated silencing mechanism in the opportunistic fungal pathogen Mucor circinelloides was first described in the early 2000. Since then, Mucor has reached an outstanding position within the fungal kingdom as a model system to achieve a deeper understanding of regulation of endogenous functions by the RNA interference (RNAi) machinery. M. circinelloides combines diverse components of its RNAi machinery to carry out functions not only limited to the defense against invasive nucleic acids, but also to regulate expression of its own genes by producing different classes of endogenous small RNA molecules (esRNAs). The recent discovery of a novel RNase that participates in a new RNA degradation pathway adds more elements to the gene silencing-mediated regulation. This review focuses on esRNAs in M. circinelloides, the different pathways involved in their biogenesis, and their roles in regulating specific physiological and developmental processes in response to environmental signals, highlighting the complexity of silencing-mediated regulation in fungi. PMID:26593631

  7. RNAi Mediated curcin precursor gene silencing in Jatropha (Jatropha curcas L.).

    PubMed

    Patade, Vikas Yadav; Khatri, Deepti; Kumar, Kamal; Grover, Atul; Kumari, Maya; Gupta, Sanjay Mohan; Kumar, Devender; Nasim, Mohammed

    2014-07-01

    Curcin, a type I ribosomal inhibiting protein-RIP, encoded by curcin precursor gene, is a phytotoxin present in Jatropha (Jatropha curcas L.). Here, we report designing of RNAi construct for the curcin precursor gene and further its genetic transformation of Jatropha to reduce its transcript expression. Curcin precursor gene was first cloned from Jatropha strain DARL-2 and part of the gene sequence was cloned in sense and antisense orientation separated by an intron sequence in plant expression binary vector pRI101 AN. The construction of the RNAi vector was confirmed by double digestion and nucleotide sequencing. The vector was then mobilized into Agrobacterium tumefaciens strain GV 3101 and used for tissue culture independent in planta transformation protocol optimized for Jatropha. Germinating seeds were injured with a needle before infection with Agrobacterium and then transferred to sterilized sand medium. The seedlings were grown for 90 days and genomic DNA was isolated from leaves for transgenic confirmation based on real time PCR with NPT II specific dual labeled probe. Result of the transgenic confirmation analysis revealed presence of the gene silencing construct in ten out of 30 tested seedlings. Further, quantitative transcript expression analysis of the curcin precursor gene revealed reduction in the transcript abundance by more than 98% to undetectable level. The transgenic plants are being grown in containment for further studies on reduction in curcin protein content in Jatropha seeds. PMID:24574003

  8. DNA vector-based RNAi approach for stable depletion of poly(ADP-ribose) polymerase-1

    SciTech Connect

    Shah, Rashmi G.; Ghodgaonkar, Medini M.; Affar, El Bachir; Shah, Girish M. . E-mail: girish.shah@crchul.ulaval.ca

    2005-05-27

    RNA-mediated interference (RNAi) is a powerful technique that is now being used in mammalian cells to specifically silence a gene. Some recent studies have used this technique to achieve variable extent of depletion of a nuclear enzyme poly(ADP-ribose) polymerase-1 (PARP-1). These studies reported either transient silencing of PARP-1 using double-stranded RNA or stable silencing of PARP-1 with a DNA vector which was introduced by a viral delivery system. In contrast, here we report that a simple RNAi approach which utilizes a pBS-U6-based DNA vector containing strategically selected PARP-1 targeting sequence, introduced in the cells by conventional CaPO{sub 4} protocol, can be used to achieve stable and specific silencing of PARP-1 in different types of cells. We also provide a detailed strategy for selection and cloning of PARP-1-targeting sequences for the DNA vector, and demonstrate that this technique does not affect expression of its closest functional homolog PARP-2.

  9. A ribonuclease coordinates siRNA amplification and mRNA cleavage during RNAi

    PubMed Central

    Tsai, Hsin-Yue; Chen, Chun-Chieh G.; Conte, Darryl; Moresco, James J.; Chaves, Daniel A.; Mitani, Shohei; Yates, John R.; Tsai, Ming-Daw; Mello, Craig C.

    2015-01-01

    SUMMARY Effective silencing by RNA-interference (RNAi) depends on mechanisms that amplify and propagate the silencing signal. In some organisms, small-interfering (si) RNAs are amplified from target mRNAs by RNA-dependent RNA polymerase (RdRP). Both RdRP recruitment and mRNA silencing require Argonaute proteins, which are generally thought to degrade RNAi targets by directly cleaving them. However in C. elegans, the enzymatic activity of the primary Argonaute, RDE-1, is not required for silencing activity. We show that RDE-1 can instead recruit an endoribonuclease, RDE-8, to target RNA. RDE-8 can cleave RNA in vitro and is needed for the production of 3′ uridylated fragments of target mRNA in vivo. We also find that RDE-8 promotes RdRP activity, thereby ensuring amplification of siRNAs. Together, our findings suggest a model in which RDE-8 cleaves target mRNAs to mediate silencing, while generating 3’ uridylated mRNA fragments to serve as templates for the RdRP-directed amplification of the silencing signal. PMID:25635455

  10. Stable association of RNAi machinery is conserved between the cytoplasm and nucleus of human cells.

    PubMed

    Kalantari, Roya; Hicks, Jessica A; Li, Liande; Gagnon, Keith T; Sridhara, Viswanadham; Lemoff, Andrew; Mirzaei, Hamid; Corey, David R

    2016-07-01

    Argonaute 2 (AGO2), the catalytic engine of RNAi, is typically associated with inhibition of translation in the cytoplasm. AGO2 has also been implicated in nuclear processes including transcription and splicing. There has been little insight into AGO2's nuclear interactions or how they might differ relative to cytoplasm. Here we investigate the interactions of cytoplasmic and nuclear AGO2 using semi-quantitative mass spectrometry. Mass spectrometry often reveals long lists of candidate proteins, complicating efforts to rigorously discriminate true interacting partners from artifacts. We prioritized candidates using orthogonal analytical strategies that compare replicate mass spectra of proteins associated with Flag-tagged and endogenous AGO2. Interactions with TRNC6A, TRNC6B, TNRC6C, and AGO3 are conserved between nuclei and cytoplasm. TAR binding protein interacted stably with cytoplasmic AGO2 but not nuclear AGO2, consistent with strand loading in the cytoplasm. Our data suggest that interactions between functionally important components of RNAi machinery are conserved between the nucleus and cytoplasm but that accessory proteins differ. Orthogonal analysis of mass spectra is a powerful approach to streamlining identification of protein partners. PMID:27198507

  11. Allele-specific RNAi Mitigates Phenotypic Progression in a Transgenic Model of Alzheimer's Disease

    PubMed Central

    Rodríguez-Lebrón, Edgardo; Gouvion, Cynthia M; Moore, Steven A; Davidson, Beverly L; Paulson, Henry L

    2009-01-01

    Despite recent advances suggesting new therapeutic targets, Alzheimer's disease (AD) remains incurable. Aberrant production and accumulation of the Aβ peptide resulting from altered processing of the amyloid precursor protein (APP) is central to the pathogenesis of disease, particularly in dominantly inherited forms of AD. Thus, modulating the production of APP is a potential route to effective AD therapy. Here, we describe the successful use of an allele-specific RNA interference (RNAi) approach targeting the Swedish variant of APP (APPsw) in a transgenic mouse model of AD. Using recombinant adeno-associated virus (rAAV), we delivered an anti-APPsw short-hairpin RNA (shRNA) to the hippocampus of AD transgenic mice (APP/PS1). In short- and long-term transduction experiments, reduced levels of APPsw transprotein were observed throughout targeted regions of the hippocampus while levels of wild-type murine APP remained unaltered. Moreover, intracellular production of transfer RNA (tRNA)-valine promoter–driven shRNAs did not lead to detectable neuronal toxicity. Finally, long-term bilateral hippocampal expression of anti-APPsw shRNA mitigated abnormal behaviors in this mouse model of AD. The difference in phenotype progression was associated with reduced levels of soluble Aβ but not with a reduced number of amyloid plaques. Our results support the development of allele-specific RNAi strategies to treat familial AD and other dominantly inherited neurodegenerative diseases. PMID:19532137

  12. Genome-wide analysis of Notch signalling in Drosophila by transgenic RNAi

    PubMed Central

    Mummery-Widmer, Jennifer L.; Yamazaki, Masakazu; Stoeger, Thomas; Novatchkova, Maria; Bhalerao, Sheetal; Chen, Doris; Dietzl, Georg; Dickson, Barry J.; Knoblich, Juergen A.

    2010-01-01

    Genome-wide RNA interference (RNAi) screens have identified near-complete sets of genes involved in cellular processes. However, this methodology has not yet been used to study complex developmental processes in a tissue-specific manner. Here we report the use of a library of Drosophila strains expressing inducible hairpin RNAi constructs to study the Notch signalling pathway during external sensory organ development. We assigned putative loss-of-function phenotypes to 21.2% of the protein-coding Drosophila genes. Using secondary assays, we identified 6 new genes involved in asymmetric cell division and 23 novel genes regulating the Notch signalling pathway. By integrating our phenotypic results with protein interaction data, we constructed a genome-wide, functionally validated interaction network governing Notch signalling and asymmetric cell division. We used clustering algorithms to identify nuclear import pathways and the COP9 signallosome as Notch regulators. Our results show that complex developmental processes can be analysed on a genome-wide level and provide a unique resource for functional annotation of the Drosophila genome. PMID:19363474

  13. Knockdown of mTOR by lentivirus‑mediated RNA interference suppresses atherosclerosis and stabilizes plaques via a decrease of macrophages by autophagy in apolipoprotein E‑deficient mice.

