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Sample records for rna expression vector

  1. Polycistronic RNA polymerase II expression vectors for RNA interference based on BIC/miR-155.

    PubMed

    Chung, Kwan-Ho; Hart, Christopher C; Al-Bassam, Sarmad; Avery, Adam; Taylor, Jennifer; Patel, Paresh D; Vojtek, Anne B; Turner, David L

    2006-01-01

    Vector-based RNA interference (RNAi) has emerged as a valuable tool for analysis of gene function. We have developed new RNA polymerase II expression vectors for RNAi, designated SIBR vectors, based upon the non-coding RNA BIC. BIC contains the miR-155 microRNA (miRNA) precursor, and we find that expression of a short region of the third exon of mouse BIC is sufficient to produce miR-155 in mammalian cells. The SIBR vectors use a modified miR-155 precursor stem-loop and flanking BIC sequences to express synthetic miRNAs complementary to target RNAs. Like RNA polymerase III driven short hairpin RNA vectors, the SIBR vectors efficiently reduce target mRNA and protein expression. The synthetic miRNAs can be expressed from an intron, allowing coexpression of a marker or other protein with the miRNAs. In addition, intronic expression of a synthetic miRNA from a two intron vector enhances RNAi. A SIBR vector can express two different miRNAs from a single transcript for effective inhibition of two different target mRNAs. Furthermore, at least eight tandem copies of a synthetic miRNA can be expressed in a polycistronic transcript to increase the inhibition of a target RNA. The SIBR vectors are flexible tools for a variety of RNAi applications.

  2. Polycistronic RNA polymerase II expression vectors for RNA interference based on BIC/miR-155

    PubMed Central

    Chung, Kwan-Ho; Hart, Christopher C.; Al-Bassam, Sarmad; Avery, Adam; Taylor, Jennifer; Patel, Paresh D.; Vojtek, Anne B.; Turner, David L.

    2006-01-01

    Vector-based RNA interference (RNAi) has emerged as a valuable tool for analysis of gene function. We have developed new RNA polymerase II expression vectors for RNAi, designated SIBR vectors, based upon the non-coding RNA BIC. BIC contains the miR-155 microRNA (miRNA) precursor, and we find that expression of a short region of the third exon of mouse BIC is sufficient to produce miR-155 in mammalian cells. The SIBR vectors use a modified miR-155 precursor stem–loop and flanking BIC sequences to express synthetic miRNAs complementary to target RNAs. Like RNA polymerase III driven short hairpin RNA vectors, the SIBR vectors efficiently reduce target mRNA and protein expression. The synthetic miRNAs can be expressed from an intron, allowing coexpression of a marker or other protein with the miRNAs. In addition, intronic expression of a synthetic miRNA from a two intron vector enhances RNAi. A SIBR vector can express two different miRNAs from a single transcript for effective inhibition of two different target mRNAs. Furthermore, at least eight tandem copies of a synthetic miRNA can be expressed in a polycistronic transcript to increase the inhibition of a target RNA. The SIBR vectors are flexible tools for a variety of RNAi applications. PMID:16614444

  3. RNA polymerase II mediated transcription from the polymerase III promoters in short hairpin RNA expression vector

    SciTech Connect

    Rumi, Mohammad; Ishihara, Shunji . E-mail: si360405@med.shimane-u.ac.jp; Aziz, Monowar; Kazumori, Hideaki; Ishimura, Norihisa; Yuki, Takafumi; Kadota, Chikara; Kadowaki, Yasunori; Kinoshita, Yoshikazu

    2006-01-13

    RNA polymerase III promoters of human ribonuclease P RNA component H1, human U6, and mouse U6 small nuclear RNA genes are commonly used in short hairpin RNA (shRNA) expression vectors due their precise initiation and termination sites. During transient transfection of shRNA vectors, we observed that H1 or U6 promoters also express longer transcripts enough to express several reporter genes including firefly luciferase, green fluorescent protein EGFP, and red fluorescent protein JRed. Expression of such longer transcripts was augmented by upstream RNA polymerase II enhancers and completely inhibited by downstream polyA signal sequences. Moreover, the transcription of firefly luciferase from human H1 promoter was sensitive to RNA polymerase II inhibitor {alpha}-amanitin. Our findings suggest that commonly used polymerase III promoters in shRNA vectors are also prone to RNA polymerase II mediated transcription, which may have negative impacts on their targeted use.

  4. Lentivirus-expressed siRNA vectors against Alzheimer disease.

    PubMed

    Peng, Kevin A; Masliah, Eliezer

    2010-01-01

    Amyloid precursor protein (APP) has been implicated in the pathogenesis of Alzheimer disease, and the accumulation of APP products ultimately leads to the familiar histopathological and clinical manifestations associated with this most common form of dementia. A protein that has been shown to promote APP accumulation is beta-secretase (beta-site APP cleaving enzyme 1, or BACE1), which is increased in the cerebrospinal fluid in those affected with Alzheimer disease. Through in vivo studies using APP transgenic mice, we demonstrated that decreasing the expression of BACE1 via lentiviral vector delivery of BACE1 siRNA has the potential for significantly reducing the cleavage of APP, accumulation of these products, and consequent neurodegeneration. As such, lentiviral-expressed siRNA against BACE1 is a therapeutic possibility in the treatment of Alzheimer disease. We detail the use of lentivirus-expressed siRNA as a method to ameliorate Alzheimer disease neuropathology in APP transgenic mice.

  5. 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.

  6. RNA viruses as vectors for the expression of heterologous proteins.

    PubMed

    Schlesinger, S

    1995-04-01

    RNA viruses comprise a wide variety of infectious agents, some of which are the cause of disease in humans, animals, and plants. Recombinant DNA technology is now making it feasible to modify these genomes and engineer them to express heterologous proteins. Several different schemes are being employed that depend on the genome organization of the virus and on the strategy of replication of the particular virus. Several different examples are illustrated and potential uses as well as possible problems are discussed. In the future reverse genetics may convert some of these viruses from agents of disease to agents of cure. PMID:7620976

  7. Use of short hairpin RNA expression vectors to study mammalian neural development.

    PubMed

    Yu, Jenn-Yah; Wang, Tsu-Wei; Vojtek, Anne B; Parent, Jack M; Turner, David L

    2005-01-01

    The use of RNA interference (RNAi) in mammalian cells has become a powerful tool for the analysis of gene function. Here we discuss the use of DNA vectors to produce short hairpin RNAs (shRNAs) and inhibit gene expression in mammalian neural progenitors and neurons. Protocols are presented for introducing shRNA vectors into mouse P19 cells differentiated as neurons in vitro and for electroporation of shRNA vectors into primary neural progenitors from the embryonic mouse dorsal telencephalon (prospective cerebral cortex). Transfected primary cortical progenitors can be differentiated in vitro either in dissociated culture or organotypic slice culture. The use of shRNA vectors for RNAi provides a versatile approach to understand gene function during mammalian neural development.

  8. Virus-Derived Gene Expression and RNA Interference Vector for Grapevine

    PubMed Central

    Kurth, Elizabeth G.; Peremyslov, Valera V.; Prokhnevsky, Alexey I.; Kasschau, Kristin D.; Miller, Marilyn; Carrington, James C.

    2012-01-01

    The improvement of the agricultural and wine-making qualities of the grapevine (Vitis vinifera) is hampered by adherence to traditional varieties, the recalcitrance of this plant to genetic modifications, and public resistance to genetically modified organism (GMO) technologies. To address these challenges, we developed an RNA virus-based vector for the introduction of desired traits into grapevine without heritable modifications to the genome. This vector expresses recombinant proteins in the phloem tissue that is involved in sugar transport throughout the plant, from leaves to roots to berries. Furthermore, the vector provides a powerful RNA interference (RNAi) capability of regulating the expression of endogenous genes via virus-induced gene-silencing (VIGS) technology. Additional advantages of this vector include superb genetic capacity and stability, as well as the swiftness of technology implementation. The most significant applications of the viral vector include functional genomics of the grapevine and disease control via RNAi-enabled vaccination against pathogens or invertebrate pests. PMID:22438553

  9. Virus-derived gene expression and RNA interference vector for grapevine.

    PubMed

    Kurth, Elizabeth G; Peremyslov, Valera V; Prokhnevsky, Alexey I; Kasschau, Kristin D; Miller, Marilyn; Carrington, James C; Dolja, Valerian V

    2012-06-01

    The improvement of the agricultural and wine-making qualities of the grapevine (Vitis vinifera) is hampered by adherence to traditional varieties, the recalcitrance of this plant to genetic modifications, and public resistance to genetically modified organism (GMO) technologies. To address these challenges, we developed an RNA virus-based vector for the introduction of desired traits into grapevine without heritable modifications to the genome. This vector expresses recombinant proteins in the phloem tissue that is involved in sugar transport throughout the plant, from leaves to roots to berries. Furthermore, the vector provides a powerful RNA interference (RNAi) capability of regulating the expression of endogenous genes via virus-induced gene-silencing (VIGS) technology. Additional advantages of this vector include superb genetic capacity and stability, as well as the swiftness of technology implementation. The most significant applications of the viral vector include functional genomics of the grapevine and disease control via RNAi-enabled vaccination against pathogens or invertebrate pests.

  10. Efficient Downregulation of Multiple mRNA Targets with a Single shRNA-Expressing Lentiviral Vector

    PubMed Central

    Chumakov, Stepan P.; Kravchenko, Julia E.; Prassolov, Vladimir S.; Frolova, Elena I.; Chumakov, Peter M.

    2010-01-01

    Gene silencing based on RNA interference is widely used in fundamental research and in practical applications. However, a commonly incomplete functional suppression represents a serious drawback of this technology. We describe a series of lentiviral vectors each containing a single or multiple shRNA expression cassette(s) driven by a RNA polymerase III specific promoter and localized within the 3′-LTR of the lentiviral DNA backbone. The vectors also contain an antibiotic-resistance gene that allows positive selection of recipient cells. The combined expression of three different shRNAs specific to a single mRNA was shown to improve dramatically the level of mRNA inhibition, while the use of three different RNA polymerase III specific promoters avoids the loss of shRNA expression cassettes through the homologous recombination. The vector system was used for successful simultaneous suppression of three related SESN1, SESN2 and SESN3 genes, which suggests its particular value for testing phenotypes of functionally redundant genes. PMID:20064551

  11. Efficient downregulation of multiple mRNA targets with a single shRNA-expressing lentiviral vector.

    PubMed

    Chumakov, Stepan P; Kravchenko, Julia E; Prassolov, Vladimir S; Frolova, Elena I; Chumakov, Peter M

    2010-05-01

    Gene silencing based on RNA interference is widely used in fundamental research and in practical applications. However, a commonly incomplete functional suppression represents a serious drawback of this technology. We describe a series of lentiviral vectors each containing a single or multiple shRNA-expression cassette(s) driven by a RNA-polymerase III specific promoter and localized within the 3'-LTR of the lentiviral DNA backbone. The vectors also contain an antibiotic-resistance gene that allows positive selection of recipient cells. The combined expression of three different shRNAs specific to a single mRNA was shown to improve dramatically the level of mRNA inhibition, while the use of three different RNA-polymerase III specific promoters avoids the loss of shRNA-expression cassettes through the homologous recombination. The vector system was used for successful simultaneous suppression of three related SESN1, SESN2 and SESN3 genes, which suggests its particular value for testing phenotypes of functionally redundant genes.

  12. Advanced Design of Dumbbell-shaped Genetic Minimal Vectors Improves Non-coding and Coding RNA Expression.

    PubMed

    Jiang, Xiaoou; Yu, Han; Teo, Cui Rong; Tan, Genim Siu Xian; Goh, Sok Chin; Patel, Parasvi; Chua, Yiqiang Kevin; Hameed, Nasirah Banu Sahul; Bertoletti, Antonio; Patzel, Volker

    2016-09-01

    Dumbbell-shaped DNA minimal vectors lacking nontherapeutic genes and bacterial sequences are considered a stable, safe alternative to viral, nonviral, and naked plasmid-based gene-transfer systems. We investigated novel molecular features of dumbbell vectors aiming to reduce vector size and to improve the expression of noncoding or coding RNA. We minimized small hairpin RNA (shRNA) or microRNA (miRNA) expressing dumbbell vectors in size down to 130 bp generating the smallest genetic expression vectors reported. This was achieved by using a minimal H1 promoter with integrated transcriptional terminator transcribing the RNA hairpin structure around the dumbbell loop. Such vectors were generated with high conversion yields using a novel protocol. Minimized shRNA-expressing dumbbells showed accelerated kinetics of delivery and transcription leading to enhanced gene silencing in human tissue culture cells. In primary human T cells, minimized miRNA-expressing dumbbells revealed higher stability and triggered stronger target gene suppression as compared with plasmids and miRNA mimics. Dumbbell-driven gene expression was enhanced up to 56- or 160-fold by implementation of an intron and the SV40 enhancer compared with control dumbbells or plasmids. Advanced dumbbell vectors may represent one option to close the gap between durable expression that is achievable with integrating viral vectors and short-term effects triggered by naked RNA. PMID:27357627

  13. Adenovirus vectors lacking virus-associated RNA expression enhance shRNA activity to suppress hepatitis C virus replication

    NASA Astrophysics Data System (ADS)

    Pei, Zheng; Shi, Guoli; Kondo, Saki; Ito, Masahiko; Maekawa, Aya; Suzuki, Mariko; Saito, Izumu; Suzuki, Tetsuro; Kanegae, Yumi

    2013-12-01

    First-generation adenovirus vectors (FG AdVs) expressing short-hairpin RNA (shRNA) effectively downregulate the expressions of target genes. However, this vector, in fact, expresses not only the transgene product, but also virus-associated RNAs (VA RNAs) that disturb cellular RNAi machinery. We have established a production method for VA-deleted AdVs lacking expression of VA RNAs. Here, we showed that the highest shRNA activity was obtained when the shRNA was inserted not at the popularly used E1 site, but at the E4 site. We then compared the activities of shRNAs against hepatitis C virus (HCV) expressed from VA-deleted AdVs or conventional AdVs. The VA-deleted AdVs inhibited HCV production much more efficiently. Therefore, VA-deleted AdVs were more effective than the currently used AdVs for shRNA downregulation, probably because of the lack of competition between VA RNAs and the shRNAs. These VA-deleted AdVs might enable more effective gene therapies for chronic hepatitis C.

  14. Construction of an artificial MicroRNA expression vector for simultaneous inhibition of multiple genes in mammalian cells.

    PubMed

    Hu, Tao; Fu, Qiong; Chen, Ping; Ma, Li; Sin, Onsam; Guo, Deyin

    2009-05-14

    Recently, artificial microRNA (amiRNA) has become a promising RNA interference (RNAi) technology. Here, we describe a flexible and reliable method for constructing both single- and multi-amiRNA expression vectors. Two universal primers, together with two specific primers carrying the encoding sequence of amiRNA were designed and utilized to synthesize the functional amiRNA cassette through a one-step PCR. With appropriate restriction sites, the synthesized amiRNA cassettes can be cloned into any site of different destination vectors. Using the method, we constructed both single- and multi-amiRNA expression vectors to target three reporter genes, which code firefly luciferase (Fluc), enhanced green fluorescent protein (EGFP) and beta-galactosidase (LacZ), respectively. The expressions of three genes were all specifically inhibited by either the corresponding single- or the multi-amiRNA expression vector in 293T cells. And the RNAi efficiency of each amiRNA produced by both single- and multi-amiRNA expression vectors was comparable.

  15. Multigenic lentiviral vectors for combined and tissue-specific expression of miRNA- and protein-based antiangiogenic factors

    PubMed Central

    Askou, Anne Louise; Aagaard, Lars; Kostic, Corinne; Arsenijevic, Yvan; Hollensen, Anne Kruse; Bek, Toke; Jensen, Thomas Gryesten; Mikkelsen, Jacob Giehm; Corydon, Thomas Juhl

    2015-01-01

    Lentivirus-based gene delivery vectors carrying multiple gene cassettes are powerful tools in gene transfer studies and gene therapy, allowing coexpression of multiple therapeutic factors and, if desired, fluorescent reporters. Current strategies to express transgenes and microRNA (miRNA) clusters from a single vector have certain limitations that affect transgene expression levels and/or vector titers. In this study, we describe a novel vector design that facilitates combined expression of therapeutic RNA- and protein-based antiangiogenic factors as well as a fluorescent reporter from back-to-back RNApolII-driven expression cassettes. This configuration allows effective production of intron-embedded miRNAs that are released upon transduction of target cells. Exploiting such multigenic lentiviral vectors, we demonstrate robust miRNA-directed downregulation of vascular endothelial growth factor (VEGF) expression, leading to reduced angiogenesis, and parallel impairment of angiogenic pathways by codelivering the gene encoding pigment epithelium-derived factor (PEDF). Notably, subretinal injections of lentiviral vectors reveal efficient retinal pigment epithelium-specific gene expression driven by the VMD2 promoter, verifying that multigenic lentiviral vectors can be produced with high titers sufficient for in vivo applications. Altogether, our results suggest the potential applicability of combined miRNA- and protein-encoding lentiviral vectors in antiangiogenic gene therapy, including new combination therapies for amelioration of age-related macular degeneration. PMID:26052532

  16. Simultaneous inhibition of GSK3alpha and GSK3beta using hairpin siRNA expression vectors.

    PubMed

    Yu, Jenn-Yah; Taylor, Jennifer; DeRuiter, Stacy L; Vojtek, Anne B; Turner, David L

    2003-02-01

    Short interfering RNAs (siRNAs) can mediate sequence-specific inhibition of gene expression in mammalian cells. We and others have recently developed expression vector-based systems for synthesizing siRNAs or hairpin siRNAs in mammalian cells. Expression vector-based RNA interference (RNAi) effectively suppresses expression of target genes and is likely to be a powerful tool for analysis of gene function. Here we compare inhibition by vectors expressing hairpin siRNA designs either with different loop sequences connecting the two siRNA strands, or with duplex regions of different lengths. Our results suggest that lengthening the 19-nucleotide duplex region of a relatively ineffective hairpin siRNA can increase inhibition, but increasing the length of an effective 19-nt hairpin siRNA does not increase inhibition. We also demonstrate that hairpin siRNA vectors can be used to inhibit two target genes simultaneously. We have targeted glycogen synthase kinase-3alpha (GSK-3alpha) and GSK-3beta, two related kinases involved in the regulation of a variety of cellular processes and also implicated in the pathogenesis of several human diseases. Inhibition of either GSK-3alpha or GSK-3beta by transfection of hairpin siRNA vectors leads to elevated expression of the GSK-3 target beta-catenin, whereas inhibition of both kinases further increases beta-catenin expression. Our results suggest that vector-based siRNA inhibition may be useful for dissecting the functional roles of GSK-3alpha and GSK-3beta in somatic cells. The ability to inhibit two or more genes simultaneously with hairpin siRNA expression vectors should facilitate studies of gene function in mammalian cells.