    PubMed

    Wang, Xiaochuang; Li, Lingxia; Li, Manxiang; Dang, Xiaoyan; Wan, Lin; Wang, Ni; Bi, Xiaoju; Gu, Changwei; Qiu, Suijuan; Niu, Xiaolin; Zhu, Xinye; Wang, Lina

    2013-11-01

    Atherosclerotic plaque destabilization and rupture leads to acute coronary syndromes which cause serious damage to human health worldwide. However, there is currently a lack of efficient therapeutic methods. Mammalian target of rapamycin (mTOR) has been suggested to be involved in the development of atherosclerotic plaques and serves as a therapeutic target. The present study was performed to determine whether RNA interference (RNAi) of mTOR in vivo by LV‑mediated small hairpin RNA (shRNA) was capable of inhibiting the progression of atherosclerotic plaques. LV‑mediated shRNA against mTOR (LV‑shmTOR) was designed and obtained. Male apolipoprotein E‑deficient mice were fed a high‑fat diet and a constrictive collar was placed around the right carotid arteries of these mice to induce plaque formation. Eight weeks after surgery, mice were randomly divided into the mTOR RNA interference (LV‑shmTOR) group, receiving treatment with LV‑mTOR‑shRNA; the LV‑shCON group, receiving treatment with LV‑non‑specific‑shRNA; and the control group, receiving treatment with phosphate‑buffered saline. Following transfection, the mice were sacrificed to evaluate the effects of mTOR expression silencing on atherosclerosis. Transfection of LV‑mTOR‑shRNA markedly inhibited the mRNA and protein expression levels. Knockdown of mTOR ameliorated dysregulated blood lipid metabolism and stabilized aortic atherosclerotic plaques by decreasing the plaque area and increasing the fibrous cap and cap‑to‑core ratio. Furthermore, macrophages were decreased by silencing mTOR in atherosclerotic plaques. In addition, western blot analysis revealed that the knockdown of mTOR increased autophagy‑related protein 13 (Atg13) dephosphorylation and light chain 3‑I/light chain 3‑II (LC3‑I/LC3‑II) ratios, both of which were associated with a high activity of autophagy, suggesting an increase of autophagy in atherosclerotic plaques. Moreover, genes including matrix

  14. Anti-angiogenic and anti-proliferative effects of inhibition of HIF-1α by p-HIF-1α RNAi in colorectal cancer.

    PubMed

    Qiu, Yan-Yan; Hu, Song-Jiao; Bao, Yi-Jie; Liang, Bo; Yan, Cui-Na; Shi, Xiao-Jing; Yu, Hui; Zou, Yu; Tang, Li-Rui; Tang, Qing-Feng; Feng, Wen; Yin, Pei-Hao

    2015-01-01

    The aim of this study was to construct an RNA-interference plasmid (p-HIF-1α RNAi) targeting the human HIF-1α gene and assess its effects on HIF-1α expression and its anti-tumour functions in vitro. p-HIF-1α RNAi was constructed and confirmed by polymerase chain reaction (PCR) and DNA sequencing. Reverse transcriptase PCR (RT-PCR) and western blot were performed to detect HIF-1α expression in HCT116 cells following transfection of p-HIF-1α RNAi and p-control. The anti-tumour effects and mechanism of action of p-HIF-1α RNAi in HCT116 cells were further investigated. p-HIF-1α RNAi significantly inhibited HIF-1α expression in the HCT116 cell line. p-HIF-1α RNAi inhibited cell viability and reduced VEGF but not bFGF expression in the supernatant of HCT116 cells, down-regulated b-catenin and VEGF expression, and altered β-catenin location in the HCT116 cell nucleus. The plasmid p-HIF-1α RNAi can effectively and specifically inhibit HIF-1α expression, inhibit cell proliferation, and alter the expression of key components in the Wnt/β-catenin signaling pathway. Thus, p-HIF-1α RNAi is a novel and extremely promising therapeutic inhibitor of HIF-1α. PMID:26339356

  15. TET1 knockdown inhibits the odontogenic differentiation potential of human dental pulp cells

    PubMed Central

    Rao, Li-Jia; Yi, Bai-Cheng; Li, Qi-Meng; Xu, Qiong

    2016-01-01

    Human dental pulp cells (hDPCs) possess the capacity to differentiate into odontoblast-like cells and generate reparative dentin in response to exogenous stimuli or injury. Ten–eleven translocation 1 (TET1) is a novel DNA methyldioxygenase that plays an important role in the promotion of DNA demethylation and transcriptional regulation in several cell lines. However, the role of TET1 in the biological functions of hDPCs is unknown. To investigate the effect of TET1 on the proliferation and odontogenic differentiation potential of hDPCs, a recombinant shRNA lentiviral vector was used to knock down TET1 expression in hDPCs. Following TET1 knockdown, TET1 was significantly downregulated at both the mRNA and protein levels. Proliferation of the hDPCs was suppressed in the TET1 knockdown groups. Alkaline phosphatase activity, the formation of mineralized nodules, and the expression levels of DSPP and DMP1 were all reduced in the TET1-knockdown hDPCs undergoing odontogenic differentiation. Based on these results, we concluded that TET1 knockdown can prevent the proliferation and odontogenic differentiation of hDPCs, which suggests that TET1 may play an important role in dental pulp repair and regeneration. PMID:27357322

  16. TET1 knockdown inhibits the odontogenic differentiation potential of human dental pulp cells.

    PubMed

    Rao, Li-Jia; Yi, Bai-Cheng; Li, Qi-Meng; Xu, Qiong

    2016-01-01

    Human dental pulp cells (hDPCs) possess the capacity to differentiate into odontoblast-like cells and generate reparative dentin in response to exogenous stimuli or injury. Ten-eleven translocation 1 (TET1) is a novel DNA methyldioxygenase that plays an important role in the promotion of DNA demethylation and transcriptional regulation in several cell lines. However, the role of TET1 in the biological functions of hDPCs is unknown. To investigate the effect of TET1 on the proliferation and odontogenic differentiation potential of hDPCs, a recombinant shRNA lentiviral vector was used to knock down TET1 expression in hDPCs. Following TET1 knockdown, TET1 was significantly downregulated at both the mRNA and protein levels. Proliferation of the hDPCs was suppressed in the TET1 knockdown groups. Alkaline phosphatase activity, the formation of mineralized nodules, and the expression levels of DSPP and DMP1 were all reduced in the TET1-knockdown hDPCs undergoing odontogenic differentiation. Based on these results, we concluded that TET1 knockdown can prevent the proliferation and odontogenic differentiation of hDPCs, which suggests that TET1 may play an important role in dental pulp repair and regeneration. PMID:27357322

  17. Knockdown resistance in pyrethroid-resistant horn fly (Diptera: Muscidae) populations in Brazil

    Technology Transfer Automated Retrieval System (TEKTRAN)

    To investigate the kdr (knockdown resistance) resistance-associated gene mutation and determine its frequency in pyrethroid-resistant horn fly (Haematobia irritans) populations, a total of 1,804 horn flies of 37 different populations from all Brazilian regions (North, Northeast, Central-West, Southe...

  18. Knockdown of EHF inhibited the proliferation, invasion and tumorigenesis of ovarian cancer cells.

    PubMed

    Cheng, Zhongping; Guo, Jing; Chen, Li; Luo, Ning; Yang, Weihong; Qu, Xiaoyan

    2016-06-01

    Ovarian cancer is the most lethal gynecologic malignancy worldwide. ETS homologous factor (EHF), a member of E26 transformation specific (ETS) transcription factors, has been reported overexpressed in ovarian cancer. However, the molecular mechanism underlying the biological function of EHF in ovarian cancer is still unclear. Here, we found that EHF was elevated in ovarian cancer tissues compared with non-tumorous tissues. Moreover, high EHF expression level was correlated with short survival time of patients with ovarian cancer. Knockdown of EHF in ovarian cancer cells, SKOV3 and OVCAR3, significantly inhibited cell proliferation and increased cells population in G1 phase. The proteins promoting cell cycles (Cyclin B1, Cyclin D1, and PCNA) were down-regulated and the protein negatively regulating cell cycle progression (P21) was up-regulated after EHF knockdown. Moreover, inhibition of EHF in ovarian cancer cells dramatically induced cell apoptosis, but impaired cell adhesion and cell invasion. Furthermore, phosphorylation levels of ERK and AKT were notably reduced in EHF knockdown cells. Finally, in vivo data showed that knockdown of EHF inhibited tumor growth in nude mice. Our data indicates that EHF could be a potential prognosis marker for ovarian cancer and work as an oncogene by targeting ERK and AKT signaling, which can serve as a new target for ovarian cancer treatment. © 2015 Wiley Periodicals, Inc. PMID:26258986

  19. Strength knock-down assessment of porosity in composites: modelling, characterising and specimen manufacture

    NASA Astrophysics Data System (ADS)

    McMillan, Alison J.; Archer, Edward; McIlhagger, Alistair; Lelong, Guillaume

    2012-08-01

    Porosity and inclusion of foreign material is known to reduce the strength of materials, and this paper addresses the particular problem of strength knock-down assessment due to porosity in composite materials. Porosity is often measured in terms of percentage of voids per unit volume of a component, because this can be related directly to ultra-sound absorption. Nevertheless, this is a poor indicator of actual strength knock-down, as it provides little information about void size, shape, orientation and whether they are evenly distributed or are clustered. Characterisation of void clustering enables a link between a cluster characteristic and the strength knock-down. Laboratory based testing achieves controlled porosity in specimens by introducing pin-holes into the RTM in-flow pipework, which entrains voids into the body of the preform within mould tooling. Specimens are manufactured to create resin regions bounded by a fibre reinforced picture frame, to allow for easy load application. Strength knock-downs from test are related to the theoretical expectations.

  20. The functional genetic link of NLGN4X knockdown and neurodevelopment in neural stem cells

    PubMed Central

    Shi, Lingling; Chang, Xiao; Zhang, Peilin; Coba, Marcelo P.; Lu, Wange; Wang, Kai

    2013-01-01

    Genetic mutations in NLGN4X (neuroligin 4), including point mutations and copy number variants (CNVs), have been associated with susceptibility to autism spectrum disorders (ASDs). However, it is unclear how mutations in NLGN4X result in neurodevelopmental defects. Here, we used neural stem cells (NSCs) as in vitro models to explore the impacts of NLGN4X knockdown on neurodevelopment. Using two shRNAmir-based vectors targeting NLGN4X and one control shRNAmir vector, we modulated NLGN4X expression and differentiated these NSCs into mature neurons. We monitored the neurodevelopmental process at Weeks 0, 0.5, 1, 2, 4 and 6, based on morphological analysis and whole-genome gene expression profiling. At the cellular level, in NSCs with NLGN4X knockdown, we observed increasingly delayed neuronal development and compromised neurite formation, starting from Week 2 through Week 6 post differentiation. At the molecular level, we identified multiple pathways, such as neurogenesis, neuron differentiation and muscle development, which are increasingly disturbed in cells with NLGN4X knockdown. Notably, several postsynaptic genes, including DLG4, NLGN1 and NLGN3, also have decreased expression. Based on in vitro models, NLGN4X knockdown directly impacts neurodevelopmental process during the formation of neurons and their connections. Our functional genomics study highlights the utility of NSCs models in understanding the functional roles of CNVs in affecting neurodevelopment and conferring susceptibility to neurodevelopmental diseases. PMID:23710042

  1. An elastic liposomal formulation for RNAi-based topical treatment of skin disorders: Proof-of-concept in the treatment of psoriasis.