  17. Design, Construction, and Validation of Artificial MicroRNA Vectors Using Agrobacterium-Mediated Transient Expression System.

    PubMed

    Bhagwat, Basdeo; Chi, Ming; Han, Dianwei; Tang, Haifeng; Tang, Guiliang; Xiang, Yu

    2016-01-01

    Artificial microRNA (amiRNA) technology utilizes microRNA (miRNA) biogenesis pathway to produce artificially selected small RNAs using miRNA gene backbone. It provides a feasible strategy for inducing loss of gene function, and has been applied in functional genomics study, improvement of crop quality and plant virus disease resistance. A big challenge in amiRNA applications is the unpredictability of silencing efficacy of the designed amiRNAs and not all constructed amiRNA candidates would be expressed effectively in plant cells. We and others found that high efficiency and specificity in RNA silencing can be achieved by designing amiRNAs with perfect or almost perfect sequence complementarity to their targets. In addition, we recently demonstrated that Agrobacterium-mediated transient expression system can be used to validate amiRNA constructs, which provides a simple, rapid and effective method to select highly expressible amiRNA candidates for stable genetic transformation. Here, we describe the methods for design of amiRNA candidates with perfect or almost perfect base-pairing to the target gene or gene groups, incorporation of amiRNA candidates in miR168a gene backbone by one step inverse PCR amplification, construction of plant amiRNA expression vectors, and assay of transient expression of amiRNAs in Nicotiana benthamiana through agro-infiltration, small RNA extraction, and amiRNA Northern blot.

  18. piRNA pathway gene expression in the malaria vector mosquito Anopheles stephensi

    PubMed Central

    Macias, V; Coleman, J; Bonizzoni, M; James, A A

    2014-01-01

    The ability of transposons to mobilize to new places in a genome enables them to introgress rapidly into populations. The piRNA pathway has been characterized recently in the germ line of the fruit fly, Drosophila melanogaster, and is responsible for downregulating transposon mobility. Transposons have been used as tools in mosquitoes to genetically transform a number of species including Anopheles stephensi, a vector of human malaria. These mobile genetic elements also have been proposed as tools to drive antipathogen effector genes into wild mosquito populations to replace pathogen-susceptible insects with those engineered genetically to be resistant to or unable to transmit a pathogen. The piRNA pathway may affect the performance of such proposed genetic engineering strategies. In the present study, we identify and describe the An. stephensi orthologues of the major genes in the piRNA pathway, Ago3, Aubergine (Aub) and Piwi. Consistent with a role in protection from transposon movement, these three genes are expressed constitutively in the germ-line cells of ovaries and induced further after a blood meal. PMID:24947897

  19. Bovine respiratory syncytial virus nucleocapsid protein: mRNA sequence analysis and expression from recombinant vaccinia virus vectors.

    PubMed

    Amann, V L; Lerch, R A; Anderson, K; Wertz, G W

    1992-04-01

    The nucleotide sequence of the mRNA encoding the nucleocapsid (N) protein of bovine respiratory syncytial (BRS) virus, strain 391-2, was determined. Recombinant vectors containing a cDNA of the complete N gene were constructed, and expression of the N protein in eukaryotic cells was demonstrated using two different vector systems. The BRS virus N mRNA was 1197 nucleotides in length, exclusive of poly(A), and had a single major open reading frame that encoded a polypeptide of 391 amino acids with a calculated M(r) of 42.6K. The nucleotide and amino acid sequences of the BRS virus N gene were compared to those of human respiratory syncytial (HRS) virus strains A2 and 18537, and to BRS virus strain A51908. The level of nucleic acid identity between the N mRNA of BRS virus 391-2 and both HRS virus subtypes was 80 to 81%, whereas the identity between the two BRS virus strains was 97%. A 93 to 94% level of identity was observed between the deduced amino acid sequences of the N protein of BRS virus 391-2 and the corresponding sequences of the two HRS virus strains. The two BRS virus N proteins differed in amino acid sequence at only three positions. Recombinant BRS virus N protein was expressed using two different vector systems: in cells from a plasmid using the vaccinia virus/T7 polymerase expression system or from a recombinant vaccinia virus. N proteins synthesized by the two vector systems migrated with an electrophoretic mobility identical to that of authentic BRS virus N protein, and were precipitated by anti-BRS virus antibodies.

  20. Construction and functional screening of a metagenomic library using a T7 RNA polymerase-based expression cosmid vector.

    PubMed

    Lussier, François-Xavier; Chambenoit, Olivier; Côté, Amélie; Hupé, Jean-François; Denis, François; Juteau, Pierre; Beaudet, Réjean; Shareck, François

    2011-09-01

    The metagenomic approach has greatly accelerated the discovery of new enzymes by giving access to the genetic potential of microorganisms from various environments. Function-based screening depends on adequate expression of the foreign genes in the heterologous host, which can be challenging in large-insert libraries. In this study, the shuttle cosmid vector pFX583 was used for the construction and screening of a metagenomic library. This vector allows T7 RNA polymerase-directed transcription of the cloned DNA and can be used in Escherichia coli and Streptomyces lividans. The DNA used for the library construction was obtained from an enriched biomass. The library was screened for lipolytic and proteolytic activities using E. coli and S. lividans as hosts. Numerous E. coli clones with lipolytic activity were detected. Unfortunately, proteases could not be detected in both hosts. From the lipolytic activity screen, a gene coding for a new lipase was isolated, and partial characterization was conducted. PMID:21108039

  1. RNA viral vectors for improved Agrobacterium-mediated transient expression of heterologous proteins in Nicotiana benthamiana cell suspensions and hairy roots

    PubMed Central

    2012-01-01

    Background Plant cell suspensions and hairy root cultures represent scalable protein expression platforms. Low protein product titers have thus far limited the application of transient protein expression in these hosts. The objective of this work was to overcome this limitation by harnessing A. tumefaciens to deliver replicating and non-replicating RNA viral vectors in plant tissue co-cultures. Results Replicating vectors derived from Potato virus X (PVX) and Tobacco rattle virus (TRV) were modified to contain the reporter gene β-glucuronidase (GUS) with a plant intron to prevent bacterial expression. In cell suspensions, a minimal PVX vector retaining only the viral RNA polymerase gene yielded 6.6-fold more GUS than an analogous full-length PVX vector. Transient co-expression of the minimal PVX vector with P19 of Tomato bushy stunt virus or HC-Pro of Tobacco etch virus to suppress post-transcriptional gene silencing increased GUS expression by 44 and 83%, respectively. A non-replicating vector containing a leader sequence from Cowpea mosaic virus (CPMV-HT) modified for enhanced translation led to 70% higher transient GUS expression than a control treatment. In hairy roots, a TRV vector capable of systemic movement increased GUS accumulation by 150-fold relative to the analogous PVX vector. Histochemical staining for GUS in TRV-infected hairy roots revealed the capacity for achieving even higher productivity per unit biomass. Conclusions For the first time, replicating PVX vectors and a non-replicating CPMV-HT vector were successfully applied toward transient heterologous protein expression in cell suspensions. A replicating TRV vector achieved transient GUS expression levels in hairy roots more than an order of magnitude higher than the highest level previously reported with a viral vector delivered by A. tumefaciens. PMID:22559055

  2. The closterovirus-derived gene expression and RNA interference vectors as tools for research and plant biotechnology

    PubMed Central

    Dolja, Valerian V.; Koonin, Eugene V.

    2013-01-01

    Important progress in understanding replication, interactions with host plants, and evolution of closteroviruses enabled engineering of several vectors for gene expression and virus-induced gene silencing. Due to the broad host range of closteroviruses, these vectors expanded vector applicability to include important woody plants such as citrus and grapevine. Furthermore, large closterovirus genomes offer genetic capacity and stability unrivaled by other plant viral vectors. These features provided immense opportunities for using closterovirus vectors for the functional genomics studies and pathogen control in economically valuable crops. This review briefly summarizes advances in closterovirus research during the last decade, explores the relationships between virus biology and vector design, and outlines the most promising directions for future application of closterovirus vectors. PMID:23596441

  3. A PCR-Based Method to Construct Lentiviral Vector Expressing Double Tough Decoy for miRNA Inhibition.

    PubMed

    Qiu, Huiling; Zhong, Jiasheng; Luo, Lan; Liu, Nian; Kang, Kang; Qu, Junle; Peng, Wenda; Gou, Deming

    2015-01-01

    DNA vector-encoded Tough Decoy (TuD) miRNA inhibitor is attracting increased attention due to its high efficiency in miRNA suppression. The current methods used to construct TuD vectors are based on synthesizing long oligonucleotides (~90 mer), which have been costly and problematic because of mutations during synthesis. In this study, we report a PCR-based method for the generation of double Tough Decoy (dTuD) vector in which only two sets of shorter oligonucleotides (< 60 mer) were used. Different approaches were employed to test the inhibitory potency of dTuDs. We demonstrated that dTuD is the most efficient method in miRNA inhibition in vitro and in vivo. Using this method, a mini dTuD library against 88 human miRNAs was constructed and used for a high-throughput screening (HTS) of AP-1 pathway-related miRNAs. Seven miRNAs (miR-18b-5p, -101-3p, -148b-3p, -130b-3p, -186-3p, -187-3p and -1324) were identified as candidates involved in AP-1 pathway regulation. This novel method allows for an accurate and cost-effective generation of dTuD miRNA inhibitor, providing a powerful tool for efficient miRNA suppression in vitro and in vivo. PMID:26624995

  4. A PCR-Based Method to Construct Lentiviral Vector Expressing Double Tough Decoy for miRNA Inhibition

    PubMed Central

    Luo, Lan; Liu, Nian; Kang, Kang; Qu, Junle; Peng, Wenda; Gou, Deming

    2015-01-01

    DNA vector-encoded Tough Decoy (TuD) miRNA inhibitor is attracting increased attention due to its high efficiency in miRNA suppression. The current methods used to construct TuD vectors are based on synthesizing long oligonucleotides (~90 mer), which have been costly and problematic because of mutations during synthesis. In this study, we report a PCR-based method for the generation of double Tough Decoy (dTuD) vector in which only two sets of shorter oligonucleotides (< 60 mer) were used. Different approaches were employed to test the inhibitory potency of dTuDs. We demonstrated that dTuD is the most efficient method in miRNA inhibition in vitro and in vivo. Using this method, a mini dTuD library against 88 human miRNAs was constructed and used for a high-throughput screening (HTS) of AP-1 pathway-related miRNAs. Seven miRNAs (miR-18b-5p, -101-3p, -148b-3p, -130b-3p, -186-3p, -187-3p and -1324) were identified as candidates involved in AP-1 pathway regulation. This novel method allows for an accurate and cost-effective generation of dTuD miRNA inhibitor, providing a powerful tool for efficient miRNA suppression in vitro and in vivo. PMID:26624995

  5. Targeted therapy via oral administration of attenuated Salmonella expression plasmid-vectored Stat3-shRNA cures orthotopically transplanted mouse HCC.

    PubMed

    Tian, Y; Guo, B; Jia, H; Ji, K; Sun, Y; Li, Y; Zhao, T; Gao, L; Meng, Y; Kalvakolanu, D V; Kopecko, D J; Zhao, X; Zhang, L; Xu, D

    2012-06-01

    The development of RNA interference-based cancer gene therapies has been delayed due to the lack of effective tumor-targeting delivery systems. Attenuated Salmonella enterica serovar Typhimurium (S. Typhimurium) has a natural tropism for solid tumors. We report here the use of attenuated S. Typhimurium as a vector to deliver shRNA directly into tumor cells. Constitutively activated signal transducer and activator of transcription 3 (Stat3) is a key transcription factor involved in both hepatocellular carcinoma (HCC) growth and metastasis. In this study, attenuated S. Typhimurium was capable of delivering shRNA-expressing vectors to the targeted cancer cells and inducing RNA interference in vivo. More importantly, a single oral dose of attenuated S. Typhimurium carrying shRNA-expressing vectors targeting Stat3 induced remarkably delayed and reduced HCC (in 70% of mice). Cancer in these cured mice did not recur over 2 years following treatment. These data demonstrated that RNA interference combined with Salmonella as a delivery system may offer a novel clinical approach for cancer gene therapy. PMID:22555509

  6. The recombinant adeno-associated virus vector (rAAV2)-mediated apolipoprotein B mRNA-specific hammerhead ribozyme: a self-complementary AAV2 vector improves the gene expression

    PubMed Central

    Zhong, Shumei; Sun, Shihua; Teng, Ba-Bie

    2004-01-01

    Background In humans, overproduction of apolipoprotein B (apoB) is positively associated with premature coronary artery diseases. To reduce the levels of apoB mRNA, we have designed an apoB mRNA-specific hammerhead ribozyme targeted at nucleotide sequences GUA6679 (RB15) mediated by adenovirus, which efficiently cleaves and decreases apoB mRNA by 80% in mouse liver and attenuates the hyperlipidemic condition. In the current study, we used an adeno-associated virus vector, serotype 2 (AAV2) and a self-complementary AAV2 vector (scAAV2) to demonstrate the effect of long-term tissue-specific gene expression of RB15 on the regulation apoB mRNA in vivo. Methods We constructed a hammerhead ribozyme RB15 driven by a liver-specific transthyretin (TTR) promoter using an AAV2 vector (rAAV2-TTR-RB15). HepG2 cells and hyperlipidemic mice deficient in both the low density lipoprotein receptor and the apoB mRNA editing enzyme genes (LDLR-/-Apobec1-/-; LDb) were transduced with rAAV2-TTR-RB15 and a control vector rAAV-TTR-RB15-mutant (inactive ribozyme). The effects of ribozyme RB15 on apoB metabolism and atherosclerosis development were determined in LDb mice at 5-month after transduction. A self-complementary AAV2 vector expressing ribozyme RB15 (scAAV2-TTR-RB15) was also engineered and used to transduce HepG2 cells. Studies were designed to compare the gene expression efficiency between rAAV2-TTR-RB15 and scAAV2-TTR-RB15. Results The effect of ribozyme RB15 RNA on reducing apoB mRNA levels in HepG2 cells was observed only on day-7 after rAAV2-TTR-RB15 transduction. And, at 5-month after rAAV2-TTR-RB15 treatment, the apoB mRNA levels in LDb mice were significantly decreased by 43%, compared to LDb mice treated with control vector rAAV2-TTR-RB15-mutant. Moreover, both the rAAV2-TTR-RB15 viral DNA and ribozyme RB15 RNA were still detectable in mice livers at 5-month after treatment. However, this rAAV2-TTR-RB15 vector mediated a prolonged but low level of ribozyme RB15 gene

  7. A Comparison of Target Gene Silencing using Synthetically Modified siRNA and shRNA That Express Recombinant Lentiviral Vectors.

    PubMed

    Spirin, P V; Baskaran, D; Rubtsov, P M; Zenkova, M A; Vlassov, V V; Chernolovskaya, E L; Prassolov, V S

    2009-07-01

    RNA-interference is an effective natural mechanism of post-transcriptional modulation of gene expression. RNA-interference mechanism exist as in high eukaryotes both animals and plants as well in lower eukaryotes and viruses. RNA-interference is now used as a powerful tool in study of functional gene activity and many essential for fundamental biology results was obtained with this approach. Also it's widely believed that RNA-interference could be used in working out of new therapeutic medicine against malignant, infectious and hereditary diseases. One of the main problems of these developments is search of effective methods of siRNA transfer into the target cells. At present time for these purpose different sorts of transfect ions or viral transduction are used. At present article the results of comparison of inhibition of expression of oncogene AML-ET O by synthetic siRNA and by recombinant lentiviruses coding for corresponding shRNA are presented.

  8. A Comparison of Target Gene Silencing using Synthetically Modified siRNA and shRNA That Express Recombinant Lentiviral Vectors

    PubMed Central

    Spirin, P.V.; Baskaran, D.; Rubtsov, P.M.; Zenkova, M.A.; Vlassov, V.V.; Chernolovskaya, E.L.

    2009-01-01

    RNA-interference is an effective natural mechanism of post-transcriptional modulation of gene expression. RNA-interference mechanism exist as in high eukaryotes both animals and plants as well in lower eukaryotes and viruses. RNA-interference is now used as a powerful tool in study of functional gene activity and many essential for fundamental biology results was obtained with this approach. Also it's widely believed that RNA-interference could be used in working out of new therapeutic medicine against malignant, infectious and hereditary diseases. One of the main problems of these developments is search of effective methods of siRNA transfer into the target cells. At present time for these purpose different sorts of transfect ions or viral transduction are used. At present article the results of comparison of inhibition of expression of oncogene AML-ET O by synthetic siRNA and by recombinant lentiviruses coding for corresponding shRNA are presented. PMID:22649608

  9. Striatal modulation of BDNF expression using microRNA124a-expressing lentiviral vectors impairs ethanol-induced conditioned-place preference and voluntary alcohol consumption.

    PubMed

    Bahi, Amine; Dreyer, Jean-Luc

    2013-07-01

    Alcohol abuse is a major health, economic and social concern in modern societies, but the exact molecular mechanisms underlying ethanol addiction remain elusive. Recent findings show that small non-coding microRNA (miRNA) signaling contributes to complex behavioral disorders including drug addiction. However, the role of miRNAs in ethanol-induced conditioned-place preference (CPP) and voluntary alcohol consumption has not yet been directly addressed. Here, we assessed the expression profile of miR124a in the dorsal striatum of rats upon ethanol intake. The results show that miR124a was downregulated in the dorso-lateral striatum (DLS) following alcohol drinking. Then, we identified brain-derived neurotrophic factor (BDNF) as a direct target of miR124a. In fact, BDNF mRNA was upregulated following ethanol drinking. We used lentiviral vector (LV) gene transfer technology to further address the role of miR124a and its direct target BDNF in ethanol-induced CPP and alcohol consumption. Results reveal that stereotaxic injection of LV-miR124a in the DLS enhances ethanol-induced CPP as well as voluntary alcohol consumption in a two-bottle choice drinking paradigm. Moreover, miR124a-silencer (LV-siR124a) as well as LV-BDNF infusion in the DLS attenuates ethanol-induced CPP as well as voluntary alcohol consumption. Importantly, LV-miR124a, LV-siR124a and LV-BDNF have no effect on saccharin and quinine intake. Our findings indicate that striatal miR124a and BDNF signaling have crucial roles in alcohol consumption and ethanol conditioned reward.