    PubMed

    Desmet, Eline; Bracke, Stefanie; Forier, Katrien; Taevernier, Lien; Stuart, Marc C A; De Spiegeleer, Bart; Raemdonck, Koen; Van Gele, Mireille; Lambert, Jo

    2016-03-16

    RNA interference (RNAi) is a rapidly emerging approach for targeted gene silencing to alleviate disease pathology. However, lack of efficient carriers for targeted delivery delays the clinical translation of RNAi. An interesting target for local RNAi therapeutics is the skin as it allows direct access to target cells. Still, applications are limited due to the effective skin barrier which hinders penetration. Herein, a description is given of a liposomal carrier, called 'DDC642', capable of delivering RNAi molecules to the epidermis of impaired and intact human skin, without targeting the dermis or circulatory system. In a psoriasis tissue model, down-regulation of the psoriasis marker human beta-defensin 2 by DDC642-delivered siRNA was confirmed, providing proof-of-concept. These liposomes thus hold great potential as topical delivery system for RNAi therapeutics in the treatment of numerous skin diseases. PMID:26806466

  2. Severe Nephrotoxic Nephritis following Conditional and Kidney-Specific Knockdown of Stanniocalcin-1

    PubMed Central

    Huang, Luping; Lou, Yahuan; Ju, Huiming; Zhang, Lin; Pan, Jenny Szu-Chin; Ross, April; Sun, Yuxiang; Truong, Luan D.; Sheikh-Hamad, David

    2015-01-01

    Background Inflammation is the hallmark of nephrotoxic nephritis. Stanniocalcin-1 (STC1), a pro-survival factor, inhibits macrophages, stabilizes endothelial barrier function, and diminishes trans-endothelial migration of leukocytes; consistently, transgenic (Tg) overexpression of STC1 protects from nephrotoxic nephritis. Herein, we sought to determine the phenotype of nephrotoxic nephritis after conditional and kidney-specific knockdown of STC1. Methods We used Tg mice that, express either STC1 shRNA (70% knockdown of STC1 within 4d) or scrambled shRNA (control) upon delivery of Cre-expressing plasmid to the kidney using ultrasound microbubble technique. Sheep anti-mouse GBM antibody was administered 4d after shRNA activation; and mice were euthanized 10 days later for analysis. Results Serum creatinine, proteinuria, albuminuria and urine output were similar 10 days after anti-GBM delivery in both groups; however, anti-GBM antibody delivery to mice with kidney-specific knockdown of STC1 produced severe nephrotoxic nephritis, characterized by severe tubular necrosis, glomerular hyalinosis/necrosis and massive cast formation, while control mice manifested mild tubular injury and crescentic glomerulonephritis. Surprisingly, the expression of cytokines/chemokines and infiltration with T-cells and macrophages were also diminished in STC1 knockdown kidneys. Staining for sheep anti-mouse GBM antibody, deposition of mouse C3 and IgG in the kidney, and antibody response to sheep IgG were equal. Conclusions nephrotoxic nephritis after kidney-specific knockdown of STC1 is characterized by severe tubular and glomerular necrosis, possibly due to loss of STC1-mediated pro-survival factors, and we attribute the paucity of inflammation to diminished release of cytokines/chemokines/growth factors from the necrotic epithelium. PMID:26393521

  3. Dimethylated H3K27 Is a Repressive Epigenetic Histone Mark in the Protist Entamoeba histolytica and Is Significantly Enriched in Genes Silenced via the RNAi Pathway.

    PubMed

    Foda, Bardees M; Singh, Upinder

    2015-08-21

    RNA interference (RNAi) is a fundamental biological process that plays a crucial role in regulation of gene expression in many organisms. Transcriptional gene silencing (TGS) is one of the important nuclear roles of RNAi. Our previous data show that Entamoeba histolytica has a robust RNAi pathway that links to TGS via Argonaute 2-2 (Ago2-2) associated 27-nucleotide small RNAs with 5'-polyphosphate termini. Here, we report the first repressive histone mark to be identified in E. histolytica, dimethylation of H3K27 (H3K27Me2), and demonstrate that it is enriched at genes that are silenced by RNAi-mediated TGS. An RNAi-silencing trigger can induce H3K27Me2 deposits at both episomal and chromosomal loci, mediating gene silencing. Our data support two phases of RNAi-mediated TGS: an active silencing phase where the RNAi trigger is present and both H3K27Me2 and Ago2-2 concurrently enrich at chromosomal loci; and an established silencing phase in which the RNAi trigger is removed, but gene silencing with H3K27Me2 enrichment persist independently of Ago2-2 deposition. Importantly, some genes display resistance to chromosomal silencing despite induction of functional small RNAs. In those situations, the RNAi-triggering plasmid that is maintained episomally gets partially silenced and has H3K27Me2 enrichment, but the chromosomal copy displays no repressive histone enrichment. Our data are consistent with a model in which H3K27Me2 is a repressive histone modification, which is strongly associated with transcriptional repression. This is the first example of an epigenetic histone modification that functions to mediate RNAi-mediated TGS in the deep-branching eukaryote E. histolytica. PMID:26149683

  4. Constraint factor graph cut–based active contour method for automated cellular image segmentation in RNAi screening

    PubMed Central

    CHEN, C.; LI, H.; ZHOU, X.; WONG, S. T. C.

    2010-01-01

    Summary Image-based, high throughput genome-wide RNA interference (RNAi) experiments are increasingly carried out to facilitate the understanding of gene functions in intricate biological processes. Automated screening of such experiments generates a large number of images with great variations in image quality, which makes manual analysis unreasonably time-consuming. Therefore, effective techniques for automatic image analysis are urgently needed, in which segmentation is one of the most important steps. This paper proposes a fully automatic method for cells segmentation in genome-wide RNAi screening images. The method consists of two steps: nuclei and cytoplasm segmentation. Nuclei are extracted and labelled to initialize cytoplasm segmentation. Since the quality of RNAi image is rather poor, a novel scale-adaptive steerable filter is designed to enhance the image in order to extract long and thin protrusions on the spiky cells. Then, constraint factor GCBAC method and morphological algorithms are combined to be an integrated method to segment tight clustered cells. Compared with the results obtained by using seeded watershed and the ground truth, that is, manual labelling results by experts in RNAi screening data, our method achieves higher accuracy. Compared with active contour methods, our method consumes much less time. The positive results indicate that the proposed method can be applied in automatic image analysis of multi-channel image screening data. PMID:18445146

  5. Effects of mutations in stem and loop regions on the structure and function of adenovirus VA RNAI.

    PubMed Central

    Mellits, K H; Mathews, M B

    1988-01-01

    Adenovirus virus-associated (VA) RNAI is required for efficient protein synthesis at late times of adenoviral infection, and in some other situations where double-stranded RNA (dsRNA) is present. It prevents inhibition of protein synthesis by a dsRNA-activated protein kinase and the secondary structure of VA RNAI is though to be important for its activity. To test this idea and to define structures and sequences responsible for VA RNAI activity, we constructed several mutant VA RNA genes and tested them in a transient expression assay. Activity is unaffected by deletions within a small region near the center of the gene, nt 72-85, but it is greatly diminished by deletion or substitution of sequences on the 3' side of this region. The structures of wild-type and mutant RNAs were examined by nuclease-sensitivity analysis. We propose a model for wild-type VA RNAI which differs from that predicted to be the most stable structure. Surprisingly disruption of the longest duplex region in the molecule is tolerated, provided that adjacent structural elements are not rearranged. However, perturbations of elements located in the center of the structure correlate well with loss of function. Images PMID:3181142

  6. The novel ABC transporter ABCH1 is a potential target for RNAi-based insect pest control and resistance management

    PubMed Central

    Guo, Zhaojiang; Kang, Shi; Zhu, Xun; Xia, Jixing; Wu, Qingjun; Wang, Shaoli; Xie, Wen; Zhang, Youjun

    2015-01-01

    Insect pests cause serious crop damage and develop high-level resistance to chemical insecticides and Bacillus thuringiensis (Bt) insecticidal Cry toxins. A new promising approach for controlling them and overcoming this resistance is RNA interference (RNAi). The RNAi-based insect control strategy depends on the selection of suitable target genes. In this study, we cloned and characterized a novel ABC transporter gene PxABCH1 in diamondback moth, Plutella xylostella (L.). Phylogenetic analysis showed that PxABCH1 is closely related to ABCA and ABCG subfamily members. Spatial-temporal expression detection revealed that PxABCH1 was expressed in all tissues and developmental stages, and highest expressed in head and male adult. Midgut sequence variation and expression analyses of PxABCH1 in all the susceptible and Bt-resistant P. xylostella strains and the functional analysis by sublethal RNAi demonstrated that Cry1Ac resistance was independent of this gene. Silencing of PxABCH1 by a relatively high dose of dsRNA dramatically reduced its expression and resulted in larval and pupal lethal phenotypes in both susceptible and Cry1Ac-resistant P. xylostella strains. To our knowledge, this study provides the first insight into ABCH1 in lepidopterans and reveals it as an excellent target for RNAi-based insect pest control and resistance management. PMID:26333918

  7. The novel ABC transporter ABCH1 is a potential target for RNAi-based insect pest control and resistance management.

    PubMed

    Guo, Zhaojiang; Kang, Shi; Zhu, Xun; Xia, Jixing; Wu, Qingjun; Wang, Shaoli; Xie, Wen; Zhang, Youjun

    2015-01-01

    Insect pests cause serious crop damage and develop high-level resistance to chemical insecticides and Bacillus thuringiensis (Bt) insecticidal Cry toxins. A new promising approach for controlling them and overcoming this resistance is RNA interference (RNAi). The RNAi-based insect control strategy depends on the selection of suitable target genes. In this study, we cloned and characterized a novel ABC transporter gene PxABCH1 in diamondback moth, Plutella xylostella (L.). Phylogenetic analysis showed that PxABCH1 is closely related to ABCA and ABCG subfamily members. Spatial-temporal expression detection revealed that PxABCH1 was expressed in all tissues and developmental stages, and highest expressed in head and male adult. Midgut sequence variation and expression analyses of PxABCH1 in all the susceptible and Bt-resistant P. xylostella strains and the functional analysis by sublethal RNAi demonstrated that Cry1Ac resistance was independent of this gene. Silencing of PxABCH1 by a relatively high dose of dsRNA dramatically reduced its expression and resulted in larval and pupal lethal phenotypes in both susceptible and Cry1Ac-resistant P. xylostella strains. To our knowledge, this study provides the first insight into ABCH1 in lepidopterans and reveals it as an excellent target for RNAi-based insect pest control and resistance management. PMID:26333918

  8. RNAi-mediated resistance against Cotton leaf curl disease in elite Indian cotton (Gossypium hirsutum) cultivar Narasimha.