  10. Lentiviral vector platform for improved erythropoietin expression concomitant with shRNA mediated host cell elastase down regulation.

    PubMed

    Dhamne, Hemant; Chande, Ajit G; Mukhopadhyaya, Robin

    2014-01-01

    Lentiviral vector (LV) mediated gene transfer holds great promise to develop stable cell lines for sustained transgene expression providing a valuable alternative to the conventional plasmid transfection based recombinant protein production methods. We report here making a third generation HIV-2 derived LV containing erythropoietin (EPO) gene expression cassette to generate a stable HEK293 cell line secreting EPO constitutively. A high producer cell clone was obtained by limiting dilution and was adapted to serum free medium. The suspension adapted cell clone stably produced milligram per liter quantities of EPO. Subsequent host metabolic engineering using lentiviral RNAi targeted to block an endogenous candidate protease elastase, identified through an in silico approach, resulted in appreciable augmentation of EPO expression above the original level. This study of LV based improved glycoprotein expression with host cell metabolic engineering for stable production of protein therapeutics thus exemplifies the versatility of LV and is of significant future biopharmaceutical importance.

  11. Lentiviral vector platform for improved erythropoietin expression concomitant with shRNA mediated host cell elastase down regulation.

    PubMed

    Dhamne, Hemant; Chande, Ajit G; Mukhopadhyaya, Robin

    2014-01-01

    Lentiviral vector (LV) mediated gene transfer holds great promise to develop stable cell lines for sustained transgene expression providing a valuable alternative to the conventional plasmid transfection based recombinant protein production methods. We report here making a third generation HIV-2 derived LV containing erythropoietin (EPO) gene expression cassette to generate a stable HEK293 cell line secreting EPO constitutively. A high producer cell clone was obtained by limiting dilution and was adapted to serum free medium. The suspension adapted cell clone stably produced milligram per liter quantities of EPO. Subsequent host metabolic engineering using lentiviral RNAi targeted to block an endogenous candidate protease elastase, identified through an in silico approach, resulted in appreciable augmentation of EPO expression above the original level. This study of LV based improved glycoprotein expression with host cell metabolic engineering for stable production of protein therapeutics thus exemplifies the versatility of LV and is of significant future biopharmaceutical importance. PMID:24325878

  12. In vitro silencing effect of chitosan nanoplexes containing siRNA expressing vector targeting VEGF in breast cancer cell lines.

    PubMed

    Salva, E; Akbuğa, J

    2010-12-01

    Small interferring RNA (siRNA) is a powerful tool for controlling cellular processes of gene silencing at post-transcriptional level due to its high sequence-specific inhibition efficiency. The aim of this study is to compare silencing effect of chitosan/shVEGF (shRNA-expressing plasmid DNA targeting vascular endothelial growth factor-A (VEGF-A)) nanoplexes between four different cell lines, two of which are MCF-7 and MDA-MB435 breast cancer cell lines. Nanoplexes were prepared using different concentrations. The morphological and physicochemical characterization were made and silencing activity of appropriate formulations were investigated in vitro. As a result of in vitro transfection studies made in different cell lines with nanoplexes in the different ratios, the highest gene inhibition (60%) was measured in MCF-7 after transfection while the lowest gene inhibition (29%) was observed in MDA-MB435. This work suggests that RNA interference has potential to be applied to delivery system studies and to the angiogenesis treatment.

  13. Long-term Expression of Apolipoprotein B mRNA-specific Hammerhead Ribozyme via scAAV8.2 Vector Inhibits Atherosclerosis in Mice

    PubMed Central

    Nischal, Hersharan; Sun, Hua; Wang, Yuchun; Ford, David A; Cao, Ying; Wei, Peng; Teng, Ba-Bie

    2013-01-01

    Target substrate-specific hammerhead ribozyme cleaves the specific mRNA efficiently and results in the inhibition of gene expression. In humans, overproduction of apolipoprotein B (apoB) is positively associated with premature coronary artery diseases. The goal of this study is to demonstrate that long-term reduction of apoB gene expression using hammerhead ribozyme would result in inhibition of atherosclerosis development. We designed two hammerhead ribozymes targeted at the nucleotides of apoB mRNA GUC2326 (designated RB1) and GUA6679 (designated RB15), and we used self-complementary adeno-associated virus 8.2 (scAAV8.2) vector to deliver these active ribozymes of RB1, RB15, combination of RB1/RB15, and an inactive hammerhead ribozyme RB15 mutant to atherosclerosis-prone LDb mice (Ldlr−/−Apobec1−/−). LDb mice lack both low density lipoproteins (LDL) receptor (Ldlr−/−) and apoB mRNA editing enzyme (Apobec1−/−) genes and develop atherosclerosis spontaneously. After the RB1, RB15, or combination of RB1/RB15 ribozymes treatment, the LDb mice had significantly decreased plasma triglyceride and apoB levels, resulting in markedly decreased of atherosclerotic lesions, Furthermore, the active ribozymes treatment decreased the levels of diacylglycerol acyltransferase 1 (Dgat1) mRNA and the levels of multiple diacylglycerol (DAG) molecular species. These results provide the first evidence that decreased apoB levels results to reduction of Dgat1 expression and triglyceride levels (TAG), which had a significant impact on the development of atherosclerosis. PMID:24084845

  14. MicroRNA-regulated viral vectors for gene therapy.

    PubMed

    Geisler, Anja; Fechner, Henry

    2016-05-20

    Safe and effective gene therapy approaches require targeted tissue-specific transfer of a therapeutic transgene. Besides traditional approaches, such as transcriptional and transductional targeting, microRNA-dependent post-transcriptional suppression of transgene expression has been emerging as powerful new technology to increase the specificity of vector-mediated transgene expression. MicroRNAs are small non-coding RNAs and often expressed in a tissue-, lineage-, activation- or differentiation-specific pattern. They typically regulate gene expression by binding to imperfectly complementary sequences in the 3' untranslated region (UTR) of the mRNA. To control exogenous transgene expression, tandem repeats of artificial microRNA target sites are usually incorporated into the 3' UTR of the transgene expression cassette, leading to subsequent degradation of transgene mRNA in cells expressing the corresponding microRNA. This targeting strategy, first shown for lentiviral vectors in antigen presenting cells, has now been used for tissue-specific expression of vector-encoded therapeutic transgenes, to reduce immune response against the transgene, to control virus tropism for oncolytic virotherapy, to increase safety of live attenuated virus vaccines and to identify and select cell subsets for pluripotent stem cell therapies, respectively. This review provides an introduction into the technical mechanism underlying microRNA-regulation, highlights new developments in this field and gives an overview of applications of microRNA-regulated viral vectors for cardiac, suicide gene cancer and hematopoietic stem cell therapy, as well as for treatment of neurological and eye diseases. PMID:27226955

  15. MicroRNA-regulated viral vectors for gene therapy

    PubMed Central

    Geisler, Anja; Fechner, Henry

    2016-01-01

    Safe and effective gene therapy approaches require targeted tissue-specific transfer of a therapeutic transgene. Besides traditional approaches, such as transcriptional and transductional targeting, microRNA-dependent post-transcriptional suppression of transgene expression has been emerging as powerful new technology to increase the specificity of vector-mediated transgene expression. MicroRNAs are small non-coding RNAs and often expressed in a tissue-, lineage-, activation- or differentiation-specific pattern. They typically regulate gene expression by binding to imperfectly complementary sequences in the 3’ untranslated region (UTR) of the mRNA. To control exogenous transgene expression, tandem repeats of artificial microRNA target sites are usually incorporated into the 3’ UTR of the transgene expression cassette, leading to subsequent degradation of transgene mRNA in cells expressing the corresponding microRNA. This targeting strategy, first shown for lentiviral vectors in antigen presenting cells, has now been used for tissue-specific expression of vector-encoded therapeutic transgenes, to reduce immune response against the transgene, to control virus tropism for oncolytic virotherapy, to increase safety of live attenuated virus vaccines and to identify and select cell subsets for pluripotent stem cell therapies, respectively. This review provides an introduction into the technical mechanism underlying microRNA-regulation, highlights new developments in this field and gives an overview of applications of microRNA-regulated viral vectors for cardiac, suicide gene cancer and hematopoietic stem cell therapy, as well as for treatment of neurological and eye diseases. PMID:27226955

  16. Viral and Synthetic RNA Vector Technologies and Applications.

    PubMed

    Schott, Juliane W; Morgan, Michael; Galla, Melanie; Schambach, Axel

    2016-09-01

    Use of RNA is an increasingly popular method to transiently deliver genetic information for cell manipulation in basic research and clinical therapy. In these settings, viral and nonviral RNA platforms are employed for delivery of small interfering RNA and protein-coding mRNA. Technological advances allowing RNA modification for increased stability, improved translation and reduced immunogenicity have led to increased use of nonviral synthetic RNA, which is delivered in naked form or upon formulation. Alternatively, highly efficient viral entry pathways are exploited to transfer genes of interest as RNA incorporated into viral particles. Current viral RNA transfer technologies are derived from Retroviruses, nonsegmented negative-strand RNA viruses or positive-stranded Alpha- and Flaviviruses. In retroviral particles, the genes of interest can either be incorporated directly into the viral RNA genome or as nonviral RNA. Nonsegmented negative-strand virus-, Alpha- and Flavivirus-derived vectors support prolonged expression windows through replication of viral RNA encoding genes of interest. Mixed technologies combining viral and nonviral components are also available. RNA transfer is ideal for all settings that do not require permanent transgene expression and excludes potentially detrimental DNA integration into the target cell genome. Thus, RNA-based technologies are successfully applied for reprogramming, transdifferentiation, gene editing, vaccination, tumor therapy, and gene therapy. PMID:27377044

  17. A stable RNA virus-based vector for citrus trees

    SciTech Connect

    Folimonov, Alexey S.; Folimonova, Svetlana Y.; Bar-Joseph, Moshe; Dawson, William O.

    2007-11-10

    Virus-based vectors are important tools in plant molecular biology and plant genomics. A number of vectors based on viruses that infect herbaceous plants are in use for expression or silencing of genes in plants as well as screening unknown sequences for function. Yet there is a need for useful virus-based vectors for woody plants, which demand much greater stability because of the longer time required for systemic infection and analysis. We examined several strategies to develop a Citrus tristeza virus (CTV)-based vector for transient expression of foreign genes in citrus trees using a green fluorescent protein (GFP) as a reporter. These strategies included substitution of the p13 open reading frame (ORF) by the ORF of GFP, construction of a self-processing fusion of GFP in-frame with the major coat protein (CP), or expression of the GFP ORF as an extra gene from a subgenomic (sg) mRNA controlled either by a duplicated CTV CP sgRNA controller element (CE) or an introduced heterologous CE of Beet yellows virus. Engineered vector constructs were examined for replication, encapsidation, GFP expression during multiple passages in protoplasts, and for their ability to infect, move, express GFP, and be maintained in citrus plants. The most successful vectors based on the 'add-a-gene' strategy have been unusually stable, continuing to produce GFP fluorescence after more than 4 years in citrus trees.

  18. U1 snRNA as an effective vector for stable expression of antisense molecules and for the inhibition of the splicing reaction.

    PubMed

    Martone, Julie; De Angelis, Fernanda Gabriella; Bozzoni, Irene

    2012-01-01

    We report the use of the U1 snRNA as a vector for the stable expression of antisense molecules against the splice junctions of specific dystrophin exons. The single-stranded 5' terminus of U1 can be replaced by unrelated sequences as long as 50 nucleotides without affecting both the stability and the ability to assemble into snRNP particles. Effective exon skipping has been obtained for different dystrophin exons by antisense sequences against 5' and 3' splice sites alone or in combination with ESE sequences. The efficacy of these molecules has been studied both in in vitro systems and in animals. In both cases the chimeric molecules, delivered as part of lentiviral or AAV vectors (De Angelis et al. Proc Natl Acad Sci USA 99:9456-9461, 2002; Denti et al. Proc Natl Acad Sci USA 103: 3758-3763, 2006; Denti et al. Hum Gene Ther 17: 565-743, 2006; Denti et al. Hum Gene Ther 19: 601-608, 2008; Incitti et al. Mol Ther 18: 1675-1682, 2010), provided high skipping activity and efficient rescue of dystrophin synthesis. Moreover, the U1-antisense molecules, delivered to mice via systemic injection of recombinant AAV viruses, displayed body wide transduction, long-term expression, dystrophin rescue as well as morphological and functional benefit (Denti et al. Hum Gene Ther 19: 601-608, 2008). In this Chapter we report methods for producing U1-antisense expression cassettes in the backbone of lentiviral constructs and for testing their activity both in patients' derived myoblasts as well as in fibroblasts reprogrammed to muscle differentiation.

  19. Sustained expression from DNA vectors.

    PubMed

    Wong, Suet Ping; Argyros, Orestis; Harbottle, Richard P

    2015-01-01

    DNA vectors have the potential to become powerful medical tools for treatment of human disease. The human body has, however, developed a range of defensive strategies to detect and silence foreign or misplaced DNA, which is more typically encountered during infection or chromosomal damage. A clinically relevant human gene therapy vector must overcome or avoid these protections whilst delivering sustained levels of therapeutic gene product without compromising the vitality of the recipient host. Many non-viral DNA vectors trigger these defense mechanisms and are subsequently destroyed or rendered silent. Thus, without modification or considered design, the clinical utility of a typical DNA vector is fundamentally limited due to the transient nature of its transgene expression. The development of safe and persistently expressing DNA vectors is a crucial prerequisite for its successful clinical application and subsequently remains, therefore, one of the main strategic tasks of non-viral gene therapy research. In this chapter we will describe our current understanding of the mechanisms that can destroy or silence DNA vectors and discuss strategies, which have been utilized to improve their sustenance and the level and duration of their transgene expression.

  20. Induction of CML28-specific cytotoxic T cell responses using co-transfected dendritic cells with CML28 DNA vaccine and SOCS1 small interfering RNA expression vector

    SciTech Connect

    Zhou Hongsheng; Zhang Donghua . E-mail: hanson2008@gmail.com; Wang Yaya; Dai Ming; Zhang Lu; Liu Wenli; Liu Dan; Tan Huo; Huang Zhenqian

    2006-08-18

    CML28 is an attractive target for antigen-specific immunotherapy. SOCS1 represents an inhibitory control mechanism for DC antigen presentation and the magnitude of adaptive immunity. In this study, we evaluated the potential for inducing CML28-specific cytotoxic T lymphocytes (CTL) responses by dendritic cells (DCs)-based vaccination. We constructed a CML28 DNA vaccine and a SOCS1 siRNA vector and then cotransfect monocyte-derived DCs. Flow cytometry analysis showed gene silencing of SOCS1 resulted in higher expressions of costimulative moleculars in DCs. Mixed lymphocyte reaction (MLR) indicated downregulation of SOCS1 stronger capability to stimulate proliferation of responder cell in DCs. The CTL assay revealed transfected DCs effectively induced autologous CML28-specific CTL responses and the lytic activities induced by SOCS1-silenced DCs were significantly higher compared with those induced by SOCS1-expressing DCs. These results in our study indicates gene silencing of SOCS1 remarkably enhanced the cytotoxicity efficiency of CML28 DNA vaccine in DCs.

  1. Antigenic structures stably expressed by recombinant TGEV-derived vectors.

    PubMed

    Becares, Martina; Sanchez, Carlos M; Sola, Isabel; Enjuanes, Luis; Zuñiga, Sonia

    2014-09-01

    Coronaviruses (CoVs) are positive-stranded RNA viruses with potential as immunization vectors, expressing high levels of heterologous genes and eliciting both secretory and systemic immune responses. Nevertheless, its high recombination rate may result in the loss of the full-length foreign gene, limiting their use as vectors. Transmissible gastroenteritis virus (TGEV) was engineered to express porcine reproductive and respiratory syndrome virus (PRRSV) small protein domains, as a strategy to improve heterologous gene stability. After serial passage in tissue cultures, stable expression of small PRRSV protein antigenic domains was achieved. Therefore, size reduction of the heterologous genes inserted in CoV-derived vectors led to the stable expression of antigenic domains. Immunization of piglets with these TGEV vectors led to partial protection against a challenge with a virulent PRRSV strain, as immunized animals showed reduced clinical signs and lung damage. Further improvement of TGEV-derived vectors will require the engineering of vectors with decreased recombination rate.

  2. Development of hybrid baculovirus vectors for artificial MicroRNA delivery and prolonged gene suppression.

    PubMed

    Chen, Chiu-Ling; Luo, Wen-Yi; Lo, Wen-Hsin; Lin, Kun-Ju; Sung, Li-Yu; Shih, Yung-Shen; Chang, Yu-Han; Hu, Yu-Chen

    2011-12-01

    MicroRNA (miRNA) plays essential roles in regulating gene expression, but miRNA delivery remains a hurdle, thus entailing a vector system for efficient transfer. Baculovirus emerges as a promising gene delivery vector but its inherent transient expression restricts its applications in some scenarios. Therefore, this study primarily aimed to develop baculovirus as a miRNA expression vector for prolonged gene suppression. We constructed recombinant baculoviruses carrying artificial egfp-targeting miRNA sequences within the miR155 backbone, which after expression by the cytomegalovirus promoter could knockdown the enhanced green fluorescent protein (EGFP) expression in a sequence- and dose-dependent manner. By swapping the mature miRNA sequences, the baculovirus miRNA shuttle effectively repressed the overexpression of endogenous TNF-α in arthritic synoviocytes without inducing apoptosis. To prolong the baculovirus-mediated expression, we further developed a hybrid baculovirus vector that exploited the Sleeping Beauty (SB) transposon for gene integration and sustained miRNA expression. The hybrid baculovirus vector that combined the miR155 scaffold and SB transposon effectively repressed the transgene expression for a prolonged period of time, hence diversifying the applications of baculovirus to indications necessitating prolonged gene regulation such as arthritis. PMID:21732325

  3. Lentiviral Vector-Mediated RNA Silencing in the Central Nervous System

    PubMed Central

    Foster, Edmund; Moon, Lawrence D.F.

    2014-01-01

    Abstract RNA silencing is an established method for investigating gene function and has attracted particular interest because of the potential for generating RNA-based therapeutics. Using lentiviral vectors as an efficient delivery system that offers stable, long-term expression in postmitotic cells further enhances the applicability of an RNA-based gene therapy for the CNS. In this review we provide an overview of both lentiviral vectors and RNA silencing along with design considerations for generating lentiviral vectors capable of RNA silencing. We go on to describe the current preclinical data regarding lentiviral vector-mediated RNA silencing for CNS disorders and discuss the concerns of side effects associated with lentiviral vectors and small interfering RNAs and how these might be mitigated. PMID:24090197

  4. Inducible and reversible suppression of Npm1 gene expression using stably integrated small interfering RNA vector in mouse embryonic stem cells

    SciTech Connect

    Wang Beibei; Lu Rui; Wang Weicheng; Jin Ying . E-mail: yjin@sibs.ac.cn

    2006-09-08

    The tetracycline (Tc)-inducible small interference RNA (siRNA) is a powerful tool for studying gene function in mammalian cells. However, the system is infrequently utilized in embryonic stem (ES) cells. Here, we present First application of the Tc-inducible, stably integrated plasmid-based siRNA system in mouse ES cells to down-regulate expression of Npm1, an essential gene for embryonic development. The physiological role of Npm1 in ES cells has not been defined. Our data show that the knock-down of Npm1 expression by this siRNA system was not only highly efficient, but also Tc- dose- and induction time-dependent. Particularly, the down-regulation of Npm1 expression was reversible. Importantly, suppression of Npm1 expression in ES cells resulted in reduced cell proliferation. Taken together, this system allows for studying gene function in a highly controlled manner, otherwise difficult to achieve in ES cells. Moreover, our results demonstrate that Npm1 is essential for ES cell proliferation.

  5. Hepatic RNA Interference: Delivery by Synthetic Vectors

    PubMed Central

    Haynes, Matthew; Huang, Leaf

    2013-01-01

    Though the pharmaceutical industry’s infatuation with the therapeutic potential of RNA interference (RNAi) technology has finally come down from its initial lofty levels,[1] hope is by no means lost for the once-burgeoning enterprise, as recent clinical trials are beginning to show efficacy in areas ranging from amyloidosis to hypercholesterolemia to muscular dystrophy. With such resurgence comes a more informed perspective on the needs of such therapeutics: a renewed focus on true RNA drug development, and a desire for enhanced site-specific delivery.[2] In this review, we will discuss the latter with regard to hepatic targeting by synthetic vectors, covering the implications of organ and cellular physiology on conjugate structure, particle morphology, and active targeting. In presenting efficacy in a variety of disease models, we emphasize as well the extraordinary degree to which synthetic formulation improves upon and coordinates efforts with oligonucleotide development. Such advances in the understanding of and the technology behind RNAi have the potential to finally stabilize the long-term prospects RNA therapeutic development. PMID:24678447

  6. [Methods for construction of transgenic plant expression vector: a review].

    PubMed

    Zhang, Yangpu; Yang, Shushen

    2015-03-01

    Construction of recombinant plasmid vector for gene expression is a key step in making transgenic plants and important to study gene function and plant genetic engineering. A right choice of gene construction method can be cost-effective and achieve more diverse recombinant plasmids. In addition to the traditional methods in construction of plant gene expression vectors, such as Gateway technology, three DNA method and one step cloning, a few novel methods have been developed in recent years. These methods include oligonucleotide synthesis-based construction of small fragment gene expression vectors via competitive connection; construction of small RNA expression vector using pre-microRNA; recombination-fusion PCR method which inserts DNA fragments of multiple restriction sites into the target vector; and insertion of a DNA fragment into any region of a linear vector via In-Fusion Kit. Construction of complex vectors with many fragments uses sequence and ligation-independent cloning method, Gibson isothermal assembly or Golden Gate assembly. This paper summarizes our working experience in the area of recombinant vector construction and reports from others with an intention to disseminate ideas about currently widely used DNA recombination methods for plant transformation.

  7. Bacteriophage lambda-based expression vectors.

    PubMed

    Christensen, A C

    2001-03-01

    Bacteriophage lambda has been in use as a cloning vector for over 25 years, and has been used extensively as an expression vector. The efficiency of packaging and infection, and the simplicity of plaque screening are advantages of lambda as a cloning vector. A number of ingenious modifications help overcome the disadvantages associated with its mode of growth and its size. Some lambda vectors have been designed to be readily converted into plasmids or phagemids, and there are a variety of promoters and fusions that can be used to drive expression of foreign genes. Screening lambda libraries with antibodies or ligands is a powerful way of identifying novel genes. PMID:11434310

  8. Antisense RNA suppression of peroxidase gene expression

    SciTech Connect

    Lagrimini, L.M.; Bradford, S.; De Leon, F.D. )

    1989-04-01

    The 5{prime} half the anionic peroxidase cDNA of tobacco was inserted into a CaMV 35S promoter/terminator expression cassette in the antisense configuration. This was inserted into the Agrobacterium-mediated plant transformation vector pCIBIO which includes kanamycin selection, transformed into two species of tobacco (N. tabacum and M. sylvestris), and plants were subsequently regenerated on kanamycin. Transgenic plants were analyzed for peroxidase expression and found to have 3-5 fold lower levels of peroxidase than wild-type plants. Isoelectric focusing demonstrated that the antisense RNA only suppressed the anionic peroxidase. Wound-induced peroxidase expression was found not to be affected by the antisense RNA. Northern blots show a greater than 5 fold suppression of anionic peroxidase mRNA in leaf tissue, and the antisense RNA was expressed at a level 2 fold over the endogenous mRNA. Plants were self-pollinated and F1 plants showed normal segregation. N. sylvestris transgenic plants with the lowest level of peroxidase are epinastic, and preliminary results indicate elevated auxin levels. Excised pith tissue from both species of transgenic plants rapidly collapse when exposed to air, while pith tissue from wild-type plants showed little change when exposed to air. Further characterization of these phenotypes is currently being made.

  9. The role of RNA interference (RNAi) in arbovirus-vector interactions.

    PubMed

    Blair, Carol D; Olson, Ken E

    2015-02-17

    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.

  10. Development of non-defective recombinant densovirus vectors for microRNA delivery in the invasive vector mosquito, Aedes albopictus

    PubMed Central

    Liu, Peiwen; Li, Xiaocong; Gu, Jinbao; Dong, Yunqiao; Liu, Yan; Santhosh, Puthiyakunnon; Chen, Xiaoguang

    2016-01-01

    We previously reported that mosquito densoviruses (MDVs) are potential vectors for delivering foreign nucleic acids into mosquito cells. However, considering existing expression strategies, recombinant viruses would inevitably become replication-defective viruses and lose their ability for secondary transmission. The packaging limitations of the virion represent a barrier for the development of MDVs for viral paratransgenesis or as high-efficiency bioinsecticides. Herein, we report the development of a non-defective recombinant Aedes aegypti densovirus (AaeDV) miRNA expression system, mediated by an artificial intron, using an intronic miRNA expression strategy. We demonstrated that this recombinant vector could be used to overexpress endogenous miRNAs or to decrease endogenous miRNAs by generating antisense sponges to explore the biological functions of miRNAs. In addition, the vector could express antisense-miRNAs to induce efficient gene silencing in vivo and in vitro. The recombinant virus effectively self-replicated and retained its secondary transmission ability, similar to the wild-type virus. The recombinant virus was also genetically stable. This study demonstrated the first construction of a non-defective recombinant MDV miRNA expression system, which represents a tool for the functional analysis of mosquito genes and lays the foundation for the application of viral paratransgenesis for dengue virus control. PMID:26879823

  11. 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

  12. Expression patterns of cotton chloroplast genes during development: implications for development of plastid transformation vectors

    Technology Transfer Automated Retrieval System (TEKTRAN)

    In order to express genes of interest in plastids, transformation vectors must be developed that include appropriate promoters to drive expression at effective levels in both green and non-green tissues. Typically, chloroplasts are transformed with vectors that contain ribosomal RNA promoters for h...

  13. Molecular interactions and immune responses between maize fine streak virus and the leafhopper vector G. nigrifrons through differential expression and RNA interference

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Maize fine streak virus (MFSV) is an emerging virus of maize that is transmitted by an insect vector, the leafhopper called Graminella nigrifrons. Virus transmission by the leafhopper requires that the virus enter into and multiply in insect cells, tissues and organs before being transmitted to a ne...

  14. Small interfering RNA pathway modulates persistent infection of a plant virus in its insect vector

    PubMed Central

    Lan, Hanhong; Wang, Haitao; Chen, Qian; Chen, Hongyan; Jia, Dongsheng; Mao, Qianzhuo; Wei, Taiyun

    2016-01-01

    Plant reoviruses, rhabdoviruses, tospoviruses, and tenuiviruses are transmitted by insect vectors in a persistent-propagative manner. How such persistent infection of plant viruses in insect vectors is established and maintained remains poorly understood. In this study, we used rice gall dwarf virus (RGDV), a plant reovirus, and its main vector leafhopper Recilia dorsalis as a virus–insect system to determine how the small interference (siRNA) pathway modulates persistent infection of a plant virus in its insect vector. We showed that a conserved siRNA antiviral response was triggered by the persistent replication of RGDV in cultured leafhopper cells and in intact insects, by appearance of virus-specific siRNAs, primarily 21-nt long, and the increased expression of siRNA pathway core components Dicer-2 and Argonaute-2. Silencing of Dicer-2 using RNA interference strongly suppressed production of virus-specific siRNAs, promoted viral accumulation, and caused cytopathological changes in vitro and in vivo. When the viral accumulation level rose above a certain threshold of viral genome copy (1.32 × 1014 copies/μg insect RNA), the infection of the leafhopper by RGDV was lethal rather than persistent. Taken together, our results revealed a new finding that the siRNA pathway in insect vector can modulate persistent infection of plant viruses. PMID:26864546

  15. Small interfering RNA pathway modulates persistent infection of a plant virus in its insect vector.

    PubMed

    Lan, Hanhong; Wang, Haitao; Chen, Qian; Chen, Hongyan; Jia, Dongsheng; Mao, Qianzhuo; Wei, Taiyun

    2016-01-01

    Plant reoviruses, rhabdoviruses, tospoviruses, and tenuiviruses are transmitted by insect vectors in a persistent-propagative manner. How such persistent infection of plant viruses in insect vectors is established and maintained remains poorly understood. In this study, we used rice gall dwarf virus (RGDV), a plant reovirus, and its main vector leafhopper Recilia dorsalis as a virus-insect system to determine how the small interference (siRNA) pathway modulates persistent infection of a plant virus in its insect vector. We showed that a conserved siRNA antiviral response was triggered by the persistent replication of RGDV in cultured leafhopper cells and in intact insects, by appearance of virus-specific siRNAs, primarily 21-nt long, and the increased expression of siRNA pathway core components Dicer-2 and Argonaute-2. Silencing of Dicer-2 using RNA interference strongly suppressed production of virus-specific siRNAs, promoted viral accumulation, and caused cytopathological changes in vitro and in vivo. When the viral accumulation level rose above a certain threshold of viral genome copy (1.32 × 10(14) copies/μg insect RNA), the infection of the leafhopper by RGDV was lethal rather than persistent. Taken together, our results revealed a new finding that the siRNA pathway in insect vector can modulate persistent infection of plant viruses. PMID:26864546

  16. Inducing RNA interference in the arbovirus vector, Culicoides sonorensis.

    PubMed

    Mills, M K; Nayduch, D; Michel, K

    2015-02-01

    Biting midges in the genus Culicoides are important vectors of arboviral diseases, including epizootic haemorrhagic disease, bluetongue and most likely Schmallenberg, which cause significant economic burdens worldwide. Research on these vectors has been hindered by the lack of a sequenced genome, the difficulty of consistent culturing of certain species and the absence of molecular techniques such as RNA interference (RNAi). Here, we report the establishment of RNAi as a research tool for the adult midge, Culicoides sonorensis. Based on previous research and transcriptome analysis, which revealed putative small interfering RNA pathway member orthologues, we hypothesized that adult C. sonorensis midges have the molecular machinery needed to perform RNA silencing. Injection of control double-stranded RNA targeting green fluorescent protein (dsGFP), into the haemocoel of 2-3-day-old adult female midges resulted in survival curves that support virus transmission. dsRNA injection targeting the newly identified C. sonorensis inhibitor of apoptosis protein 1 (CsIAP1) orthologue resulted in a 40% decrease of transcript levels and 73% shorter median survivals as compared with dsGFP-injected controls. These results reveal the conserved function of IAP1. Importantly, they also demonstrate the feasibility of RNAi by dsRNA injection in adult midges, which will greatly facilitate studies of the underlying mechanisms of vector competence in C. sonorensis.

  17. A novel intranuclear RNA vector system for long-term stem cell modification.

    PubMed

    Ikeda, Y; Makino, A; Matchett, W E; Holditch, S J; Lu, B; Dietz, A B; Tomonaga, K

    2016-03-01

    Genetically modified stem and progenitor cells have emerged as a promising regenerative platform in the treatment of genetic and degenerative disorders, highlighted by their successful therapeutic use in inherent immunodeficiencies. However, biosafety concerns over insertional mutagenesis resulting from integrating recombinant viral vectors have overshadowed the widespread clinical applications of genetically modified stem cells. Here, we report an RNA-based episomal vector system, amenable for long-term transgene expression in stem cells. Specifically, we used a unique intranuclear RNA virus, borna disease virus (BDV), as the gene transfer vehicle, capable of persistent infections in various cell types. BDV-based vectors allowed for long-term transgene expression in mesenchymal stem cells (MSCs) without affecting cellular morphology, cell surface CD105 expression or the adipogenicity of MSCs. Similarly, replication-defective BDV vectors achieved long-term transduction of human induced pluripotent stem cells, while maintaining the ability to differentiate into three embryonic germ layers. Thus, the BDV-based vectors offer a genomic modification-free, episomal RNA delivery system for sustained stem cell transduction. PMID:26632671

  18. A novel intranuclear RNA vector system for long-term stem cell modification

    PubMed Central

    Ikeda, Yasuhiro; Makino, Akiko; Matchett, William E.; Holditch, Sara J.; Lu, Brian; Dietz, Allan B.; Tomonaga, Keizo

    2015-01-01

    Genetically modified stem and progenitor cells have emerged as a promising regenerative platform in the treatment of genetic and degenerative disorders, highlighted by their successful therapeutic use in inherent immunodeficiencies. However, biosafety concerns over insertional mutagenesis resulting from integrating recombinant viral vectors have overshadowed the widespread clinical applications of genetically modified stem cells. Here, we report an RNA-based episomal vector system, amenable for long-term transgene expression in stem cells. Specifically, we used a unique intranuclear RNA virus, Borna disease virus (BDV), as the gene transfer vehicle, capable of persistent infections in various cell types. BDV-based vectors allowed for long-term transgene expression in mesenchymal stem cells (MSCs) without affecting cellular morphology, cell surface CD105 expression, or the adipogenicity of MSCs. Similarly, replication-defective BDV vectors achieved long-term transduction of human induced pluripotent stem cells (iPSCs), while maintaining the ability to differentiate into three embryonic germ layers. Thus, the BDV-based vectors offer a genomic modification-free, episomal RNA delivery system for sustained stem cell transduction. PMID:26632671

  19. Novel Recombinant Hepatitis B Virus Vectors Efficiently Deliver Protein and RNA Encoding Genes into Primary Hepatocytes

    PubMed Central

    Hong, Ran; Bai, Weiya; Zhai, Jianwei; Liu, Wei; Li, Xinyan; Zhang, Jiming; Cui, Xiaoxian; Zhao, Xue; Ye, Xiaoli; Deng, Qiang; Tiollais, Pierre; Wen, Yumei

    2013-01-01

    Hepatitis B virus (HBV) has extremely restricted host and hepatocyte tropism. HBV-based vectors could form the basis of novel therapies for chronic hepatitis B and other liver diseases and would also be invaluable for the study of HBV infection. Previous attempts at developing HBV-based vectors encountered low yields of recombinant viruses and/or lack of sufficient infectivity/cargo gene expression in primary hepatocytes, which hampered follow-up applications. In this work, we constructed a novel vector based on a naturally occurring, highly replicative HBV mutant with a 207-bp deletion in the preS1/polymerase spacer region. By applying a novel insertion strategy that preserves the continuity of the polymerase open reading frame (ORF), recombinant HBV (rHBV) carrying protein or small interfering RNA (siRNA) genes were obtained that replicated and were packaged efficiently in cultured hepatocytes. We demonstrated that rHBV expressing a fluorescent reporter (DsRed) is highly infective in primary tree shrew hepatocytes, and rHBV expressing HBV-targeting siRNA successfully inhibited antigen expression from coinfected wild-type HBV. This novel HBV vector will be a powerful tool for hepatocyte-targeting gene delivery, as well as the study of HBV infection. PMID:23552416

  20. Inhibition of simian immunodeficiency virus by foamy virus vectors expressing siRNAs

    SciTech Connect

    Park, Jeonghae; Nadeau, Peter; Zucali, James R.; Johnson, Calvin M.; Mergia, Ayalew . E-mail: mergiaa@mail.vetmed.ufl.edu

    2005-12-20

    Viral vectors available for gene therapy are either inefficient or suffer from safety concerns for human applications. Foamy viruses are non-pathogenic retroviruses that offer several unique opportunities for gene transfer in various cell types from different species. In this report, we describe the use of simian foamy virus type 1 (SFV-1) vector to examine the efficacy of therapeutic genes. Hairpin short-interfering RNA (siRNA) that targets the simian immunodeficiency virus (SIV) rev/env was placed under the control of the PolIII U6 snRNA promoter for expression and screened for silencing target genes using cognate target-reporter fusions. We have identified an effective siRNA (designated R2) which reduces the rev and env gene expression by 89% and 95%, respectively. Using the simian foamy virus type 1 (SFV-1) based vector, we delivered the PolIII expressed R2 siRNA into cultured cells and challenged with SIV. The results show that the R2 siRNA is a potent inhibitor of SIV replication as determined by p27 expression and reverse transcriptase assays. Vectors based on a non-pathogenic SFV-1 vector may provide a safe and efficient alternative to currently available vectors, and the SIV model will help devise protocols for effective anti-HIV gene therapy.