    PubMed

    Khatoon, Sameena; Kumar, Abhinav; Sarin, Neera B; Khan, Jawaid A

    2016-08-01

    Cotton leaf curl disease (CLCuD) is caused by several distinct begomovirus species in association with disease-specific betasatellite essential for induction of disease symptoms. CLCuD is a serious threat for the cultivation of cotton (Gossypium sp.) and several species in the family Malvaceae. In this study, RNAi-based approach was applied to generate transgenic cotton (Gossypium hirsutum) plants resistant to Cotton leaf curl Rajasthan virus (CLCuRV). An intron hairpin (ihp) RNAi construct capable of expressing dsRNA homologous to the intergenic region (IR) of CLCuRV was designed and developed. Following Agrobacterium tumefaciens-mediated transformation of cotton (G. hirsutum cv. Narasimha) plants with the designed ihpRNAi construct, a total of 9 independent lines of transformed cotton were obtained. The presence of the potential stretch of IR in the transformed cotton was confirmed by PCR coupled with Southern hybridization. Upon inoculation with viruliferous whiteflies, the transgenic plants showed high degree of resistance. None of them displayed any CLCuD symptoms even after 90 days post inoculation. The transformed cotton plants showed the presence of siRNAs. The present study demonstrated that ihp dsRNA-mediated resistance strategy of RNAi is an effective means to combat the CLCuD infection in cotton. PMID:27028623

  9. Agrobacterium-mediated transformation of the β-subunit gene in 7S globulin protein in soybean using RNAi technology.

    PubMed

    Qu, J; Liu, S Y; Wang, P W; Guan, S Y; Fan, Y G; Yao, D; Zhang, L; Dai, J L

    2016-01-01

    The objective of this study was to use RNA interference (RNAi) to improve protein quality and decrease anti-nutritional effects in soybean. Agrobacterium tumefaciens-mediated transformation was conducted using RNAi and an expression vector containing the 7S globulin β-subunit gene. The BAR gene was used as the selective marker and cotyledonary nodes of soybean genotype Jinong 27 were chosen as explant material. Regenerated plants were detected by molecular biology techniques. Transformation of the β-subunit gene in the 7S protein was detected by PCR, Southern blot, and q-PCR. Positive plants (10 T0, and 6 T1, and 13 T2) were tested by PCR. Hybridization bands were detected by Southern blot analysis in two of the T1 transgenic plants. RNAi expression vectors containing the soybean 7S protein β-subunit gene were successfully integrated into the genome of transgenic plants. qRT-PCR analysis in soybean seeds showed a clear decrease in expression of the soybean β-subunit gene. The level of 7S protein β-subunit expression in transgenic plants decreased by 77.5% as compared to that of the wild-type plants. This study has established a basis for the application of RNAi to improve the anti-nutritional effects of soybean. PMID:27173254

  10. Control of larval and egg development in Aedes aegypti with Ribonucleic acid interference (RNAi) against juvenile hormone acid methyl transferase

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Ribonucleic acid interference (RNAi) is a powerful approach for elucidating gene functions in a variety of organisms, including mosquitoes and many other insects. Little has been done, however, to harness this approach in order to control adult and larval mosquitoes. Juvenile hormone (JH) plays a pi...

  11. Neuronal Migration Is Regulated by Endogenous RNAi and Chromatin-Binding Factor ZFP-1/AF10 in Caenorhabditis elegans

    PubMed Central

    Kennedy, Lisa M.; Grishok, Alla

    2014-01-01

    Endogenous short RNAs and the conserved plant homeodomain (PHD) zinc-finger protein ZFP-1/AF10 regulate overlapping sets of genes in Caenorhabditis elegans, which suggests that they control common biological pathways. We have shown recently that the RNAi factor RDE-4 and ZFP-1 negatively modulate transcription of the insulin/PI3 signaling-dependent kinase PDK-1 to promote C. elegans fitness. Moreover, we have demonstrated that the insulin/IGF-1-PI3K-signaling pathway regulates the activity of the DAF-16/FOXO transcription factor in the hypodermis to nonautonomously promote the anterior migrations of the hermaphrodite-specific neurons (HSNs) during embryogenesis of C. elegans. In this study, we implicate the PHD-containing isoform of ZFP-1 and endogenous RNAi in the regulation of HSN migration. ZFP-1 affects HSN migration in part through its negative effect on pdk-1 transcription and modulation of downstream DAF-16 activity. We also identify a novel role for ZFP-1 and RNAi pathway components, including RDE-4, in the regulation of HSN migration in parallel with DAF-16. Therefore, the coordinated activities of DAF-16, ZFP-1, and endogenous RNAi contribute to gene regulation during development to ensure proper neuronal positioning. PMID:24558261

  12. An efficient transgenic system by TA cloning vectors and RNAi for C. elegans

    SciTech Connect

    Gengyo-Ando, Keiko; Yoshina, Sawako; Inoue, Hideshi; Mitani, Shohei . E-mail: mitani1@research.twmu.ac.jp

    2006-11-03

    In the nematode, transgenic analyses have been performed by microinjection of DNA from various sources into the syncytium gonad. To expedite these transgenic analyses, we solved two potential problems in this work. First, we constructed an efficient TA-cloning vector system which is useful for any promoter. By amplifying the genomic DNA fragments which contain regulatory sequences with or without the coding region, we could easily construct plasmids expressing fluorescent protein fusion without considering restriction sites. We could dissect motor neurons with three colors in a single animal. Second, we used feeding RNAi to isolate transgenic strains which express lag-2::venus fusion gene. We found that the fusion protein is toxic when ectopically expressed in embryos but is functional to rescue a loss of function mutant in the lag-2 gene. Thus, the transgenic system described here should be useful to examine the protein function in the nematode.

  13. A Genome-Wide RNAi Screen to Dissect Centriole Duplication and Centrosome Maturation in Drosophila

    PubMed Central

    Dobbelaere, Jeroen; Josué, Filipe; Suijkerbuijk, Saskia; Baum, Buzz; Tapon, Nicolas; Raff, Jordan

    2008-01-01

    Centrosomes comprise a pair of centrioles surrounded by an amorphous pericentriolar material (PCM). Here, we have performed a microscopy-based genome-wide RNA interference (RNAi) screen in Drosophila cells to identify proteins required for centriole duplication and mitotic PCM recruitment. We analysed 92% of the Drosophila genome (13,059 genes) and identified 32 genes involved in centrosome function. An extensive series of secondary screens classified these genes into four categories: (1) nine are required for centriole duplication, (2) 11 are required for centrosome maturation, (3) nine are required for both functions, and (4) three genes regulate centrosome separation. These 32 hits include several new centrosomal components, some of which have human homologs. In addition, we find that the individual depletion of only two proteins, Polo and Centrosomin (Cnn) can completely block centrosome maturation. Cnn is phosphorylated during mitosis in a Polo-dependent manner, suggesting that the Polo-dependent phosphorylation of Cnn initiates centrosome maturation in flies. PMID:18798690

  14. RNAi screening reveals a large signaling network controlling the Golgi apparatus in human cells

    PubMed Central

    Chia, Joanne; Goh, Germaine; Racine, Victor; Ng, Susanne; Kumar, Pankaj; Bard, Frederic

    2012-01-01

    The Golgi apparatus has many important physiological functions, including sorting of secretory cargo and biosynthesis of complex glycans. These functions depend on the intricate and compartmentalized organization of the Golgi apparatus. To investigate the mechanisms that regulate Golgi architecture, we developed a quantitative morphological assay using three different Golgi compartment markers and quantitative image analysis, and performed a kinome- and phosphatome-wide RNAi screen in HeLa cells. Depletion of 159 signaling genes, nearly 20% of genes assayed, induced strong and varied perturbations in Golgi morphology. Using bioinformatics data, a large regulatory network could be constructed. Specific subnetworks are involved in phosphoinositides regulation, acto-myosin dynamics and mitogen activated protein kinase signaling. Most gene depletion also affected Golgi functions, in particular glycan biosynthesis, suggesting that signaling cascades can control glycosylation directly at the Golgi level. Our results provide a genetic overview of the signaling pathways that control the Golgi apparatus in human cells. PMID:23212246

  15. INDUCIBLE RNAi-MEDIATED GENE SILENCING USING NANOSTRUCTURED GENE DELIVERY ARRAYS

    SciTech Connect

    Mann, David George James; McKnight, Timothy E; Mcpherson, Jackson; Hoyt, Peter R; Melechko, Anatoli Vasilievich; Simpson, Michael L; Sayler, Gary Steven

    2008-01-01

    RNA interference has become a powerful biological tool over the last decade. In this study, a tetracycline-inducible shRNA vector system was designed for silencing CFP expression and introduced alongside the yfp marker gene into Chinese hamster ovary cells using spatially indexed vertically aligned carbon nanofiber arrays (VACNFs) in a gene delivery process termed impalefection. The VACNF architecture provided simultaneous delivery of multiple genes, subsequent adherence and proliferation of interfaced cells, and repeated monitoring of single cells over time. 24 hours after nanofiber-mediated delivery, 53.1% 10.4% of the cells that expressed the yfp marker gene were also fully silenced by the inducible CFP-silencing shRNA vector. Additionally, efficient CFP-silencing was observed in single cells among a population of cells that remained CFP-expressing. This effective transient expression system enables rapid analysis of gene silencing effects using RNAi in single cells and cell populations.

  16. INDUCIBLE RNAi-MEDIATED GENE SILENCING USING NANOSTRUCTURED GENE DELIVERY ARRAYS

    SciTech Connect

    Mann, David George James; McKnight, Timothy E; Mcpherson, Jackson; Hoyt, Peter R; Melechko, Anatoli Vasilievich; Simpson, Michael L; Sayler, Gary Steven

    2008-01-01

    RNA interference has become a powerful biological tool over the last decade. In this study, a tetracycline-inducible shRNA vector system was designed for silencing CFP expression and delivered alongside the yfp marker gene into Chinese hamster ovary cells using impalefection on spatially indexed vertically aligned carbon nanofiber arrays (VACNFs). The VACNF architecture provided simultaneous delivery of multiple genes, subsequent adherence and proliferation of interfaced cells, and repeated monitoring of single cells over time. Following impalefection and tetracycline induction, 53.1% 10.4% of impalefected cells were fully silenced by the inducible CFP-silencing shRNA vector. Additionally, efficient CFP-silencing was observed in single cells among a population of cells that remained CFP-expressing. This effective transient expression system enables rapid analysis of gene silencing effects using RNAi in single cells and cell populations.