  1. Transfer and Expression of Small Interfering RNAs in Mammalian Cells Using Lentiviral Vectors.

    PubMed

    Lebedev, T D; Spirin, P V; Prassolov, V S

    2013-04-01

    RNA interference is a convenient tool for modulating gene expression. The widespread application of RNA interference is made difficult because of the imperfections of the methods used for efficient target cell delivery of whatever genes are under study. One of the most convenient and efficient gene transfer and expression systems is based on the use of lentiviral vectors, which direct the synthesis of small hairpin RNAs (shRNAs), the precursors of siRNAs. The application of these systems enables one to achieve sustainable and long-term shRNA expression in cells. This review considers the adaptation of the processing of artificial shRNA to the mechanisms used by cellular microRNAs and simultaneous expression of several shRNAs as potential approaches for producing lentiviral vectors that direct shRNA synthesis. Approaches to using RNA interference for the treatment of cancer, as well as hereditary and viral diseases, are under active development today. The improvement made to the methods for constructing lentiviral vectors and the investigation into the mechanisms of processing of small interfering RNA allow one to now consider lentiviral vectors that direct shRNA synthesis as one of the most promising tools for delivering small interfering RNAs.

  2. Transfer and Expression of Small Interfering RNAs in Mammalian Cells Using Lentiviral Vectors

    PubMed Central

    Lebedev, T. D.; Spirin, P. V.; Prassolov, V. S.

    2013-01-01

    RNA interference is a convenient tool for modulating gene expression. The widespread application of RNA interference is made difficult because of the imperfections of the methods used for efficient target cell delivery of whatever genes are under study. One of the most convenient and efficient gene transfer and expression systems is based on the use of lentiviral vectors, which direct the synthesis of small hairpin RNAs (shRNAs), the precursors of siRNAs. The application of these systems enables one to achieve sustainable and long-term shRNA expression in cells. This review considers the adaptation of the processing of artificial shRNA to the mechanisms used by cellular microRNAs and simultaneous expression of several shRNAs as potential approaches for producing lentiviral vectors that direct shRNA synthesis. Approaches to using RNA interference for the treatment of cancer, as well as hereditary and viral diseases, are under active development today. The improvement made to the methods for constructing lentiviral vectors and the investigation into the mechanisms of processing of small interfering RNA allow one to now consider lentiviral vectors that direct shRNA synthesis as one of the most promising tools for delivering small interfering RNAs. PMID:23819033

  3. Vector insert-targeted integrative antisense expression system for plasmid stabilization.

    PubMed

    Luke, Jeremy M; Carnes, Aaron E; Hodgson, Clague P; Williams, James A

    2011-01-01

    Some DNA vaccine and gene therapy vector-encoded transgenes are toxic to the E. coli plasmid production host resulting in poor production yields. For plasmid products undergoing clinical evaluation, sequence modification to eliminate toxicity is undesirable because an altered vector is a new chemical entity. We hypothesized that: (1) insert-encoded toxicity is mediated by unintended expression of a toxic insert-encoded protein from spurious bacterial promoters; and (2) that toxicity could be eliminated with antisense RNA-mediated translation inhibition. We developed the pINT PR PL vector, a chromosomally integrable RNA expression vector, and utilized it to express insert-complementary (anti-insert) RNA from a single defined site in the bacterial chromosome. Anti-insert RNA eliminated leaky fluorescent protein expression from a target plasmid. A toxic retroviral gag pol helper plasmid produced in a gag pol anti-insert strain had fourfold improved plasmid fermentation yields. Plasmid fermentation yields were also fourfold improved when a DNA vaccine plasmid containing a toxic Influenza serotype H1 hemagglutinin transgene was grown in an H1 sense strand anti-insert production strain, suggesting that in this case toxicity was mediated by an antisense alternative reading frame-encoded peptide. This anti-insert chromosomal RNA expression technology is a general approach to improve production yields with plasmid-based vectors that encode toxic transgenes, or toxic alternative frame peptides. PMID:20607625

  4. MicroRNA expression profiling using microarrays.

    PubMed

    Love, Cassandra; Dave, Sandeep

    2013-01-01

    MicroRNAs are small noncoding RNAs which are able to regulate gene expression at both the transcriptional and translational levels. There is a growing recognition of the role of microRNAs in nearly every tissue type and cellular process. Thus there is an increasing need for accurate quantitation of microRNA expression in a variety of tissues. Microarrays provide a robust method for the examination of microRNA expression. In this chapter, we describe detailed methods for the use of microarrays to measure microRNA expression and discuss methods for the analysis of microRNA expression data. PMID:23666707

  5. Mitochondrial RNA granules: Compartmentalizing mitochondrial gene expression

    PubMed Central

    Jourdain, Alexis A.; Boehm, Erik; Maundrell, Kinsey

    2016-01-01

    In mitochondria, DNA replication, gene expression, and RNA degradation machineries coexist within a common nondelimited space, raising the question of how functional compartmentalization of gene expression is achieved. Here, we discuss the recently characterized “mitochondrial RNA granules,” mitochondrial subdomains with an emerging role in the regulation of gene expression. PMID:26953349

  6. [Construction and tobacco transformation of COR and BBE genes hairpin RNA vector of Papaver somniferum].

    PubMed

    Lu, Yan-Mei; Zhang, Jin-Wen; Wei, Yu-Jie; Wei, Gui-Min; Zhang, Yan-Hong; Gao, Yi-Feng

    2013-07-01

    The gene expressions of codeinone reductase (COR) and berberine bridge enzyme (BBE) in Papaver somniferum were blocked by RNA hairpin of RNA interference (RNAi). The complete sequences of COR and BBE genes were cloned by reverse transcription-polymerase chain reaction (RT-PCR), the results of homology comparison revealed that the cloned COR and BBE genes had high homology with the other gene family members reported in the GenBank. The target sequences of COR and BBE genes were screened in accordance with the design principle of RNAi, a 643 bp fusion gene was obtained by the method of overlapping PCR, then plant expression vector ihpRNA was constructed based on intermediate vector pHANNIBAL and plant expression vector pCEPSPS. With that 78 transgenic plants were obtained through Agrobacterium-mediated and 17 positive plants were screened by PCR, that could initially indicate that the target fragments of COR and BBE gene had been integrated into tobacco genome.

  7. Virus-Derived Vectors for the Expression of Multiple Proteins in Plants.

    PubMed

    Saxena, Pooja; Thuenemann, Eva C; Sainsbury, Frank; Lomonossoff, George P

    2016-01-01

    This chapter constitutes a practical guide to using the "pEAQ" vector series for transient or stable expression of one or more protein(s) in Nicotiana benthamiana plants. The pEAQ vectors are a series of small binary vectors designed for controlled expression of multiple proteins in plants. To achieve high levels of expression, an expression system based on translational enhancement by the untranslated regions of RNA-2 from cowpea mosaic virus (CPMV), named CPMV-HT, is used. The expression vector pEAQ-HT combines the user-friendly pEAQ plasmid with CPMV-HT to provide a system for high-level expression of proteins in plants. PMID:26614280

  8. Packaging of HCV-RNA into lentiviral vector

    SciTech Connect

    Caval, Vincent; Piver, Eric; Ivanyi-Nagy, Roland; Darlix, Jean-Luc; Pages, Jean-Christophe

    2011-11-04

    Highlights: Black-Right-Pointing-Pointer Description of HCV-RNA Core-D1 interactions. Black-Right-Pointing-Pointer In vivo evaluation of the packaging of HCV genome. Black-Right-Pointing-Pointer Determination of the role of the three basic sub-domains of D1. Black-Right-Pointing-Pointer Heterologous system involving HIV-1 vector particles to mobilise HCV genome. Black-Right-Pointing-Pointer Full length mobilisation of HCV genome and HCV-receptor-independent entry. -- Abstract: The advent of infectious molecular clones of Hepatitis C virus (HCV) has unlocked the understanding of HCV life cycle. However, packaging of the genomic RNA, which is crucial to generate infectious viral particles, remains poorly understood. Molecular interactions of the domain 1 (D1) of HCV Core protein and HCV RNA have been described in vitro. Since compaction of genetic information within HCV genome has hampered conventional mutational approach to study packaging in vivo, we developed a novel heterologous system to evaluate the interactions between HCV RNA and Core D1. For this, we took advantage of the recruitment of Vpr fusion-proteins into HIV-1 particles. By fusing HCV Core D1 to Vpr we were able to package and transfer a HCV subgenomic replicon into a HIV-1 based lentiviral vector. We next examined how deletion mutants of basic sub-domains of Core D1 influenced HCV RNA recruitment. The results emphasized the crucial role of the first and third basic regions of D1 in packaging. Interestingly, the system described here allowed us to mobilise full-length JFH1 genome in CD81 defective cells, which are normally refractory to HCV infection. This finding paves the way to an evaluation of the replication capability of HCV in various cell types.

  9. siRNA Therapy, Challenges and Underlying Perspectives of Dendrimer as Delivery Vector.

    PubMed

    Tekade, Rakesh Kumar; Maheshwari, Rahul G S; Sharma, Piyoosh A; Tekade, Muktika; Chauhan, Abhay Singh

    2015-01-01

    siRNA technology presents a helpful means of gene silencing in mammalian cells. Advancement in the field includes enhanced attentiveness in the characterization of target and off-target effects employing suitable controls and gene expression microarrays. These will permit expansion in the measurement of single and multiple target combinations and also permit comprehensive efforts to understand mammalian cell processes. Another fact is that the delivery of siRNA requires the creation of a nanoparticulate vector with controlled structural geometry and surface modalities inside the targeted cells. On the other hand, dendrimers represent the class of carrier system where massive control over size, shape and physicochemical properties makes this delivery vector exceptional and favorable in genetic transfection applications. The siRNA therapeutics may be incorporated inside the geometry of the density controlled dendrimers with the option of engineering the structure to the specific needs of the genetic material and its indication. The existing reports on the siRNA carrying and deliverance potential of dendrimers clearly suggest the significance of this novel class of polymeric architecture and certainly elevate the futuristic use of this highly branched vector as genetic material delivery system.

  10. Double-stranded RNA transcribed from vector-based oligodeoxynucleotide acts as transcription factor decoy

    SciTech Connect

    Xiao, Xiao; Gang, Yi; Wang, Honghong; Wang, Jiayin; Zhao, Lina; Xu, Li; Liu, Zhiguo

    2015-02-06

    Highlights: • A shRNA vector based transcription factor decoy, VB-ODN, was designed. • VB-ODN for NF-κB inhibited cell viability in HEK293 cells. • VB-ODN inhibited expression of downstream genes of target transcription factors. • VB-ODN may enhance nuclear entry ratio for its feasibility of virus production. - Abstract: In this study, we designed a short hairpin RNA vector-based oligodeoxynucleotide (VB-ODN) carrying transcription factor (TF) consensus sequence which could function as a decoy to block TF activity. Specifically, VB-ODN for Nuclear factor-κB (NF-κB) could inhibit cell viability and decrease downstream gene expression in HEK293 cells without affecting expression of NF-κB itself. The specific binding between VB-ODN produced double-stranded RNA and NF-κB was evidenced by electrophoretic mobility shift assay. Moreover, similar VB-ODNs designed for three other TFs also inhibit their downstream gene expression but not that of themselves. Our study provides a new design of decoy for blocking TF activity.

  11. Selective silencing of gene target expression by siRNA expression plasmids in human cervical cancer cells.

    PubMed

    Peralta-Zaragoza, Oscar; De-la-O-Gómez, Faustino; Deas, Jessica; Fernández-Tilapa, Gloria; Fierros-Zárate, Geny Del Socorro; Gómez-Cerón, Claudia; Burguete-García, Ana; Torres-Poveda, Kirvis; Bermúdez-Morales, Victor Hugo; Rodríguez-Dorantes, Mauricio; Pérez-Plasencia, Carlos; Madrid-Marina, Vicente

    2015-01-01

    RNA interference is a natural mechanism to silence post-transcriptional gene expression in eukaryotic cells in which microRNAs act to cleave or halt the translation of target mRNAs at specific target sequences. Mature microRNAs, 19-25 nucleotides in length, mediate their effect at the mRNA level by inhibiting translation, or inducing cleavage of the mRNA target. This process is directed by the degree of complementary nucleotides between the microRNAs and the target mRNA; perfect complementary base pairing induces cleavage of mRNA, whereas several mismatches lead to translational arrest. Biological effects of microRNAs can be manipulated through the use of small interference RNAs (siRNAs) generated by chemical synthesis, or by cloning in molecular vectors. The cloning of a DNA insert in a molecular vector that will be transcribed into the corresponding siRNAs is an approach that has been developed using siRNA expression plasmids. These vectors contain DNA inserts designed with software to generate highly efficient siRNAs which will assemble into RNA-induced silencing complexes (RISC), and silence the target mRNA. In addition, the DNA inserts may be contained in cloning cassettes, and introduced in other molecular vectors. In this chapter we describe an attractive technology platform to silence cellular gene expression using specific siRNA expression plasmids, and evaluate its biological effect on target gene expression in human cervical cancer cells. PMID:25348304

  12. A single-plasmid vector for transgene amplification using short hairpin RNA targeting the 3'-UTR of amplifiable dhfr.

    PubMed

    Kang, Shin-Young; Kim, Yeon-Gu; Lee, Hong Weon; Lee, Eun Gyo

    2015-12-01

    Gene amplification using dihydrofolate reductase gene (dhfr) and methotrexate (MTX) is widely used for recombinant protein production in mammalian cells and is typically conducted in DHFR-deficient Chinese hamster ovary (CHO) cell lines. Generation of DHFR-deficient cells can be achieved by an expression vector incorporating short hairpin RNA (shRNA) that targets the 3'-untranslated region (UTR) of endogenous dhfr. Thus, shRNAs were designed to target the 3'-UTR of endogenous dhfr, and shRNA-2 efficiently down-regulated dhfr expression in CHO-K1 cells. A single gene copy of shRNA-2 also decreased the translational level of DHFR by 80% in Flp-In CHO cells. shRNA-2 was then incorporated into a plasmid vector expressing human erythropoietin (EPO) and an exogenous DHFR to develop EPO-producing cells in the Flp-In system. The specific EPO productivity (q EPO) was enhanced by stepwise increments of MTX concentration, and differences in the amplification rate were observed in Flp-In CHO cells that expressed shRNA-2. In addition, the q EPO increased by more than 2.5-fold in the presence of 500 nM MTX. The mRNA expression level and gene copy numbers of dhfr were correlated with increased productivity in the cells, which is influenced by inhibition of endogenous dhfr. This study reveals that an expression vector including shRNA that targets the 3'-UTR of endogenous dhfr can enhance the transgene amplification rate and productivity by generating DHFR-deficient cells. This approach may be applied for amplifying the foreign gene in wild-type cell lines as a versatile single-plasmid vector.

  13. A single-plasmid vector for transgene amplification using short hairpin RNA targeting the 3'-UTR of amplifiable dhfr.

    PubMed

    Kang, Shin-Young; Kim, Yeon-Gu; Lee, Hong Weon; Lee, Eun Gyo

    2015-12-01

    Gene amplification using dihydrofolate reductase gene (dhfr) and methotrexate (MTX) is widely used for recombinant protein production in mammalian cells and is typically conducted in DHFR-deficient Chinese hamster ovary (CHO) cell lines. Generation of DHFR-deficient cells can be achieved by an expression vector incorporating short hairpin RNA (shRNA) that targets the 3'-untranslated region (UTR) of endogenous dhfr. Thus, shRNAs were designed to target the 3'-UTR of endogenous dhfr, and shRNA-2 efficiently down-regulated dhfr expression in CHO-K1 cells. A single gene copy of shRNA-2 also decreased the translational level of DHFR by 80% in Flp-In CHO cells. shRNA-2 was then incorporated into a plasmid vector expressing human erythropoietin (EPO) and an exogenous DHFR to develop EPO-producing cells in the Flp-In system. The specific EPO productivity (q EPO) was enhanced by stepwise increments of MTX concentration, and differences in the amplification rate were observed in Flp-In CHO cells that expressed shRNA-2. In addition, the q EPO increased by more than 2.5-fold in the presence of 500 nM MTX. The mRNA expression level and gene copy numbers of dhfr were correlated with increased productivity in the cells, which is influenced by inhibition of endogenous dhfr. This study reveals that an expression vector including shRNA that targets the 3'-UTR of endogenous dhfr can enhance the transgene amplification rate and productivity by generating DHFR-deficient cells. This approach may be applied for amplifying the foreign gene in wild-type cell lines as a versatile single-plasmid vector. PMID:26245680

  14. Double-stranded RNA transcribed from vector-based oligodeoxynucleotide acts as transcription factor decoy.

    PubMed

    Xiao, Xiao; Gang, Yi; Wang, Honghong; Wang, Jiayin; Zhao, Lina; Xu, Li; Liu, Zhiguo

    2015-02-01

    In this study, we designed a short hairpin RNA vector-based oligodeoxynucleotide (VB-ODN) carrying transcription factor (TF) consensus sequence which could function as a decoy to block TF activity. Specifically, VB-ODN for Nuclear factor-κB (NF-κB) could inhibit cell viability and decrease downstream gene expression in HEK293 cells without affecting expression of NF-κB itself. The specific binding between VB-ODN produced double-stranded RNA and NF-κB was evidenced by electrophoretic mobility shift assay. Moreover, similar VB-ODNs designed for three other TFs also inhibit their downstream gene expression but not that of themselves. Our study provides a new design of decoy for blocking TF activity.

  15. Determinants of effective lentivirus-driven microRNA expression in vivo

    PubMed Central

    Mishima, Takuya; Sadovsky, Elena; Gegick, Margaret E.; Sadovsky, Yoel

    2016-01-01

    Manipulation of microRNA (miRNA) levels, including overexpression of mature species, has become an important biological tool, even motivating miRNA-based therapeutics. To assess key determinants of miRNA overexpression in a mammalian system in vivo, we sought to bypass the laborious generation of a transgenic animal by exploiting placental trophoblast-specific gene manipulation using lentiviral vectors, which has been instrumental in elucidating trophoblast biology. We examined the impact of several key components of miRNA stem loops and their flanking sequences on the efficiency of mature miRNA expression in vivo. By combining established and novel approaches for miRNA expression, we engineered lentivirus-driven miRNA expression plasmids, which we tested in the mouse placenta. We found that reverse sense inserts minimized single-strand splicing and degradation, and that maintaining longer, poly-A-containing arms flanking the miRNA stem-loop markedly enhanced transgenic miRNA expression. Additionally, we accomplished overexpression of diverse mammalian, drosophila, or C. elegans miRNAs, either based on native context or using a “cassette” replacement of the mature miRNA sequence. Together, we have identified primary miRNA sequences that are paramount for effective expression of mature miRNAs, and validated their role in mice. Principles established by our findings may guide the design of efficient miRNA vectors for in vivo use. PMID:27627961

  16. Determinants of effective lentivirus-driven microRNA expression in vivo.

    PubMed

    Mishima, Takuya; Sadovsky, Elena; Gegick, Margaret E; Sadovsky, Yoel

    2016-01-01

    Manipulation of microRNA (miRNA) levels, including overexpression of mature species, has become an important biological tool, even motivating miRNA-based therapeutics. To assess key determinants of miRNA overexpression in a mammalian system in vivo, we sought to bypass the laborious generation of a transgenic animal by exploiting placental trophoblast-specific gene manipulation using lentiviral vectors, which has been instrumental in elucidating trophoblast biology. We examined the impact of several key components of miRNA stem loops and their flanking sequences on the efficiency of mature miRNA expression in vivo. By combining established and novel approaches for miRNA expression, we engineered lentivirus-driven miRNA expression plasmids, which we tested in the mouse placenta. We found that reverse sense inserts minimized single-strand splicing and degradation, and that maintaining longer, poly-A-containing arms flanking the miRNA stem-loop markedly enhanced transgenic miRNA expression. Additionally, we accomplished overexpression of diverse mammalian, drosophila, or C. elegans miRNAs, either based on native context or using a "cassette" replacement of the mature miRNA sequence. Together, we have identified primary miRNA sequences that are paramount for effective expression of mature miRNAs, and validated their role in mice. Principles established by our findings may guide the design of efficient miRNA vectors for in vivo use. PMID:27627961

  17. Gene expression: RNA interference in adult mice

    NASA Astrophysics Data System (ADS)

    McCaffrey, Anton P.; Meuse, Leonard; Pham, Thu-Thao T.; Conklin, Douglas S.; Hannon, Gregory J.; Kay, Mark A.

    2002-07-01

    RNA interference is an evolutionarily conserved surveillance mechanism that responds to double-stranded RNA by sequence-specific silencing of homologous genes. Here we show that transgene expression can be suppressed in adult mice by synthetic small interfering RNAs and by small-hairpin RNAs transcribed in vivo from DNA templates. We also show the therapeutic potential of this technique by demonstrating effective targeting of a sequence from hepatitis C virus by RNA interference in vivo.

  18. Transient gene expression mediated by integrase-defective retroviral vectors.

    PubMed

    Yu, Seung Shin; Dan, Kazuyuki; Chono, Hideto; Chatani, Emi; Mineno, Junichi; Kato, Ikunoshin

    2008-04-18

    Nonintegrating retroviral vectors were produced from a Moloney murine leukemia virus (MoMLV)-based retroviral vector system by introducing a point mutation into the integrase (IN) gene of the packaging plasmid. The efficacy of IN-defective retroviral vectors was measured through the transient expression of ZsGreen or luciferase in human cell lines. The IN-defective retroviral vectors could transduce target cells efficiently, but their gene expression was transient and lower than that seen with the integrating vectors. IN-defective retroviral vector gene expression decreased to background levels in fewer than 10 days. Southern blot analysis of transduced K562 cells confirmed the loss of a detectable vector sequence by 15 days. The residual integration activity of the IN-defective vector was 1000- to 10,000-fold lower than that of the integrating vector. These results demonstrate that the IN-defective retroviral vectors can provide a useful tool for efficient transient gene expression targeting of primary hematopoietic stem cells and lymphoid cells.

  19. Silencing of Inducible Immunoproteasome Subunit Expression by Chemically Modified siRNA and shRNA.

    PubMed

    Gvozdeva, Olga V; Prassolov, Vladimir S; Zenkova, Marina A; Vlassov, Valentin V; Chernolovskaya, Elena L

    2016-08-01

    Overexpression of inducible subunits of immunoproteasome is related to pathogenesis of some chronic diseases. Specific inhibition of the immunosubunits may be used for the treatment of these diseases and RNA interference is one of the potent methods used in this area. We designed 2'-O-methyl modified siRNAs with selectively protected nuclease-sensitive sites, which efficiently silence LMP2, LMP7, and MECL-1 genes expression. To provide stable long-lasting inhibition of target genes, short-hairpin RNAs (shRNA) expressed by lentiviral vectors were constructed. Our results demonstrated that chemically modified siRNAs inhibited the expression of target genes with similar efficiency or with efficiency exceeding that of corresponding shRNAs and provide silencing effect for 5 days.

  20. Development of a new noncytopathic Semliki Forest virus vector providing high expression levels and stability

    SciTech Connect

    Casales, Erkuden; Rodriguez-Madoz, Juan R.; Ruiz-Guillen, Marta; Razquin, Nerea; Cuevas, Yolanda; Prieto, Jesus; Smerdou, Cristian

    2008-06-20

    Alphavirus vectors express high levels of recombinant proteins in mammalian cells, but their cytopathic nature makes this expression transient. In order to generate a Semliki Forest virus (SFV) noncytopathic vector we introduced mutations previously described to turn Sindbis virus noncytopathic into a conserved position in an SFV vector expressing LacZ. Interestingly, mutant P718T in replicase nsp2 subunit was able to replicate in only a small percentage of BHK cells, producing {beta}-gal-expressing colonies without selection. Puromycin N-acetyl-transferase (pac) gene was used to replace LacZ in this mutant allowing selection of an SFV noncytopathic replicon containing a second mutation in nsp2 nuclear localization signal (R649H). This latter mutation did not confer a noncytopathic phenotype by itself and did not alter nsp2 nuclear translocation. Replicase synthesis was diminished in the SFV double mutant, leading to genomic and subgenomic RNA levels that were 125-fold and 66-fold lower than in wild-type vector, respectively. Interestingly, this mutant expressed {beta}-gal levels similar to parental vector. By coexpressing pac and LacZ from independent subgenomic promoters this vector was able to generate stable cell lines maintaining high expression levels during at least 10 passages, indicating that it could be used as a powerful system for protein production in mammalian cells.

  1. Development of a new noncytopathic Semliki Forest virus vector providing high expression levels and stability.

    PubMed

    Casales, Erkuden; Rodriguez-Madoz, Juan R; Ruiz-Guillen, Marta; Razquin, Nerea; Cuevas, Yolanda; Prieto, Jesus; Smerdou, Cristian

    2008-06-20

    Alphavirus vectors express high levels of recombinant proteins in mammalian cells, but their cytopathic nature makes this expression transient. In order to generate a Semliki Forest virus (SFV) noncytopathic vector we introduced mutations previously described to turn Sindbis virus noncytopathic into a conserved position in an SFV vector expressing LacZ. Interestingly, mutant P718T in replicase nsp2 subunit was able to replicate in only a small percentage of BHK cells, producing beta-gal-expressing colonies without selection. Puromycin N-acetyl-transferase (pac) gene was used to replace LacZ in this mutant allowing selection of an SFV noncytopathic replicon containing a second mutation in nsp2 nuclear localization signal (R649H). This latter mutation did not confer a noncytopathic phenotype by itself and did not alter nsp2 nuclear translocation. Replicase synthesis was diminished in the SFV double mutant, leading to genomic and subgenomic RNA levels that were 125-fold and 66-fold lower than in wild-type vector, respectively. Interestingly, this mutant expressed beta-gal levels similar to parental vector. By coexpressing pac and LacZ from independent subgenomic promoters this vector was able to generate stable cell lines maintaining high expression levels during at least 10 passages, indicating that it could be used as a powerful system for protein production in mammalian cells.

  2. Hygromycin-resistance vectors for gene expression in Pichia pastoris.

    PubMed

    Yang, Junjie; Nie, Lei; Chen, Biao; Liu, Yingmiao; Kong, Yimeng; Wang, Haibin; Diao, Liuyang

    2014-04-01

    Pichia pastoris is a common host organism for heterologous protein expression and metabolic engineering. Zeocin-, G418-, nourseothricin- and blasticidin-resistance genes are the only dominant selectable markers currently available for selecting P. pastoris transformants. We describe here new P. pastoris expression vectors that confer a hygromycin resistance base on the Klebsiella pneumoniae hph gene. To demonstrate the application of the vectors for intracellular and secreted protein expression, green fluorescent protein (GFP) and human serum albumin (HSA) were cloned into the vectors and transformed into P. pastoris cells. The resulting strains expressed GFP and HSA constitutively or inducibly. The hygromycin resistance marker was also suitable for post-transformational vector amplication (PTVA) for obtaining strains with high plasmid copy numbers. A strain with multiple copies of the HSA expression cassette after PTVA had increased HSA expression compared with a strain with a single copy of the plasmid. To demonstrate compatibility of the new vectors with other vectors bearing antibiotic-resistance genes, P. pastoris was transformed with the Saccharomyces cerevisiae genes GSH1, GSH2 or SAM2 on plasmids containing genes for resistance to Zeocin, G418 or hygromycin. The resulting strain produced glutathione and S-adenosyl-L-methionine at levels approximately twice those of the parent strain. The new hygromycin-resistance vectors allow greater flexibility and potential applications in recombinant protein production and other research using P. pastoris. PMID:24822243

  3. Viral vectors: from virology to transgene expression

    PubMed Central

    Bouard, D; Alazard-Dany, N; Cosset, F-L

    2009-01-01

    In the late 1970s, it was predicted that gene therapy would be applied to humans within a decade. However, despite some success, gene therapy has still not become a routine practise in medicine. In this review, we will examine the problems, both experimental and clinical, associated with the use of viral material for transgenic insertion. We shall also discuss the development of viral vectors involving the most important vector types derived from retroviruses, adenoviruses, herpes simplex viruses and adeno-associated viruses. This article is part of a themed section on Vector Design and Drug Delivery. For a list of all articles in this section see the end of this paper, or visit: http://www3.interscience.wiley.com/journal/121548564/issueyear?year=2009 PMID:18776913

  4. [Development of transgenic maize with anti-rough dwarf virus artificial miRNA vector and their disease resistance].

    PubMed

    Xuan, Ning; Zhao, Chuanzhi; Peng, Zhenying; Chen, Gao; Bian, Fei; Lian, Mingzheng; Liu, Guoxia; Wang, Xingjun; Bi, Yuping

    2015-09-01

    Maize is one of the most important food crops. Rice black-streaked dwarf virus is a maize rough dwarf disease pathogen. The occurrence and transmission of maize rough dwarf disease brings great damage to maize production. The technology of using artificial miRNA to build antiviral plant has been proven effective in a variety of plants. However, such trials in maize have not been reported. We designed primers based on the sequence of maize zea-miR159a precursor and sequence of function protein genes and silencing RBSDV coding genes in RBSDV genome. We constructed amiRNA (artificial miRNA) gene for silencing RBSDV coding gene and gene silencing suppressor. We constructed pCAMBIA3301-121-amiRNA plant expression vector for transforming maize inbred lines Z31 by using agrobacterium mediated method. After molecular analysis of transgenic maize, homozygous lines with high miRNA expression were selected by molecular detection for a subsequent natural infection experiment. We studied the severity of maize rough dwarf disease according to a grading standard (grade 0 to 4). The experiment results showed that the disease resistance of transgenic homozygous maize with the anti-rough dwarf virus amiRNA vector was better than that of wild type. Among the transgenic maize, S6-miR159 transgenic maize had high disease resistance. It is feasible to create new maize variety by the use of artificial miRNA. PMID:26955715

  5. [Construction of PPENK-MIDGE-NLS gene vector and the expression in rat].

    PubMed

    Chen, Xi; Xu, Xuemin; Peng, Xijuan; Jiang, Wei; Yao, Linong

    2015-02-01

    Increasing the production and secretion of endogenous opioid peptide by immune cell can significantly induce myocardial protective effects against ischemia-reperfusion injury. Gene therapy is promising to increase endogenous enkephalin (ENK). However, classical viral and plasmid vectors for gene delivery are hampered by immunogenicity, gene recombination, oncogene activation, the production of antibacterial antibody and changes in physiological gene expression. Minimalistic immunologically defined gene expression (MIDGE) can overcome all the deficients of viral and plasmid vectors. The exon of rat's preproenkephalin (PPENK) gene was amplified by PCR and the fragments were cloned into pEGFP-N1 plasmids. The recombined plasmids were digested with enzymes to obtain a linear vector contained promoter, preproenkephalin gene, RNA stable sequences and oligodesoxy nucleotides (ODNs) added to both ends of the gene vector to protect gene vector from exonuclease degradation. A nuclear localization sequence (NLS) was attached to an ODN to ensure the effective transport to the nucleus and transgene expression. Flow cytometry, laser confocal microscopy and Western blotting demonstrated that PPENK-MIDGE-NLS can transfect leukocyte of rat in vivo, increase the expression of proenkephalin (PENK) in tissue, and the transfection efficiency depends on gene vector's dosage. These results indicate that PPENK-MIDGE-NLS could be an innovative method to protect and treatment of myocardial ischemia-reperfusion injury.

  6. Development of expression vectors based on pepino mosaic virus

    PubMed Central

    2011-01-01

    Background Plant viruses are useful expression vectors because they can mount systemic infections allowing large amounts of recombinant protein to be produced rapidly in differentiated plant tissues. Pepino mosaic virus (PepMV) (genus Potexvirus, family Flexiviridae), a widespread plant virus, is a promising candidate expression vector for plants because of its high level of accumulation in its hosts and the absence of severe infection symptoms. We report here the construction of a stable and efficient expression vector for plants based on PepMV. Results Agroinfectious clones were produced from two different PepMV genotypes (European and Chilean), and these were able to initiate typical PepMV infections. We explored several strategies for vector development including coat protein (CP) replacement, duplication of the CP subgenomic promoter (SGP) and the creation of a fusion protein using the foot-and-mouth disease virus (FMDV) 2A catalytic peptide. We found that CP replacement vectors were unable to move systemically and that vectors with duplicated SGPs (even heterologous SGPs) suffered from significant transgene instability. The fusion protein incorporating the FMDV 2A catalytic peptide gave by far the best results, maintaining stability through serial passages and allowing the accumulation of GFP to 0.2-0.4 g per kg of leaf tissue. The possible use of PepMV as a virus-induced gene silencing vector to study gene function was also demonstrated. Protocols for the use of this vector are described. Conclusions A stable PepMV vector was generated by expressing the transgene as a CP fusion using the sequence encoding the foot-and-mouth disease virus (FMDV) 2A catalytic peptide to separate them. We have generated a novel tool for the expression of recombinant proteins in plants and for the functional analysis of virus and plant genes. Our experiments have also highlighted virus requirements for replication in single cells as well as intercellular and long

  7. Model-driven engineering of gene expression from RNA replicons.

    PubMed

    Beal, Jacob; Wagner, Tyler E; Kitada, Tasuku; Azizgolshani, Odisse; Parker, Jordan Moberg; Densmore, Douglas; Weiss, Ron

    2015-01-16

    RNA replicons are an emerging platform for engineering synthetic biological systems. Replicons self-amplify, can provide persistent high-level expression of proteins even from a small initial dose, and, unlike DNA vectors, pose minimal risk of chromosomal integration. However, no quantitative model sufficient for engineering levels of protein expression from such replicon systems currently exists. Here, we aim to enable the engineering of multigene expression from more than one species of replicon by creating a computational model based on our experimental observations of the expression dynamics in single- and multireplicon systems. To this end, we studied fluorescent protein expression in baby hamster kidney (BHK-21) cells using a replicon derived from Sindbis virus (SINV). We characterized expression dynamics for this platform based on the dose-response of a single species of replicon over 50 h and on a titration of two cotransfected replicons expressing different fluorescent proteins. From this data, we derive a quantitative model of multireplicon expression and validate it by designing a variety of three-replicon systems, with profiles that match desired expression levels. We achieved a mean error of 1.7-fold on a 1000-fold range, thus demonstrating how our model can be applied to precisely control expression levels of each Sindbis replicon species in a system.

  8. Model-driven engineering of gene expression from RNA replicons.

    PubMed

    Beal, Jacob; Wagner, Tyler E; Kitada, Tasuku; Azizgolshani, Odisse; Parker, Jordan Moberg; Densmore, Douglas; Weiss, Ron

    2015-01-16

    RNA replicons are an emerging platform for engineering synthetic biological systems. Replicons self-amplify, can provide persistent high-level expression of proteins even from a small initial dose, and, unlike DNA vectors, pose minimal risk of chromosomal integration. However, no quantitative model sufficient for engineering levels of protein expression from such replicon systems currently exists. Here, we aim to enable the engineering of multigene expression from more than one species of replicon by creating a computational model based on our experimental observations of the expression dynamics in single- and multireplicon systems. To this end, we studied fluorescent protein expression in baby hamster kidney (BHK-21) cells using a replicon derived from Sindbis virus (SINV). We characterized expression dynamics for this platform based on the dose-response of a single species of replicon over 50 h and on a titration of two cotransfected replicons expressing different fluorescent proteins. From this data, we derive a quantitative model of multireplicon expression and validate it by designing a variety of three-replicon systems, with profiles that match desired expression levels. We achieved a mean error of 1.7-fold on a 1000-fold range, thus demonstrating how our model can be applied to precisely control expression levels of each Sindbis replicon species in a system. PMID:24877739

  9. Silencing Status Epilepticus-Induced BDNF Expression with Herpes Simplex Virus Type-1 Based Amplicon Vectors.

    PubMed

    Falcicchia, Chiara; Trempat, Pascal; Binaschi, Anna; Perrier-Biollay, Coline; Roncon, Paolo; Soukupova, Marie; Berthommé, Hervé; Simonato, Michele

    2016-01-01

    Brain-derived neurotrophic factor (BDNF) has been found to produce pro- but also anti-epileptic effects. Thus, its validity as a therapeutic target must be verified using advanced tools designed to block or to enhance its signal. The aim of this study was to develop tools to silence the BDNF signal. We generated Herpes simplex virus type 1 (HSV-1) derived amplicon vectors, i.e. viral particles containing a genome of 152 kb constituted of concatameric repetitions of an expression cassette, enabling the expression of the gene of interest in multiple copies. HSV-1 based amplicon vectors are non-pathogenic and have been successfully employed in the past for gene delivery into the brain of living animals. Therefore, amplicon vectors should represent a logical choice for expressing a silencing cassette, which, in multiple copies, is expected to lead to an efficient knock-down of the target gene expression. Here, we employed two amplicon-based BDNF silencing strategies. The first, antisense, has been chosen to target and degrade the cytoplasmic mRNA pool of BDNF, whereas the second, based on the convergent transcription technology, has been chosen to repress transcription at the BDNF gene. Both these amplicon vectors proved to be effective in down-regulating BDNF expression in vitro, in BDNF-expressing mesoangioblast cells. However, only the antisense strategy was effective in vivo, after inoculation in the hippocampus in a model of status epilepticus in which BDNF mRNA levels are strongly increased. Interestingly, the knocking down of BDNF levels induced with BDNF-antisense was sufficient to produce significant behavioral effects, in spite of the fact that it was produced only in a part of a single hippocampus. In conclusion, this study demonstrates a reliable effect of amplicon vectors in knocking down gene expression in vitro and in vivo. Therefore, this approach may find broad applications in neurobiological studies.