  17. RNAi Screen Reveals Potentially Novel Roles of Cytokines in Myoblast Differentiation

    PubMed Central

    Ge, Yejing; Waldemer, Rachel J.; Nalluri, Ramakrishna; Nuzzi, Paul D.; Chen, Jie

    2013-01-01

    Cytokines are cell-secreted signaling molecules that modulate various cellular functions, with the best-characterized roles in immune responses. The expression of numerous cytokines in skeletal muscle tissues and muscle cells has been reported, but their function in skeletal myogenesis, the formation of skeletal muscle, has been largely underexplored. To systematically examine the potential roles of cytokines in skeletal myogenesis, we undertook an RNAi screen of 134 mouse cytokine genes for their involvement in the differentiation of C2C12 myoblasts. Our results have uncovered 29 cytokines as strong candidates for novel myogenic regulators, potentially conferring positive and negative regulation at distinct stages of myogenesis. These candidates represent a diverse collection of cytokine families, including interleukins, TNF-related factors, and chemokines. Our findings suggest the fundamental importance of cytokines in the cell-autonomous regulation of myoblast differentiation, and may facilitate future identification of novel therapeutic targets for improving muscle regeneration and growth in health and diseases. PMID:23844157

  18. Patterns of molecular evolution of RNAi genes in social and socially parasitic bumblebees.

    PubMed

    Helbing, Sophie; Lattorff, H Michael G

    2016-08-01

    The high frequency of interactions amongst closely related individuals in social insect colonies enhances pathogen transmission. Group-mediated behavior supporting immune defenses tends to decrease selection acting on immune genes. Along with low effective population sizes this might result in relaxed constraint and rapid evolution of immune system genes. Here, we show that antiviral siRNA genes show high rates of molecular evolution with argonaute 2, armitage and maelstrom evolving faster in social bumblebees compared to their socially parasitic cuckoo bumblebees that lack a worker caste. RNAi genes show frequent positive selection at the codon level additionally supported by the occurrence of parallel evolution. Their evolutionary rate is linked to their pathway specific position with genes directly interacting with viruses showing the highest rates of molecular evolution. We suggest that higher pathogen load in social insects indeed drives the molecular evolution of immune genes including antiviral siRNA, if not compensated by behavior. PMID:27117935

  19. Spherical Nucleic Acid Nanoparticle Conjugates as an RNAi-Based Therapy for Glioblastoma

    PubMed Central

    Jensen, Samuel A.; Day, Emily S.; Ko, Caroline H.; Hurley, Lisa A.; Luciano, Janina P.; Kouri, Fotini M.; Merkel, Timothy J.; Luthi, Andrea J.; Patel, Pinal C.; Cutler, Joshua I.; Daniel, Weston L.; Scott, Alexander W.; Rotz, Matthew W.; Meade, Thomas J.; Giljohann, David A.; Mirkin, Chad A.; Stegh, Alexander H.

    2014-01-01

    Glioblastoma multiforme (GBM) is a neurologically debilitating disease that culminates in death 14 to 16 months after diagnosis. An incomplete understanding of how cataloged genetic aberrations promote therapy resistance, combined with ineffective drug delivery to the central nervous system, has rendered GBM incurable. Functional genomics efforts have implicated several oncogenes in GBM pathogenesis but have rarely led to the implementation of targeted therapies. This is partly because many “undruggable” oncogenes cannot be targeted by small molecules or antibodies. We preclinically evaluate an RNA interference (RNAi)–based nanomedicine platform, based on spherical nucleic acid (SNA) nanoparticle conjugates, to neutralize oncogene expression in GBM. SNAs consist of gold nanoparticles covalently functionalized with densely packed, highly oriented small interfering RNA duplexes. In the absence of auxiliary transfection strategies or chemical modifications, SNAs efficiently entered primary and transformed glial cells in vitro. In vivo, the SNAs penetrated the blood-brain barrier and blood-tumor barrier to disseminate throughout xenogeneic glioma explants. SNAs targeting the oncoprotein Bcl2Like12 (Bcl2L12)—an effector caspase and p53 inhibitor overexpressed in GBM relative to normal brain and low-grade astrocytomas—were effective in knocking down endogenous Bcl2L12 mRNA and protein levels, and sensitized glioma cells toward therapy-induced apoptosis by enhancing effector caspase and p53 activity. Further, systemically delivered SNAs reduced Bcl2L12 expression in intracerebral GBM, increased intratumoral apoptosis, and reduced tumor burden and progression in xenografted mice, without adverse side effects. Thus, silencing antiapoptotic signaling using SNAs represents a new approach for systemic RNAi therapy for GBM and possibly other lethal malignancies. PMID:24174328

  20. Genome-wide RNAi screen reveals a role for the ESCRT complex in rotavirus cell entry.

    PubMed

    Silva-Ayala, Daniela; López, Tomás; Gutiérrez, Michelle; Perrimon, Norbert; López, Susana; Arias, Carlos F

    2013-06-18

    Rotavirus (RV) is the major cause of childhood gastroenteritis worldwide. This study presents a functional genome-scale analysis of cellular proteins and pathways relevant for RV infection using RNAi. Among the 522 proteins selected in the screen for their ability to affect viral infectivity, an enriched group that participates in endocytic processes was identified. Within these proteins, subunits of the vacuolar ATPase, small GTPases, actinin 4, and, of special interest, components of the endosomal sorting complex required for transport (ESCRT) machinery were found. Here we provide evidence for a role of the ESCRT complex in the entry of simian and human RV strains in both monkey and human epithelial cells. In addition, the ESCRT-associated ATPase VPS4A and phospholipid lysobisphosphatidic acid, both crucial for the formation of intralumenal vesicles in multivesicular bodies, were also found to be required for cell entry. Interestingly, it seems that regardless of the molecules that rhesus RV and human RV strains use for cell-surface attachment and the distinct endocytic pathway used, all these viruses converge in early endosomes and use multivesicular bodies for cell entry. Furthermore, the small GTPases RHOA and CDC42, which regulate different types of clathrin-independent endocytosis, as well as early endosomal antigen 1 (EEA1), were found to be involved in this process. This work reports the direct involvement of the ESCRT machinery in the life cycle of a nonenveloped virus and highlights the complex mechanism that these viruses use to enter cells. It also illustrates the efficiency of high-throughput RNAi screenings as genetic tools for comprehensively studying the interaction between viruses and their host cells. PMID:23733942

  1. RNAi-mediated oncogene silencing confers resistance to crown gall tumorigenesis.

    PubMed

    Escobar, M A; Civerolo, E L; Summerfelt, K R; Dandekar, A M

    2001-11-01

    Crown gall disease, caused by the soil bacterium Agrobacterium tumefaciens, results in significant economic losses in perennial crops worldwide. A. tumefaciens is one of the few organisms with a well characterized horizontal gene transfer system, possessing a suite of oncogenes that, when integrated into the plant genome, orchestrate de novo auxin and cytokinin biosynthesis to generate tumors. Specifically, the iaaM and ipt oncogenes, which show approximately 90% DNA sequence identity across studied A. tumefaciens strains, are required for tumor formation. By expressing two self-complementary RNA constructions designed to initiate RNA interference (RNAi) of iaaM and ipt, we generated transgenic Arabidopsis thaliana and Lycopersicon esculentum plants that are highly resistant to crown gall disease development. In in vitro root inoculation bioassays with two biovar I strains of A. tumefaciens, transgenic Arabidopsis lines averaged 0.0-1.5% tumorigenesis, whereas wild-type controls averaged 97.5% tumorigenesis. Similarly, several transformed tomato lines that were challenged by stem inoculation with three biovar I strains, one biovar II strain, and one biovar III strain of A. tumefaciens displayed between 0.0% and 24.2% tumorigenesis, whereas controls averaged 100% tumorigenesis. This mechanism of resistance, which is based on mRNA sequence homology rather than the highly specific receptor-ligand binding interactions characteristic of traditional plant resistance genes, should be highly durable. If successful and durable under field conditions, RNAi-mediated oncogene silencing may find broad applicability in the improvement of tree crop and ornamental rootstocks. PMID:11687652

  2. RNAiFOLD: a constraint programming algorithm for RNA inverse folding and molecular design.

    PubMed

    Garcia-Martin, Juan Antonio; Clote, Peter; Dotu, Ivan

    2013-04-01

    Synthetic biology is a rapidly emerging discipline with long-term ramifications that range from single-molecule detection within cells to the creation of synthetic genomes and novel life forms. Truly phenomenal results have been obtained by pioneering groups--for instance, the combinatorial synthesis of genetic networks, genome synthesis using BioBricks, and hybridization chain reaction (HCR), in which stable DNA monomers assemble only upon exposure to a target DNA fragment, biomolecular self-assembly pathways, etc. Such work strongly suggests that nanotechnology and synthetic biology together seem poised to constitute the most transformative development of the 21st century. In this paper, we present a Constraint Programming (CP) approach to solve the RNA inverse folding problem. Given a target RNA secondary structure, we determine an RNA sequence which folds into the target structure; i.e. whose minimum free energy structure is the target structure. Our approach represents a step forward in RNA design--we produce the first complete RNA inverse folding approach which allows for the specification of a wide range of design constraints. We also introduce a Large Neighborhood Search approach which allows us to tackle larger instances at the cost of losing completeness, while retaining the advantages of meeting design constraints (motif, GC-content, etc.). Results demonstrate that our software, RNAiFold, performs as well or better than all state-of-the-art approaches; nevertheless, our approach is unique in terms of completeness, flexibility, and the support of various design constraints. The algorithms presented in this paper are publicly available via the interactive webserver http://bioinformatics.bc.edu/clotelab/RNAiFold; additionally, the source code can be downloaded from that site. PMID:23600819

  3. TRM6/61 connects PKCα with translational control through tRNAi(Met) stabilization: impact on tumorigenesis.

    PubMed

    Macari, F; El-Houfi, Y; Boldina, G; Xu, H; Khoury-Hanna, S; Ollier, J; Yazdani, L; Zheng, G; Bièche, I; Legrand, N; Paulet, D; Durrieu, S; Byström, A; Delbecq, S; Lapeyre, B; Bauchet, L; Pannequin, J; Hollande, F; Pan, T; Teichmann, M; Vagner, S; David, A; Choquet, A; Joubert, D

    2016-04-01

    Accumulating evidence suggests that changes of the protein synthesis machinery alter translation of specific mRNAs and participate in malignant transformation. Here we show that protein kinase C α (PKCα) interacts with TRM61, the catalytic subunit of the TRM6/61 tRNA methyltransferase. The TRM6/61 complex is known to methylate the adenosine 58 of the initiator methionine tRNA (tRNAi(Met)), a nuclear post-transcriptional modification associated with the stabilization of this crucial component of the translation-initiation process. Depletion of TRM6/61 reduced proliferation and increased death of C6 glioma cells, effects that can be partially rescued by overexpression of tRNAi(Met). In contrast, elevated TRM6/61 expression regulated the translation of a subset of mRNAs encoding proteins involved in the tumorigenic process and increased the ability of C6 cells to form colonies in soft agar or spheres when grown in suspension. In TRM6/61/tRNAi(Met)-overexpressing cells, PKCα overexpression decreased tRNAi(Met) expression and both colony- and sphere-forming potentials. A concomitant increase in TRM6/TRM61 mRNA and tRNAi(Met) expression with decreased expression of PKCα mRNA was detected in highly aggressive glioblastoma multiforme as compared with Grade II/III glioblastomas, highlighting the clinical relevance of our findings. Altogether, we suggest that PKCα tightly controls TRM6/61 activity to prevent translation deregulation that would favor neoplastic development. PMID:26234676

  4. Lentivirus-Mediated knockdown of tectonic family member 1 inhibits medulloblastoma cell proliferation

    PubMed Central

    Jing, Junjie; Wang, Chengfeng; Liang, Qinchuan; Zhao, Yang; Zhao, Qingshuang; Wang, Shousen; Ma, Jie