  10. Silencing Status Epilepticus-Induced BDNF Expression with Herpes Simplex Virus Type-1 Based Amplicon Vectors

    PubMed Central

    Falcicchia, Chiara; Trempat, Pascal; Binaschi, Anna; Perrier-Biollay, Coline; Roncon, Paolo; Soukupova, Marie; Berthommé, Hervé; Simonato, Michele

    2016-01-01

    Brain-derived neurotrophic factor (BDNF) has been found to produce pro- but also anti-epileptic effects. Thus, its validity as a therapeutic target must be verified using advanced tools designed to block or to enhance its signal. The aim of this study was to develop tools to silence the BDNF signal. We generated Herpes simplex virus type 1 (HSV-1) derived amplicon vectors, i.e. viral particles containing a genome of 152 kb constituted of concatameric repetitions of an expression cassette, enabling the expression of the gene of interest in multiple copies. HSV-1 based amplicon vectors are non-pathogenic and have been successfully employed in the past for gene delivery into the brain of living animals. Therefore, amplicon vectors should represent a logical choice for expressing a silencing cassette, which, in multiple copies, is expected to lead to an efficient knock-down of the target gene expression. Here, we employed two amplicon-based BDNF silencing strategies. The first, antisense, has been chosen to target and degrade the cytoplasmic mRNA pool of BDNF, whereas the second, based on the convergent transcription technology, has been chosen to repress transcription at the BDNF gene. Both these amplicon vectors proved to be effective in down-regulating BDNF expression in vitro, in BDNF-expressing mesoangioblast cells. However, only the antisense strategy was effective in vivo, after inoculation in the hippocampus in a model of status epilepticus in which BDNF mRNA levels are strongly increased. Interestingly, the knocking down of BDNF levels induced with BDNF-antisense was sufficient to produce significant behavioral effects, in spite of the fact that it was produced only in a part of a single hippocampus. In conclusion, this study demonstrates a reliable effect of amplicon vectors in knocking down gene expression in vitro and in vivo. Therefore, this approach may find broad applications in neurobiological studies. PMID:26954758

  11. Inducing RNA interference in the arbovirus vector, Culicoides sonorensis

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Biting midges in the genus Culicoides are important vectors of arboviral diseases, including Epizootic Hemorrhagic Disease, Bluetongue, and likely Schmallenberg, which cause significant economic burden worldwide. Research on these vectors has been hindered by the lack of a sequenced genome, the diff...

  12. Inhibition of Human Immunodeficiency Virus Type 1 Replication in Primary Macrophages by Using Tat- or CCR5-Specific Small Interfering RNAs Expressed from a Lentivirus Vector

    PubMed Central

    Lee, Ming-Ta M.; Coburn, Glen A.; McClure, Myra O.; Cullen, Bryan R.

    2003-01-01

    Although several groups have demonstrated that RNA interference, induced by transfection of small interfering RNA (siRNA) duplexes, can protect cells against a viral challenge in culture, this protection is transient. Here, we describe lentivirus expression vectors that can stably express siRNAs at levels sufficient to block virus replication. We have used these vectors to stably express siRNAs specific for the essential human immunodeficiency virus type 1 (HIV-1) Tat transcription factor or specific for a cellular coreceptor, CCR5, that is required for infection by the majority of primary HIV-1 isolates. These lentivirus vectors are shown to protect cells, including primary macrophages, against HIV-1 infection in culture by inducing selective degradation of their target mRNA species. These data suggest that it should be possible to block the expression of specific viral or cellular genes in vivo by using viral vectors to stably express the appropriate siRNAs. PMID:14581533

  13. Differential nanotoxicological and neuroinflammatory liabilities of non-viral vectors for RNA interference in the central nervous system.

    PubMed

    Godinho, Bruno M D C; McCarthy, David J; Torres-Fuentes, Cristina; Beltrán, Caroll J; McCarthy, Joanna; Quinlan, Aoife; Ogier, Julien R; Darcy, Raphael; O'Driscoll, Caitriona M; Cryan, John F

    2014-01-01

    Progression of RNA interference-based gene silencing technologies for the treatment of disorders of the central nervous system (CNS) depends on the availability of efficient non-toxic nanocarriers. Despite advances in the field of nanotechnology undesired and non-specific interactions with different brain-cell types occur and are poorly investigated. To this end, we studied the cytotoxic and neuroinflammatory effects of widely-used transfection reagents and modified amphiphilic β-cyclodextrins (CDs). All non-viral vectors formed positively charged nanoparticles with distinctive physicochemical properties. Differential and significant cytotoxic effects were observed among commercially available cationic vectors, whereas CDs induced limited disruptions of cellular membrane integrity and mitochondrial dehydrogenase activity. Interestingly, murine derived BV2 microglia cells and a rat striatal in vitro model of Huntington's disease (ST14A-HTT120Q) were more susceptible to toxicity than human U87 astroglioma cells. BV2 microglia presented significant increases in cytokine, toll-like receptor 2 and cyclooxygenase-2 gene expression after transfection with selected commercial vectors but not with CD.siRNA nanoparticles. Non-viral siRNA nanoparticles formulated with G6 polyamidoamine (PAMAM) also significantly increased cytokine gene expression in the brain following injections into the mouse striatum. Together our data identify modified CDs as nanosystems that enable siRNA delivery to the brain with low levels of cytotoxicity and immunological activation. PMID:24138827

  14. Correlations of microRNA:microRNA expression patterns reveal insights into microRNA clusters and global microRNA expression patterns.

    PubMed

    Chaulk, S G; Ebhardt, H A; Fahlman, R P

    2016-01-01

    MicroiRNAs are genome encoded small double stranded RNAs that regulate expression of homologous mRNAs. With approximately 2500 human miRNAs and each having hundreds of potential mRNA targets, miRNA based gene regulation is quite pervasive in both development and disease. While there are numerous studies investigating miRNA:mRNA and miRNA:protein target expression correlations, there are relatively few studies of miRNA:miRNA co-expression. Here we report on our analysis of miRNA:miRNA co-expression using expression data from the miRNA expression atlas of Landgraf et al. Our analysis indicates that many, but not all, genomically clustered miRNAs are co-expressed as a single pri-miRNA transcript. We have also identified co-expression groups that have similar biological activity. Further, the non-correlative miRNAs we have uncovered have been shown to be of utility in establishing miRNA biomarkers and signatures for certain tumours and cancers.

  15. Gamma-Retroviral Vector Design for the Co-Expression of Artificial microRNAs and Therapeutic Proteins

    PubMed Central

    Park, Tristen S.; Zhang, Ling; Zheng, Zhili; Morgan, Richard A.

    2014-01-01

    To generate γ-retroviral vectors for stable conjoint expression of artificial microRNAs (amiR) and therapeutic genes in primary human lymphocytes, and to identify the design parameters that are key for successful vector generation. Gamma-retroviral vectors were designed to co-express both amiRs and a linked reporter gene, truncated CD34 (tCD34). Artificial miRs based on microRNAs miR-16, miR-142, miR-146b, miR-150, miR155, and miR-223 were inserted into sites within the intron of the vector and tested for tCD34 expression by flow cytometry (FACS). Different constructs were assembled with amiRs targeted to knockdown expression of suppressor of cytokine signaling 1 (SOCS1) or programmed cell death 1 (PDCD1, PD-1). Three of the six amiRs maintained tCD34 expression. Expansion of primary human T cells transduced with these amiR vectors, as well as transgene expression, were equivalent to control engineered T cells over a 40-day period. Knockdown of SOCS1 RNA and PD-1 expression by FACS was shown to vary between constructs, dependent on either the specific short interfering RNA sequence used in the amiR, or the microRNA backbone and location in the vector intron. Gamma-retroviral vectors that both efficiently knockdown endogenous gene expression and maintain linked transgene production can be produced, but empirical vector evaluations were best suited for optimal construct analysis. PMID:25019196

  16. Efficient Gene Suppression in Dorsal Root Ganglia and Spinal Cord Using Adeno-Associated Virus Vectors Encoding Short-Hairpin RNA.

    PubMed

    Enomoto, Mitsuhiro; Hirai, Takashi; Kaburagi, Hidetoshi; Yokota, Takanori

    2016-01-01

    RNA interference is a powerful tool used to induce loss-of-function phenotypes through post-transcriptional gene silencing. Small interfering RNA (siRNA) molecules have been used to target the central nervous system (CNS) and are expected to have clinical utility against refractory neurodegenerative diseases. However, siRNA is characterized by low transduction efficiency, insufficient inhibition of gene expression, and short duration of therapeutic effects, and is thus not ideal for treatment of neural tissues and diseases. To address these problems, viral delivery of short-hairpin RNA (shRNA) expression cassettes that support more efficient and long-lasting transduction into target tissues is expected to be a promising delivery tool. Various types of gene therapy vectors have been developed, such as adenovirus, adeno-associated virus (AAV), herpes simplex virus and lentivirus; however, AAV is particularly advantageous because of its relative lack of immunogenicity and lack of chromosomal integration. In human clinical trials, recombinant AAV vectors are relatively safe and well-tolerated. In particular, serotype 9 of AAV (AAV9) vectors show the highest tropism for neural tissue and can cross the blood-brain barrier, and we have shown that intrathecal delivery of AAV9 yields relatively high gene transduction into dorsal root ganglia or spinal cord. This chapter describes how to successfully use AAV vectors encoding shRNA in vivo, particularly for RNA interference in the central and peripheral nervous system. PMID:26472458

  17. Efficient Gene Suppression in Dorsal Root Ganglia and Spinal Cord Using Adeno-Associated Virus Vectors Encoding Short-Hairpin RNA.

    PubMed

    Enomoto, Mitsuhiro; Hirai, Takashi; Kaburagi, Hidetoshi; Yokota, Takanori

    2016-01-01

    RNA interference is a powerful tool used to induce loss-of-function phenotypes through post-transcriptional gene silencing. Small interfering RNA (siRNA) molecules have been used to target the central nervous system (CNS) and are expected to have clinical utility against refractory neurodegenerative diseases. However, siRNA is characterized by low transduction efficiency, insufficient inhibition of gene expression, and short duration of therapeutic effects, and is thus not ideal for treatment of neural tissues and diseases. To address these problems, viral delivery of short-hairpin RNA (shRNA) expression cassettes that support more efficient and long-lasting transduction into target tissues is expected to be a promising delivery tool. Various types of gene therapy vectors have been developed, such as adenovirus, adeno-associated virus (AAV), herpes simplex virus and lentivirus; however, AAV is particularly advantageous because of its relative lack of immunogenicity and lack of chromosomal integration. In human clinical trials, recombinant AAV vectors are relatively safe and well-tolerated. In particular, serotype 9 of AAV (AAV9) vectors show the highest tropism for neural tissue and can cross the blood-brain barrier, and we have shown that intrathecal delivery of AAV9 yields relatively high gene transduction into dorsal root ganglia or spinal cord. This chapter describes how to successfully use AAV vectors encoding shRNA in vivo, particularly for RNA interference in the central and peripheral nervous system.

  18. Further evaluation of a novel nano-scale gene vector for in vivo transfection of siRNA.

    PubMed

    Liu, Fan; Qiao, Fang-Fang; Tong, Man-Li; Liu, Li-Li; Fu, Zuo-Gen; Dan, Bing; Lin, Li-Rong; Yang, Tian-Ci; Zhang, Zhong-Ying

    2011-05-01

    In this research, a lipid-cationic polymer (LCP) containing the side-chain branching of brassidic acid was synthesized using chemical methods. As a gene vector for small interfering ribonucleic acid (siRNA) transfection, the efficiency and biosafety of LCP were preliminarily evaluated to investigate its possible application on tumor gene therapy. The toxicity, side-effects, and biosafety of LCP were investigated in animals based on the results of in vitro experiments. The siRNA against cyclooxygenase-2 (COX-2) was transfected by LCP to interfere with the COX-2 expression in nude-transplanted tumors. Hematoxylin and eosin stains, immunohistochemistry, reverse transcription-polymerase chain reaction, and Western blot were performed to evaluate the efficiency of LCP for siRNA transfection. The animal toxicity experiment showed that a high concentration of LCP had a low toxic effect on animals and did not induce allergic or pyrogenic reactions. The results from the in vivo transfection indicated that LCP could efficiently transfect siRNA and silence the target gene expression. The LCP gene vector for siRNA transfection is highly efficient during in vivo transfection and had low toxicity. From all aspects of tumor gene therapy and basic research, LCP is valuable for scientific research and medical applications.

  19. Co-transduction of lentiviral and adenoviral vectors for co-delivery of growth factor and shRNA genes in mesenchymal stem cells-based chondrogenic system.

    PubMed

    Zhang, Feng; Yao, Yongchang; Su, Kai; Fang, Yu; Citra, Fudiman; Wang, Dong-An

    2015-09-01

    Gene delivery takes advantage of cellular mechanisms to express gene products and is an efficient way to deliver them into cells, influencing cellular behaviours and expression patterns. Among the delivery methods, viral vectors are applied due to their high efficiency. Two typical viral vectors for gene delivery include lentiviral vector for integrative transduction and adenoviral vector for transient episomal transduction, respectively. The selection and formulation of proper viral vectors applied to cells can modulate gene expression profiles and further impact the downstream pathways. In this study, recombinant lentiviral and adenoviral vectors were co-transduced in a synovial mesenchymal stem cells (SMSCs)-based articular chondrogenic system by which two transgenes were co-delivered - the gene for transforming growth factor (TGF)β3, to facilitate SMSC chondrogenesis, and the gene for small hairpin RNA (shRNA), targeting the mRNA of type I collagen (Col I) α1 chain to silence Col I expression and minimize fibrocartilage formation. Delivery of either gene could be achieved with either lentiviral or adenoviral vectors. Therefore, co-delivery of the two transgenes via the two types of vectors was performed to determine which combination was optimal for three-dimensional (3D) articular chondrogenesis to construct articular hyaline cartilage tissue. Suppression of Col I and expression of cartilage markers, including type II collagen, aggrecan and cartilage oligomeric matrix protein (COMP), were assessed at both the transcriptome and protein phenotypic levels. It was concluded that the combination of lentiviral-mediated TGFβ3 release and adenoviral-mediated shRNA expression (LV-T + Ad-sh) generally demonstrated optimal efficacy in engineered articular cartilage with SMSCs.

  20. Heterologous expression of human interleukin-6 in Streptomyces lividans TK24 using novel secretory expression vectors.

    PubMed

    Zhu, Yuanjun; Wang, Lifei; Du, Yu; Wang, Songmei; Yu, Tengfei; Hong, Bin

    2011-02-01

    Streptomyces is an attractive host for heterologous protein secretion. To further optimize its expression capacity, better expression vectors will be helpful. Here, based on pSGL1, a high copy number plasmid present in Streptomyces globisporus C-1027, we constructed a series of novel E. coli-Streptomyces shuttle expression vectors pIMB2-4. These vectors, which are compatible with pIJ-derived vectors, contain the strong promoter ermE*p and signal sequence SP (MelC1) of the first ORF of melanin operon in S. antibiotics (pIMB2), SP (CagA) of C-1027 apoprotein in S. globisporus C-1027 (pIMB3 and pIMB4). Using these vectors, human interleukin-6 (IL-6) could successfully be expressed and secreted using S. lividans TK24 as host. Furthermore, replacement of a rare leucine codon TTA with CTG in SP (CagA) enhanced IL-6 production.

  1. Construction and expression of prokaryotic expression vectors fused with genes of Magnaporthe oryzae effector proteins and mCherry.

    PubMed

    Yang, Y Q; Wang, H; Liang, M L; Yan, J L; Liu, L; Li, C Y; Yang, J

    2015-09-09

    The aim of the current study was to investigate the prokaryotic expression of the Magnaporthe oryzae effector genes BAS1 and BAS4 fused to the fluorescent protein mCherry. Based on previous polymorphic analysis of BAS1 and BAS4 in rice blast strains using PCR, blast strains containing the PCR products of BAS1 and BAS4 were selected for liquid culture for total RNA extraction. For PCR analysis, cDNA was selected as a template to amplify the coding region of BAS1 and BAS4, the plasmid pXY201 was selected as template to amplify the mCherry sequence, and the three sequences were cloned into pMD®19-T vectors. Positive recombinant plasmids were digested using two restriction enzymes and the cleaved fragments of BAS1 and mCherry and BAS4 and mCherry were ligated to pGEX-4T-1 vectors and expression was induced using IPTG. The PCR results showed that the sequence sizes of BAS1, BAS4, and mCherry were 348, 309, and 711 bp, respectively, and these were cloned into pMD®19-T vectors. After digestion and gel purification, the fragments of BAS1 and mCherry, BAS4 and mCherry were ligated into pGEX-4T-1 vectors and expressed in Escherichia coli BL21 competent cells. The expressed proteins were approximately 60 kDa, corresponding to their theoretical size. Prokaryotic expression products of BAS1 and BAS4 fused to mCherry were presented in this study, providing a base for constructing prokaryotic expression vectors of pathogen effector genes fused to mCherry, which will contribute to further study of the subcellular localization, function, and protein interactions of these effectors.