    2015-01-01

    Tectonic family member 1 (TCTN1) encodes a member of the tectonic family which are evolutionarily conserved secreted and transmembrane proteins, involving in a diverse variety of developmental processes. It has been demonstrated that tectonics expressed in regions that participate in Hedgehog (Hh) signaling during mouse embryonic development and was imperative for Hh-mediated patterning of the ventral neural tube. However, the expression and regulation of tectonics in human tumor is still not clear. In this study, shRNA-expressing lentivirus was constructed to knockdown TCTN1 in medulloblastoma cell line Daoy. The results showed that knockdown of TCTN1 inhibited cell proliferation and colony formation in Daoy cell line, also caused cell cycle arrest at the G2/M boundary. Taken all together, our data suggest that TCTN1 might play an important role in the progression of medulloblastoma. PMID:26550235

  5. Cathepsin L knockdown enhances curcumin-mediated inhibition of growth, migration, and invasion of glioma cells.

    PubMed

    Fei, Yao; Xiong, Yajie; Zhao, Yifan; Wang, Wenjuan; Han, Meilin; Wang, Long; Tan, Caihong; Liang, Zhongqin

    2016-09-01

    Curcumin can be used to prevent and treat cancer. However, its exact underlying molecular mechanisms remain poorly understood. Cathepsin L, a lysosomal cysteine protease, is overexpressed in several cancer types. This study aimed to determine the role of cathepsin L in curcumin-mediated inhibition of growth, migration, and invasion of glioma cells. Results revealed that the activity of cathepsin L was enhanced in curcumin-treated glioma cells. Cathepsin L knockdown induced by RNA interference significantly promoted curcumin-induced cytotoxicity, apoptosis, and cell cycle arrest. The knockdown also inhibited the migration and invasion of glioma cells. Our results suggested that the inhibition of cathepsin L can enhance the sensitivity of glioma cells to curcumin. Therefore, cathepsin L may be a new target to enhance the efficacy of curcumin against cancers. PMID:27373979

  6. Effects of Shell-Buckling Knockdown Factors in Large Cylindrical Shells

    NASA Technical Reports Server (NTRS)

    Hrinda, Glenn A.

    2012-01-01

    Shell-buckling knockdown factors (SBKF) have been used in large cylindrical shell structures to account for uncertainty in buckling loads. As the diameter of the cylinder increases, achieving the manufacturing tolerances becomes increasingly more difficult. Knockdown factors account for manufacturing imperfections in the shell geometry by decreasing the allowable buckling load of the cylinder. In this paper, large-diameter (33 ft) cylinders are investigated by using various SBKF's. An investigation that is based on finite-element analysis (FEA) is used to develop design sensitivity relationships. Different manufacturing imperfections are modeled into a perfect cylinder to investigate the effects of these imperfections on buckling. The analysis results may be applicable to large- diameter rockets, cylindrical tower structures, bulk storage tanks, and silos.

  7. AAV-mediated RLBP1 gene therapy improves the rate of dark adaptation in Rlbp1 knockout mice.

    PubMed

    Choi, Vivian W; Bigelow, Chad E; McGee, Terri L; Gujar, Akshata N; Li, Hui; Hanks, Shawn M; Vrouvlianis, Joanna; Maker, Michael; Leehy, Barrett; Zhang, Yiqin; Aranda, Jorge; Bounoutas, George; Demirs, John T; Yang, Junzheng; Ornberg, Richard; Wang, Yu; Martin, Wendy; Stout, Kelly R; Argentieri, Gregory; Grosenstein, Paul; Diaz, Danielle; Turner, Oliver; Jaffee, Bruce D; Police, Seshidhar R; Dryja, Thaddeus P

    2015-01-01

    Recessive mutations in RLBP1 cause a form of retinitis pigmentosa in which the retina, before its degeneration leads to blindness, abnormally slowly recovers sensitivity after exposure to light. To develop a potential gene therapy for this condition, we tested multiple recombinant adeno-associated vectors (rAAVs) composed of different promoters, capsid serotypes, and genome conformations. We generated rAAVs in which sequences from the promoters of the human RLBP1, RPE65, or BEST1 genes drove the expression of a reporter gene (green fluorescent protein). A promoter derived from the RLBP1 gene mediated expression in the retinal pigment epithelium and Müller cells (the intended target cell types) at qualitatively higher levels than in other retinal cell types in wild-type mice and monkeys. With this promoter upstream of the coding sequence of the human RLBP1 gene, we compared the potencies of vectors with an AAV2 versus an AAV8 capsid in transducing mouse retinas, and we compared vectors with a self-complementary versus a single-stranded genome. The optimal vector (scAAV8-pRLBP1-hRLBP1) had serotype 8 capsid and a self-complementary genome. Subretinal injection of scAAV8-pRLBP1-hRLBP1 in Rlbp1 nullizygous mice improved the rate of dark adaptation based on scotopic (rod-plus-cone) and photopic (cone) electroretinograms (ERGs). The effect was still present after 1 year. PMID:26199951

  8. AAV-mediated RLBP1 gene therapy improves the rate of dark adaptation in Rlbp1 knockout mice

    PubMed Central

    Choi, Vivian W; Bigelow, Chad E; McGee, Terri L; Gujar, Akshata N; Li, Hui; Hanks, Shawn M; Vrouvlianis, Joanna; Maker, Michael; Leehy, Barrett; Zhang, Yiqin; Aranda, Jorge; Bounoutas, George; Demirs, John T; Yang, Junzheng; Ornberg, Richard; Wang, Yu; Martin, Wendy; Stout, Kelly R; Argentieri, Gregory; Grosenstein, Paul; Diaz, Danielle; Turner, Oliver; Jaffee, Bruce D; Police, Seshidhar R; Dryja, Thaddeus P

    2015-01-01

    Recessive mutations in RLBP1 cause a form of retinitis pigmentosa in which the retina, before its degeneration leads to blindness, abnormally slowly recovers sensitivity after exposure to light. To develop a potential gene therapy for this condition, we tested multiple recombinant adeno-associated vectors (rAAVs) composed of different promoters, capsid serotypes, and genome conformations. We generated rAAVs in which sequences from the promoters of the human RLBP1, RPE65, or BEST1 genes drove the expression of a reporter gene (green fluorescent protein). A promoter derived from the RLBP1 gene mediated expression in the retinal pigment epithelium and Müller cells (the intended target cell types) at qualitatively higher levels than in other retinal cell types in wild-type mice and monkeys. With this promoter upstream of the coding sequence of the human RLBP1 gene, we compared the potencies of vectors with an AAV2 versus an AAV8 capsid in transducing mouse retinas, and we compared vectors with a self-complementary versus a single-stranded genome. The optimal vector (scAAV8-pRLBP1-hRLBP1) had serotype 8 capsid and a self-complementary genome. Subretinal injection of scAAV8-pRLBP1-hRLBP1 in Rlbp1 nullizygous mice improved the rate of dark adaptation based on scotopic (rod-plus-cone) and photopic (cone) electroretinograms (ERGs). The effect was still present after 1 year. PMID:26199951

  9. AAV-mediated gene delivery in a feline model of Sandhoff disease corrects lysosomal storage in the central nervous system.

    PubMed

    Rockwell, Hannah E; McCurdy, Victoria J; Eaton, Samuel C; Wilson, Diane U; Johnson, Aime K; Randle, Ashley N; Bradbury, Allison M; Gray-Edwards, Heather L; Baker, Henry J; Hudson, Judith A; Cox, Nancy R; Sena-Esteves, Miguel; Seyfried, Thomas N; Martin, Douglas R

    2015-01-01

    Sandhoff disease (SD) is an autosomal recessive neurodegenerative disease caused by a mutation in the gene for the β-subunit of β-N-acetylhexosaminidase (Hex), resulting in the inability to catabolize ganglioside GM2 within the lysosomes. SD presents with an accumulation of GM2 and its asialo derivative GA2, primarily in the central nervous system. Myelin-enriched glycolipids, cerebrosides and sulfatides, are also decreased in SD corresponding with dysmyelination. At present, no treatment exists for SD. Previous studies have shown the therapeutic benefit of adeno-associated virus (AAV) vector-mediated gene therapy in the treatment of SD in murine and feline models. In this study, we treated presymptomatic SD cats with AAVrh8 vectors expressing feline Hex in the thalamus combined with intracerebroventricular (Thal/ICV) injections. Treated animals showed clearly improved neurologic function and quality of life, manifested in part by prevention or attenuation of whole-body tremors characteristic of untreated animals. Hex activity was significantly elevated, whereas storage of GM2 and GA2 was significantly decreased in tissue samples taken from the cortex, cerebellum, thalamus, and cervical spinal cord. Treatment also increased levels of myelin-enriched cerebrosides and sulfatides in the cortex and thalamus. This study demonstrates the therapeutic potential of AAV for feline SD and suggests a similar potential for human SD patients. PMID:25873306

  10. AAV-Mediated Gene Delivery in a Feline Model of Sandhoff Disease Corrects Lysosomal Storage in the Central Nervous System

    PubMed Central

    Rockwell, Hannah E.; McCurdy, Victoria J.; Eaton, Samuel C.; Wilson, Diane U.; Johnson, Aime K.; Randle, Ashley N.; Bradbury, Allison M.; Gray-Edwards, Heather L.; Baker, Henry J.; Hudson, Judith A.; Cox, Nancy R.; Sena-Esteves, Miguel; Seyfried, Thomas N.

    2015-01-01

    Sandhoff disease (SD) is an autosomal recessive neurodegenerative disease caused by a mutation in the gene for the β-subunit of β-N-acetylhexosaminidase (Hex), resulting in the inability to catabolize ganglioside GM2 within the lysosomes. SD presents with an accumulation of GM2 and its asialo derivative GA2, primarily in the central nervous system. Myelin-enriched glycolipids, cerebrosides and sulfatides, are also decreased in SD corresponding with dysmyelination. At present, no treatment exists for SD. Previous studies have shown the therapeutic benefit of adeno-associated virus (AAV) vector-mediated gene therapy in the treatment of SD in murine and feline models. In this study, we treated presymptomatic SD cats with AAVrh8 vectors expressing feline Hex in the thalamus combined with intracerebroventricular (Thal/ICV) injections. Treated animals showed clearly improved neurologic function and quality of life, manifested in part by prevention or attenuation of whole-body tremors characteristic of untreated animals. Hex activity was significantly elevated, whereas storage of GM2 and GA2 was significantly decreased in tissue samples taken from the cortex, cerebellum, thalamus, and cervical spinal cord. Treatment also increased levels of myelin-enriched cerebrosides and sulfatides in the cortex and thalamus. This study demonstrates the therapeutic potential of AAV for feline SD and suggests a similar potential for human SD patients. PMID:25873306

  11. AAV-Mediated Clarin-1 Expression in the Mouse Retina: Implications for USH3A Gene Therapy

    PubMed Central

    Deng, Wen-Tao; Dyka, Frank M.; Min, Seok-Hong; Boye, Sanford L.; Chiodo, Vince A.; Abrahan, Carolina E.; Zhu, Ping; Li, Qiuhong; Strettoi, Enrica; Novelli, Elena; Nagel-Wolfrum, Kerstin; Wolfrum, Uwe; Smith, W. Clay; Hauswirth, William W.

    2016-01-01

    Usher syndrome type III (USH3A) is an autosomal recessive disorder caused by mutations in clarin-1 (CLRN1) gene, leading to progressive retinal degeneration and sensorineural deafness. Efforts to develop therapies for preventing photoreceptor cell loss are hampered by the lack of a retinal phenotype in the existing USH3 mouse models and by conflicting reports regarding the endogenous retinal localization of clarin-1, a transmembrane protein of unknown function. In this study, we used an AAV-based approach to express CLRN1 in the mouse retina in order to determine the pattern of its subcellular localization in different cell types. We found that all major classes of retinal cells express AAV-delivered CLRN1 driven by the ubiquitous, constitutive small chicken β-actin promoter, which has important implications for the design of future USH3 gene therapy studies. Within photoreceptor cells, AAV-expressed CLRN1 is mainly localized at the inner segment region and outer plexiform layer, similar to the endogenous expression of other usher proteins. Subretinal delivery using a full strength viral titer led to significant loss of retinal function as evidenced by ERG analysis, suggesting that there is a critical limit for CLRN1 expression in photoreceptor cells. Taken together, these results suggest that CLRN1 expression is potentially supported by a variety of retinal cells, and the right combination of AAV vector dose, promoter, and delivery method needs to be selected to develop safe therapies for USH3 disorder. PMID:26881841

  12. AAV Mediated GDNF Secretion From Retinal Glia Slows Down Retinal Degeneration in a Rat Model of Retinitis Pigmentosa

    PubMed Central

    Dalkara, Deniz; Kolstad, Kathleen D; Guerin, Karen I; Hoffmann, Natalie V; Visel, Meike; Klimczak, Ryan R; Schaffer, David V; Flannery, John G

    2011-01-01

    Mutations in over 80 identified genes can induce apoptosis in photoreceptors, resulting in blindness with a prevalence of 1 in 3,000 individuals. This broad genetic heterogeneity of disease impacting a wide range of photoreceptor functions renders the design of gene-specific therapies for photoreceptor degeneration impractical and necessitates the development of mutation-independent treatments to slow photoreceptor cell death. One promising strategy for photoreceptor neuroprotection is neurotrophin secretion from Müller cells, the primary retinal glia. Müller glia are excellent targets for secreting neurotrophins as they span the entire tissue, ensheath all neuronal populations, are numerous, and persist through retinal degeneration. We previously engineered an adeno-associated virus (AAV) variant (ShH10) capable of efficient and selective glial cell transduction through intravitreal injection. ShH10-mediated glial-derived neurotrophic factor (GDNF) secretion from glia, generates high GDNF levels in treated retinas, leading to sustained functional rescue for over 5 months. This GDNF secretion from glia following intravitreal vector administration is a safe and effective means to slow the progression of retinal degeneration in a rat model of retinitis pigmentosa (RP) and shows significant promise as a gene therapy to treat human retinal degenerations. These findings also demonstrate for the first time that glia-mediated secretion of neurotrophins is a promising treatment that may be applicable to other neurodegenerative conditions. PMID:21522134

  13. Modulation of tolerance to the transgene product in a nonhuman primate model of AAV-mediated gene transfer to liver

    PubMed Central

    Mingozzi, Federico; Hasbrouck, Nicole C.; Basner-Tschakarjan, Etiena; Edmonson, Shyrie A.; Hui, Daniel J.; Sabatino, Denise E.; Zhou, Shangzhen; Wright, J. Fraser; Jiang, Haiyan; Pierce, Glenn F.; Arruda, Valder R.

    2007-01-01

    Adeno-associated virus (AAV)–mediated gene transfer of factor IX (F.IX) to the liver results in long-term expression of transgene in experimental animals, but only short-term expression in humans. Loss of F.IX expression is likely due to a cytotoxic immune response to the AAV capsid, which results in clearance of transduced hepatocytes. We used a nonhuman primate model to assess the safety of AAV gene transfer coupled with an anti–T-cell regimen designed to block this immune response. Administration of a 3-drug regimen consisting of mycophenolate mofetil (MMF), sirolimus, and the anti–IL-2 receptor antibody daclizumab consistently resulted in formation of inhibitory antibodies to human F.IX following hepatic artery administration of an AAV-hF.IX vector, whereas a 2-drug regimen consisting only of MMF and sirolimus did not. Administration of daclizumab was accompanied by a dramatic drop in the population of CD4+CD25+FoxP3+ regulatory T cells (Tregs). We conclude that choice of immunosuppression (IS) regimen can modulate immune responses to the transgene product upon hepatic gene transfer in subjects not fully tolerant; and that induction of transgene tolerance may depend on a population of antigen-specific Tregs. PMID:17609423

  14. Ab-Externo AAV-Mediated Gene Delivery to the Suprachoroidal Space Using a 250 Micron Flexible Microcatheter

    PubMed Central

    Peden, Marc C.; Min, Jeff; Meyers, Craig; Lukowski, Zachary; Li, Qiuhong; Boye, Sanford L.; Levine, Monica; Hauswirth, William W.; Ratnakaram, Ramakrishna; Dawson, William; Smith, Wesley C.; Sherwood, Mark B.

    2011-01-01

    Background The current method of delivering gene replacement to the posterior segment of the eye involves a three-port pars plana vitrectomy followed by injection of the agent through a 37-gauge cannula, which is potentially wrought with retinal complications. In this paper we investigate the safety and efficacy of delivering adeno-associated viral (AAV) vector to the suprachoroidal space using an ab externo approach that utilizes an illuminated microcatheter. Methods 6 New Zealand White rabbits and 2 Dutch Belted rabbits were used to evaluate the ab externo delivery method. sc-AAV5-smCBA-hGFP vector was delivered into the suprachoroidal space using an illuminated iTrackTM 250A microcatheter. Six weeks after surgery, the rabbits were sacrificed and their eyes evaluated for AAV transfection using immunofluorescent antibody staining of GFP. Results Immunostaining of sectioned and whole-mounted eyes demonstrated robust transfection in all treated eyes, with no fluorescence in untreated control eyes. Transfection occurred diffusely and involved both the choroid and the retina. No apparent adverse effects caused by either the viral vector or the procedure itself could be seen either clinically or histologically. Conclusions The ab externo method of delivery using a microcatheter was successful in safely and effectively delivering a gene therapy agent to the suprachoroidal space. This method presents a less invasive alternative to the current method of virally vectored gene delivery. PMID:21347253

  15. AAV-mediated, optogenetic ablation of Müller Glia leads to structural and functional changes in the mouse retina.

    PubMed

    Byrne, Leah C; Khalid, Fakhra; Lee, Trevor; Zin, Emilia A; Greenberg, Kenneth P; Visel, Meike; Schaffer, David V; Flannery, John G

    2013-01-01

    Müller glia, the primary glial cell in the retina, provide structural and metabolic support for neurons and are essential for retinal integrity. Müller cells are closely involved in many retinal degenerative diseases, including macular telangiectasia type 2, in which impairment of central vision may be linked to a primary defect in Müller glia. Here, we used an engineered, Müller-specific variant of AAV, called ShH10, to deliver a photo-inducibly toxic protein, KillerRed, to Müller cells in the mouse retina. We characterized the results of specific ablation of these cells on visual function and retinal structure. ShH10-KillerRed expression was obtained following intravitreal injection and eyes were then irradiated with green light to induce toxicity. Induction of KillerRed led to loss of Müller cells and a concomitant decrease of Müller cell markers glutamine synthetase and cellular retinaldehyde-binding protein, reduction of rhodopsin and cone opsin, and upregulation of glial fibrillary acidic protein. Loss of Müller cells also resulted in retinal disorganization, including thinning of the outer nuclear layer and the photoreceptor inner and outer segments. High resolution imaging of thin sections revealed displacement of photoreceptors from the ONL, formation of rosette-like structures and the presence of phagocytic cells. Furthermore, Müller cell ablation resulted in increased area and volume of retinal blood vessels, as well as the formation of tortuous blood vessels and vascular leakage. Electrophysiologic measures demonstrated reduced retinal function, evident in decreased photopic and scotopic electroretinogram amplitudes. These results show that loss of Müller cells can cause progressive retinal degenerative disease, and suggest that AAV delivery of an inducibly toxic protein in Müller cells may be useful to create large animal models of retinal dystrophies. PMID:24086689

  16. A single intramuscular injection of rAAV-mediated mutant erythropoietin protects against MPTP-induced parkinsonism

    PubMed Central

    Dhanushkodi, A.; Akano, E. O.; Roguski, E. E.; Xue, Y.; Rao, S. K.; Matta, S. G.; Rex, T. S.; McDonald, M. P.

    2015-01-01

    Erythropoietin (Epo) is neuroprotective in a number of preparations, but can lead to unacceptably high and even lethal hematocrit levels. Recent reports show that modified Epo variants confer neuroprotection in models of glaucoma and retinal degeneration without raising hematocrit. In this study, neuroprotective effects of two Epo variants (EpoR76E and EpoS71E) were assessed in a model of Parkinson’s disease. The constructs were packaged in recombinant adeno-associated viral (rAAV) vectors and injected intramuscularly. After 3 weeks, mice received five daily injections of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and were killed 5 weeks later. The MPTP-lesioned mice pretreated with rAAV.eGFP (negative control) exhibited a 7- to 9-Hz tremor and slower latencies to move on a grid test (akinesia). Both of these symptomatic features were absent in mice pretreated with either modified Epo construct. The rAAV.eGFP-treated mice lesioned with MPTP exhibited a 41% reduction in tyrosine hydroxylase (TH)-positive neurons in the substantia nigra. The rAAV.EpoS71E construct did not protect nigral neurons, but neuronal loss in mice pretreated with rAAV.EpoR76E was only half that of rAAV.eGFP controls. Although dopamine levels were normal in all groups, 3,4-dihydroxyphenylacetic acid (DOPAC) was significantly reduced only in MPTP-lesioned mice pre-treated with rAAV.eGFP, indicating reduced dopamine turnover. Analysis of TH-positive fibers in the striatum showed normalized density in MPTP-lesioned mice pretreated with rAAV.EpoS71E, suggesting that enhanced sprouting induced by EpoS71E may have been responsible for normal behavior and dopaminergic tone in these mice. These results show that systemically administered rAAV-generated non-erythropoietic Epo may protect against MPTP-induced parkinsonism by a combination of neuroprotection and enhanced axonal sprouting. PMID:23190369

  17. Disruption of follistatin by RNAi increases apoptosis, arrests S-phase of cell cycle and decreases estradiol production in bovine granulosa cells.

    PubMed

    Chong, Zhenlu; Dong, Ping; Riaz, Hasan; Shi, Lei; Yu, Xue; Cheng, Ying; Yang, Liguo

    2015-04-01

    Follistatin (FST), a local regulator of gonadal functions is a powerful inhibitor of follicle stimulating hormone (FSH) secretion. In the present study, the expression of FST was partially silenced at both transcriptional and translational levels by RNAi-Ready pSIREN-RetroQ-ZsGreen Vector mediated recombinant pshRNA vectors in bovine granulosa cells (bGCs). The results showed that transfection with FST-1 and FST-2 vectors significantly down-regulated mRNA and protein expressions of follistatin by 51% (P = 0.0093) and 72% (P = 0.0078) respectively. After down-regulation of FST in bGCs, cell cycle was arrested at S-phase (9.2 ± 0.6 vs 12.5 ± 0.2, P = 0.0055), and apoptosis was significantly (21.3 ± 2.7 vs 13.9 ± 2.5, P = 0.0051) increased. These findings were further verified by down-regulation of protein level of B-cell leukemia/lymphoma 2 (Bcl2, P = 0.0423), and up-regulation of caspase-3 (P = 0.0362), p21 (P = 0.0067) and mRNA levels of Bcl2-associated X protein (Bax, P = 0.041). Knockdown of FST in bGCs significantly increased activin A concentration in culture medium, while level of estradiol (E2) was suppressed without affecting progesterone production. In addition, mRNA levels of all activin receptor subtypes [activin receptor types I (ACRI) and II (ACRIIA and ACRIIB)] and inhibin α-subunit were augmented (P < 0.05) without altering both inhibin β-subunits. These findings suggest that follistatin may participate in caspase3-dependent apoptosis through Bcl2/Bax gene family in bovine GCs, whereas, activin and its receptors are associated with its regulation. Activin-induced up-regulation of inhibin-α subunit in bGCs seems to be involved in the regulation of steroidogenesis. PMID:25728901

  18. Knockdown of MLO genes reduces susceptibility to powdery mildew in grapevine.

    PubMed

    Pessina, Stefano; Lenzi, Luisa; Perazzolli, Michele; Campa, Manuela; Dalla Costa, Lorenza; Urso, Simona; Valè, Giampiero; Salamini, Francesco; Velasco, Riccardo; Malnoy, Mickael

    2016-01-01

    Erysiphe necator is the causal agent of powdery mildew (PM), one of the most destructive diseases of grapevine. PM is controlled by sulfur-based and synthetic fungicides, which every year are dispersed into the environment. This is why PM-resistant varieties should become a priority for sustainable grapevine and wine production. PM resistance can be achieved in other crops by knocking out susceptibility S-genes, such as those residing at genetic loci known as MLO (Mildew Locus O). All MLO S-genes of dicots belong to the phylogenetic clade V, including grapevine genes VvMLO7, 11 and 13, which are upregulated during PM infection, and VvMLO6, which is not upregulated. Before adopting a gene-editing approach to knockout candidate S-genes, the evidence that loss of function of MLO genes can reduce PM susceptibility is necessary. This paper reports the knockdown through RNA interference of VvMLO6, 7, 11 and 13. The knockdown of VvMLO6, 11 and 13 did not decrease PM severity, whereas the knockdown of VvMLO7 in combination with VvMLO6 and VvMLO11 reduced PM severity up to 77%. The knockdown of VvMLO7 and VvMLO6 seemed to be important for PM resistance, whereas a role for VvMLO11 does not seem likely. Cell wall appositions (papillae) were present in both resistant and susceptible lines in response to PM attack. Thirteen genes involved in defense were less upregulated in infected mlo plants, highlighting the early mlo-dependent disruption of PM invasion. PMID:27390621

  19. Knockdown of MLO genes reduces susceptibility to powdery mildew in grapevine

    PubMed Central

    Pessina, Stefano; Lenzi, Luisa; Perazzolli, Michele; Campa, Manuela; Dalla Costa, Lorenza; Urso, Simona; Valè, Giampiero; Salamini, Francesco; Velasco, Riccardo; Malnoy, Mickael

    2016-01-01

    Erysiphe necator is the causal agent of powdery mildew (PM), one of the most destructive diseases of grapevine. PM is controlled by sulfur-based and synthetic fungicides, which every year are dispersed into the environment. This is why PM-resistant varieties should become a priority for sustainable grapevine and wine production. PM resistance can be achieved in other crops by knocking out susceptibility S-genes, such as those residing at genetic loci known as MLO (Mildew Locus O). All MLO S-genes of dicots belong to the phylogenetic clade V, including grapevine genes VvMLO7, 11 and 13, which are upregulated during PM infection, and VvMLO6, which is not upregulated. Before adopting a gene-editing approach to knockout candidate S-genes, the evidence that loss of function of MLO genes can reduce PM susceptibility is necessary. This paper reports the knockdown through RNA interference of VvMLO6, 7, 11 and 13. The knockdown of VvMLO6, 11 and 13 did not decrease PM severity, whereas the knockdown of VvMLO7 in combination with VvMLO6 and VvMLO11 reduced PM severity up to 77%. The knockdown of VvMLO7 and VvMLO6 seemed to be important for PM resistance, whereas a role for VvMLO11 does not seem likely. Cell wall appositions (papillae) were present in both resistant and susceptible lines in response to PM attack. Thirteen genes involved in defense were less upregulated in infected mlo plants, highlighting the early mlo-dependent disruption of PM invasion. PMID:27390621

  20. LRRK2 knockdown in zebrafish causes developmental defects, neuronal loss, and synuclein aggregation.

    PubMed

    Prabhudesai, Shubhangi; Bensabeur, Fatima Zahra; Abdullah, Rashed; Basak, Indranil; Baez, Solange; Alves, Guido; Holtzman, Nathalia G; Larsen, Jan Petter; Møller, Simon Geir

    2016-08-01

    Although mutations in the leucine-rich repeat kinase 2 (LRRK2) gene are the most common cause of genetic Parkinson's disease, their function is largely unknown. LRRK2 is pleiotropic in nature, shown to be involved in neurodegeneration and in more peripheral processes, including kidney functions, in rats and mice. Recent studies in zebrafish have shown conflicting evidence that removal of the LRRK2 WD40 domain may or may not affect dopaminergic neurons and/or locomotion. This study shows that ∼50% LRRK2 knockdown in zebrafish causes not only neuronal loss but also developmental perturbations such as axis curvature defects, ocular abnormalities, and edema in the eyes, lens, and otic vesicles. We further show that LRRK2 knockdown results in significant neuronal loss, including a reduction of dopaminergic neurons. Immunofluorescence demonstrates that endogenous LRRK2 is expressed in the lens, brain, heart, spinal cord, and kidney (pronephros), which mirror the LRRK2 morphant phenotypes observed. LRRK2 knockdown results further in the concomitant upregulation of β-synuclein, PARK13, and SOD1 and causes β-synuclein aggregation in the diencephalon, midbrain, hindbrain, and postoptic commissure. LRRK2 knockdown causes mislocalization of the Na(+) /K(+) ATPase protein in the pronephric ducts, suggesting that the edema might be linked to renal malfunction and that LRRK2 might be associated with pronephric duct epithelial cell differentiation. Combined, our study shows that LRRK2 has multifaceted roles in zebrafish and that zebrafish represent a complementary model to further our understanding of this central protein. © 2016 Wiley Periodicals, Inc. PMID:27265751

  1. Knockdown of NBCe1 In Vivo Compromises the Corneal Endothelial Pump

    PubMed Central

    Liu, Cailing; Cheng, Qiang; Nguyen, Tracy

    2010-01-01

    Purpose. To evaluate the role of the sodium bicarbonate cotransporter (NBCe1) as a component of the corneal endothelial pump in the in vivo rabbit eye. Methods. Lentiviruses with NBCe1 shRNA and GFP expression cassettes were injected intracamerally. Knockdown efficacy was determined 1 week to 4 weeks later by immunofluorescence, Western blot analysis, and PCR. Functional effects were monitored by corneal thickness (CT) and brinzolamide sensitivity. Results. Within 24 hours there was a modest anterior chamber inflammation that resolved within 48 hours. At 4 × 106 IFU, more than 95% of the corneal endothelial surface showed GFP fluorescence above background within 7 days. At 14 to 21 days, signs of anterior chamber inflammation reemerged, and endothelial cell GFP fluorescence disappeared within 40 days after injection. The second phase of inflammation could be avoided by using GFP-less viruses. There was no significant difference in CT between scrambled sequence and NBCe1 shRNA–injected eyes over 3 weeks. Two drops of 1% brinzolamide produced 7.85% ± 3.3% corneal swelling within 5 hours of topical instillation. However, in corneas showing more than 25% NBCe1 knockdown (30 of 42 rabbits; 59% ± 15% knockdown), corneal swelling was significantly higher (10.1% ± 2.9%) relative to control eyes. Conclusions. FIV-based lentiviral vectors can transfect CE with shRNA in rabbits. The response to GFP is consistent, with previous studies showing the production of anti-GFP antibodies. Partial knockdown of NBCe1 did not affect baseline CT, which is consistent with the corneal endothelium having a substantial functional reserve. Provocative testing using, brinzolamide, however, revealed an underlying deficiency, confirming the importance of NBCe1 bicarbonate transport and demonstrating the concerted action between NBCe1 and carbonic anhydrases. PMID:20445126

  2. Stable knockdown of Kif5b in MDCK cells leads to epithelial–mesenchymal transition

    SciTech Connect

    Cui, Ju; Jin, Guoxiang; Yu, Bin; Wang, Zai; Lin, Raozhou; Huang, Jian-Dong

    2015-07-17

    Polarization of epithelial cells requires vectorial sorting and transport of polarity proteins to apical or basolateral domains. Kif5b is the mouse homologue of the human ubiquitous Kinesin Heavy Chain (uKHC). To investigate the function of Kif5b in epithelial cells, we examined the phenotypes of Kif5b-deficient MDCK cells. Stable knockdown of Kif5b in MDCK cells resulted in reduced cell proliferation rate, profound changes in cell morphology, loss of epithelial cell marker, and gain of mesenchymal marker, as well as increased cell migration, invasion, and tumorigenesis abilities. E-cadherin and NMMIIA could interact with Kif5b in polarized MDCK cells, and their expression levels were decreased in Kif5b-deficient MDCK cells. Overexpression of E-cadherin and NMMIIA in Kif5b depleted MDCK cells could decrease mesenchymal marker expression and cell migration ability. These results indicate that stable knockdown of Kif5b in MDCK cells can lead to epithelial–mesenchymal transition, which is mediated by defective E-cadherin and NMMIIA expression. - Highlights: • Knockdown of Kif5b in MDCK cells resulted in reduced cell proliferation rate. • Kif5b deficient MDCK cells underwent epithelial–mesenchymal transition. • E-cadherin and NMMIIA could interact with Kif5b in polarized MDCK cells. • Decreased E-cadherin and NMMIIA levels mediate EMT in Kif5b deficient MDCK cells. • Overexpression of E-cadherin and NMMIIA reverse the effects of Kif5b knockdown.

  3. Knockdown of pre-mRNA cleavage factor Im 25 kDa promotes neurite outgrowth

    SciTech Connect

    Fukumitsu, Hidefumi; Soumiya, Hitomi; Furukawa, Shoei

    2012-09-07

    Hi