  2. A universal expression/silencing vector in plants.

    PubMed

    Peretz, Yuval; Mozes-Koch, Rita; Akad, Fuad; Tanne, Edna; Czosnek, Henryk; Sela, Ilan

    2007-12-01

    A universal vector (IL-60 and auxiliary constructs), expressing or silencing genes in every plant tested to date, is described. Plants that have been successfully manipulated by the IL-60 system include hard-to-manipulate species such as wheat (Triticum duram), pepper (Capsicum annuum), grapevine (Vitis vinifera), citrus, and olive (Olea europaea). Expression or silencing develops within a few days in tomato (Solanum lycopersicum), wheat, and most herbaceous plants and in up to 3 weeks in woody trees. Expression, as tested in tomato, is durable and persists throughout the life span of the plant. The vector is, in fact, a disarmed form of Tomato yellow leaf curl virus, which is applied as a double-stranded DNA and replicates as such. However, the disarmed virus does not support rolling-circle replication, and therefore viral progeny single-stranded DNA is not produced. IL-60 does not integrate into the plant's genome, and the construct, including the expressed gene, is not heritable. IL-60 is not transmitted by the Tomato yellow leaf curl virus's natural insect vector. In addition, artificial satellites were constructed that require a helper virus for replication, movement, and expression. With IL-60 as the disarmed helper "virus," transactivation occurs, resulting in an inducible expressing/silencing system. The system's potential is demonstrated by IL-60-derived suppression of a viral-silencing suppressor of Grapevine virus A, resulting in Grapevine virus A-resistant/tolerant plants.

  3. Regulated expression of a complete human beta-globin gene encoded by a transmissible retrovirus vector.

    PubMed Central

    Cone, R D; Weber-Benarous, A; Baorto, D; Mulligan, R C

    1987-01-01

    We introduced a human beta-globin gene into murine erythroleukemia (MEL) cells by infection with recombinant retroviruses containing the complete genomic globin sequence. The beta-globin gene was correctly regulated during differentiation, steady-state mRNA levels being induced 5- to 30-fold after treatment of the cells with the chemical inducer dimethyl sulfoxide. Studies using vectors which yield integrated proviruses lacking transcriptional enhancer sequences indicated that neither retroviral transcription nor the retroviral enhancer sequences themselves had any obvious effect on expression of the globin gene. Viral RNA expression also appeared inducible, being considerably depressed in uninduced MEL cells but approaching normal wild-type levels after dimethyl sulfoxide treatment. We provide data which suggest that the control point for both repression and subsequent activation of virus expression in MEL cells lies in the viral enhancer element. Images PMID:3029570

  4. Characteristic element of matrix attachment region mediates vector attachment and enhances nerve growth factor expression in Chinese hamster ovary cells.

    PubMed

    Wang, X Y; Zhang, J H; Sun, Q L; Yao, Z Y; Deng, B G; Guo, W Y; Wang, L; Dong, W H; Wang, F; Zhao, C P; Wang, T Y

    2015-08-07

    Preliminary studies have suggested that a characteristic element of the matrix attachment region (MAR) in human interferon-β mediates the adhesion of vectors to Chinese hamster ovary (CHO) cells. In this study, we investigated if vector adhesion increased nerve growth factor (NGF) expression in CHO cells. The MAR characteristic element sequence of human interferon-β was inserted into the multiple-cloning site of the pEGFP-C1 vector. The target NGF gene was inserted upstream of the MAR characteristic element sequence to construct the MAR/NGF expression vector. The recombinant plasmid was transfected into CHO cells and stable monoclonal cells were selected using G418. NGF mRNA and protein expression was detected by reverse transcriptase-polymerase chain reaction and enzyme-linked immunosorbent assay, respectively. Plasmid reduction experiments were used to determine the state of transfected plasmid in mammalian cells. The insertion of MAR into the vector increased NGF expression levels in CHO cells (1.93- fold) compared to the control. The recombinant plasmid expressing the MAR sequence was digested into a linear space vector. The inserted MAR and NGF sequences were consistent with those inserted into the plasmid before recombination. Therefore, we concluded that the MAR characteristic element mediates vector adhesion to CHO cells and enhances the stability and efficiency of the target gene expression.

  5. Circular RNA Expression: Its Potential Regulation and Function

    PubMed Central

    Salzman, Julia

    2016-01-01

    In 2012, a new feature of eukaryotic gene expression emerged: ubiquitous expression of circular RNA (circRNA) from genes traditionally thought to express messenger or linear noncoding (nc)RNA only. CircRNAs are covalently closed, circular RNA molecules that typically comprise exonic sequences and are spliced at canonical splice sites. This feature of gene expression was first recognized in humans and mouse, but it quickly emerged that it was common across essentially all eukaryotes studied by molecular biologists. CircRNA abundance, and even which alternatively spliced circRNA isoforms are expressed, varies by cell type and can exceed the abundance of the traditional linear mRNA or ncRNA transcript. CircRNAs are enriched in the brain and increase in abundance during fetal development. Together, these features raise fundamental questions regarding the regulation of circRNA in cis and in trans, and its function. PMID:27050930

  6. Chitosan/interfering RNA nanoparticle mediated gene silencing in disease vector mosquito larvae.

    PubMed

    Zhang, Xin; Mysore, Keshava; Flannery, Ellen; Michel, Kristin; Severson, David W; Zhu, Kun Yan; Duman-Scheel, Molly

    2015-03-25

    Vector mosquitoes inflict more human suffering than any other organism-and kill more than one million people each year. The mosquito genome projects facilitated research in new facets of mosquito biology, including functional genetic studies in the primary African malaria vector Anopheles gambiae and the dengue and yellow fever vector Aedes aegypti. RNA interference- (RNAi-) mediated gene silencing has been used to target genes of interest in both of these disease vector mosquito species. Here, we describe a procedure for preparation of chitosan/interfering RNA nanoparticles that are combined with food and ingested by larvae. This technically straightforward, high-throughput, and relatively inexpensive methodology, which is compatible with long double stranded RNA (dsRNA) or small interfering RNA (siRNA) molecules, has been used for the successful knockdown of a number of different genes in A. gambiae and A. aegypti larvae. Following larval feedings, knockdown, which is verified through qRT-PCR or in situ hybridization, can persist at least through the late pupal stage. This methodology may be applicable to a wide variety of mosquito and other insect species, including agricultural pests, as well as other non-model organisms. In addition to its utility in the research laboratory, in the future, chitosan, an inexpensive, non-toxic and biodegradable polymer, could potentially be utilized in the field.

  7. RNAi and viral vectors as useful tools in the functional genomics of plants. Construction of BMV-based vectors for RNA delivery into plant cells.

    PubMed

    Wojtkowiak, Agnieszka; Siek, Anna; Alejska, Magdalena; Jarmołowski, Artur; Szweykowska-Kulińska, Zofia; Figlerowicz, Marek

    2002-01-01

    The sequencing of several complete genomes and the development of a DNA microarray technology are among the most important achievements of molecular biology. They gave the proper grounds for the development of modern functional genomics. However, there is one additional condition which needs to be satisfied to truely enable the study of how a genome works: a suitable method of selectively inducing and silencing the expression of each individual gene. The methods used so far have usually only permitted the influencing of gene expression through genetic manipulations at the DNA level (genetically modified plants). The discovery of RNA interference (RNAi) opens up completely new possibilities of research on the functioning of particular plant genes, without the necessity of altering the genome structure. In this case, interference takes place at the transcript level. Thus, at any given moment during plant development, the expression of a specific gene (or several genes) can be inhibited, even if it is important for the survival of the organism under study. To this end, a double-stranded RNA inducing the RNAi phenomenon has to be delivered into the plant cell. Here we describe the construction of four brome mosaic virus-based vectors, which, as our preliminary data indicate, can be used to transfer RNA into barley cells.

  8. Gene silencing and gene expression in phytopathogenic fungi using a plant virus vector

    PubMed Central

    Mascia, Tiziana; Nigro, Franco; Abdallah, Alì; Ferrara, Massimo; De Stradis, Angelo; Faedda, Roberto; Palukaitis, Peter; Gallitelli, Donato

    2014-01-01

    RNA interference (RNAi) is a powerful approach for elucidating gene functions in a variety of organisms, including phytopathogenic fungi. In such fungi, RNAi has been induced by expressing hairpin RNAs delivered through plasmids, sequences integrated in fungal or plant genomes, or by RNAi generated in planta by a plant virus infection. All these approaches have some drawbacks ranging from instability of hairpin constructs in fungal cells to difficulties in preparing and handling transgenic plants to silence homologous sequences in fungi grown on these plants. Here we show that RNAi can be expressed in the phytopathogenic fungus Colletotrichum acutatum (strain C71) by virus-induced gene silencing (VIGS) without a plant intermediate, but by using the direct infection of a recombinant virus vector based on the plant virus, tobacco mosaic virus (TMV). We provide evidence that a wild-type isolate of TMV is able to enter C71 cells grown in liquid medium, replicate, and persist therein. With a similar approach, a recombinant TMV vector carrying a gene for the ectopic expression of the green fluorescent protein (GFP) induced the stable silencing of the GFP in the C. acutatum transformant line 10 expressing GFP derived from C71. The TMV-based vector also enabled C. acutatum to transiently express exogenous GFP up to six subcultures and for at least 2 mo after infection, without the need to develop transformation technology. With these characteristics, we anticipate this approach will find wider application as a tool in functional genomics of filamentous fungi. PMID:24594602

  9. Overcoming expression and purification problems of RhoGDI using a family of "parallel" expression vectors.

    PubMed

    Sheffield, P; Garrard, S; Derewenda, Z

    1999-02-01

    We describe the construction of expression vectors based on three of the most frequently used gene fusion affinity tags [glutathione S-transferase (GST), maltose binding protein (MBP), and the His6 peptide]. The polylinkers of pGEX4T1, pMal-c2, and a pET vector were replaced with the polylinker isolated from the baculovirus expression plasmid pFastBac. Once appropriate restriction sites have been introduced into a gene, it can be fused to all three affinity tags with little effort, allowing expression-screening experiments to be performed efficiently. We discuss the development and use of these vectors with respect to overcoming purification problems encountered for the RhoA GDP/GTP nucleotide dissociation inhibitor (RhoGDI) and their advantages over commercially available expression vectors.

  10. High level protein expression in mammalian cells using a safe viral vector: modified vaccinia virus Ankara.

    PubMed

    Hebben, Matthias; Brants, Jan; Birck, Catherine; Samama, Jean-Pierre; Wasylyk, Bohdan; Spehner, Danièle; Pradeau, Karine; Domi, Arban; Moss, Bernard; Schultz, Patrick; Drillien, Robert

    2007-12-01

    Vaccinia virus vectors are attractive tools to direct high level protein synthesis in mammalian cells. In one of the most efficient strategies developed so far, the gene to be expressed is positioned downstream of a bacteriophage T7 promoter within the vaccinia genome and transcribed by the T7 RNA polymerase, also encoded by the vaccinia virus genome. Tight regulation of transcription and efficient translation are ensured by control elements of the Escherichia coli lactose operon and the encephalomyocarditis virus leader sequence, respectively. We have integrated such a stringently controlled expression system, previously used successfully in a standard vaccinia virus backbone, into the modified vaccinia virus Ankara strain (MVA). In this manner, proteins of interest can be produced in mammalian cells under standard laboratory conditions because of the inherent safety of the MVA strain. Using this system for expression of beta-galactosidase, about 15 mg protein could be produced from 10(8) BHK21 cells over a 24-h period, a value 4-fold higher than the amount produced from an identical expression system based on a standard vaccinia virus strain. In another application, we employed the MVA vector to produce human tubulin tyrosine ligase and demonstrate that this protein becomes a major cellular protein upon induction conditions and displays its characteristic enzymatic activity. The MVA vector should prove useful for many other applications in which mammalian cells are required for protein production. PMID:17892951

  11. Heterologous viral expression systems in fosmid vectors increase the functional analysis potential of metagenomic libraries

    PubMed Central

    Terrón-González, L.; Medina, C.; Limón-Mortés, M. C.; Santero, E.

    2013-01-01

    The extraordinary potential of metagenomic functional analyses to identify activities of interest present in uncultured microorganisms has been limited by reduced gene expression in surrogate hosts. We have developed vectors and specialized E. coli strains as improved metagenomic DNA heterologous expression systems, taking advantage of viral components that prevent transcription termination at metagenomic terminators. One of the systems uses the phage T7 RNA-polymerase to drive metagenomic gene expression, while the other approach uses the lambda phage transcription anti-termination protein N to limit transcription termination. A metagenomic library was constructed and functionally screened to identify genes conferring carbenicillin resistance to E. coli. The use of these enhanced expression systems resulted in a 6-fold increase in the frequency of carbenicillin resistant clones. Subcloning and sequence analysis showed that, besides β-lactamases, efflux pumps are not only able contribute to carbenicillin resistance but may in fact be sufficient by themselves to convey carbenicillin resistance. PMID:23346364

  12. Heterologous viral expression systems in fosmid vectors increase the functional analysis potential of metagenomic libraries.

    PubMed

    Terrón-González, L; Medina, C; Limón-Mortés, M C; Santero, E

    2013-01-01

    The extraordinary potential of metagenomic functional analyses to identify activities of interest present in uncultured microorganisms has been limited by reduced gene expression in surrogate hosts. We have developed vectors and specialized E. coli strains as improved metagenomic DNA heterologous expression systems, taking advantage of viral components that prevent transcription termination at metagenomic terminators. One of the systems uses the phage T7 RNA-polymerase to drive metagenomic gene expression, while the other approach uses the lambda phage transcription anti-termination protein N to limit transcription termination. A metagenomic library was constructed and functionally screened to identify genes conferring carbenicillin resistance to E. coli. The use of these enhanced expression systems resulted in a 6-fold increase in the frequency of carbenicillin resistant clones. Subcloning and sequence analysis showed that, besides β-lactamases, efflux pumps are not only able contribute to carbenicillin resistance but may in fact be sufficient by themselves to convey carbenicillin resistance.

  13. [Construction of Fat-1 eukaryotic expression vector of excision markers and the establishment of transgenic sheep cell lines].

    PubMed

    Lima, A; Zhu, Heping; Wang, Ruiyao; Yan, Tao; Su, Xiaohu; Li, Lu; Wang, Bingping; Na, Shunwendoule; Qi, Guichun; Zhou, Huanmin

    2016-02-01

    In order to establish marker-free transgenic cell lines, we cloned Fat-1 gene, attB and Loxp sequences by PCR. Then we inserted these sequences to pN1-EGFP vector and got pEGFP-N1-Fat-1 expression vector. PhiC31 integrase mRNA which was generated by in vitro transcription and a pEGFP-N1-Fat-1 expression vector co-electroporated into sheep fetal fibroblasts, and then we got transgenic cell lines expressing green fluorescence. Prokaryotic expression and purification of Cre recombinant protein was performed. Cre recombinant protein was transducted into stably-transfected cell colonies. We identified cell colonies by sequencing and established marker-free transgenic cell lines and eventually- established marker-free transgenic cell lines which were building more safely basic for producing Fat-1 transgenic animals. PMID:27382771

  14. [Construction of Fat-1 eukaryotic expression vector of excision markers and the establishment of transgenic sheep cell lines].

    PubMed

    Lima, A; Zhu, Heping; Wang, Ruiyao; Yan, Tao; Su, Xiaohu; Li, Lu; Wang, Bingping; Na, Shunwendoule; Qi, Guichun; Zhou, Huanmin

    2016-02-01

    In order to establish marker-free transgenic cell lines, we cloned Fat-1 gene, attB and Loxp sequences by PCR. Then we inserted these sequences to pN1-EGFP vector and got pEGFP-N1-Fat-1 expression vector. PhiC31 integrase mRNA which was generated by in vitro transcription and a pEGFP-N1-Fat-1 expression vector co-electroporated into sheep fetal fibroblasts, and then we got transgenic cell lines expressing green fluorescence. Prokaryotic expression and purification of Cre recombinant protein was performed. Cre recombinant protein was transducted into stably-transfected cell colonies. We identified cell colonies by sequencing and established marker-free transgenic cell lines and eventually- established marker-free transgenic cell lines which were building more safely basic for producing Fat-1 transgenic animals.

  15. Data presenting a modified bacterial expression vector for expressing and purifying Nus solubility-tagged proteins.

    PubMed

    Gupta, Nidhi; Wu, Heng; Terman, Jonathan R

    2016-09-01

    Bacteria are the predominant source for producing recombinant proteins but while many exogenous proteins are expressed, only a fraction of those are soluble. We have found that a new actin regulatory enzyme Mical is poorly soluble when expressed in bacteria but the use of a Nus fusion protein tag greatly increases its solubility. However, available vectors containing a Nus tag have been engineered in a way that hinders the separation of target proteins from the Nus tag during protein purification. We have now used recombinant DNA approaches to overcome these issues and reengineer a Nus solubility tag-containing bacterial expression vector. The data herein present a modified bacterial expression vector useful for expressing proteins fused to the Nus solubility tag and separating such target proteins from the Nus tag during protein purification. PMID:27547802

  16. Efficient expression of protein coding genes from the murine U1 small nuclear RNA promoters.

    PubMed Central

    Bartlett, J S; Sethna, M; Ramamurthy, L; Gowen, S A; Samulski, R J; Marzluff, W F

    1996-01-01

    Few promoters are active at high levels in all cells. Of these, the majority encode structural RNAs transcribed by RNA polymerases I or III and are not accessible for the expression of proteins. An exception are the small nuclear RNAs (snRNAs) transcribed by RNA polymerase II. Although snRNA biosynthesis is unique and thought not to be compatible with synthesis of functional mRNA, we have tested these promoters for their ability to express functional mRNAs. We have used the murine U1a and U1b snRNA gene promoters to express the Escherichia coli lacZ gene and the human alpha-globin gene from either episomal or integrated templates by transfection, or infection into a variety of mammalian cell types. Equivalent expression of beta-galactosidase was obtained from < 250 nucleotides of 5'-flanking sequence containing the complete promoter of either U1 snRNA gene or from the 750-nt cytomegalovirus promoter and enhancer regions. The mRNA was accurately initiated at the U1 start site, efficiently spliced and polyadenylylated, and localized to polyribosomes. Recombinant adenovirus containing the U1b-lacZ chimeric gene transduced and expressed beta-galactosidase efficiently in human 293 cells and airway epithelial cells in culture. Viral vectors containing U1 snRNA promoters may be an attractive alternative to vectors containing viral promoters for persistent high-level expression of therapeutic genes or proteins. Images Fig. 2 Fig. 3 Fig. 4 Fig. 5 PMID:8799116

  17. Inhibitory effect of modified 5'-capped short RNA fragments on influenza virus RNA polymerase gene expression.

    PubMed

    Tado, M; Abe, T; Hatta, T; Ishikawa, M; Nakada, S; Yokota, T; Takaku, H

    2001-11-01

    We have shown previously that the 5'-capped short phosphodiester RNA fragments, Cap decoy, (Gm 12 nt) are potent inhibitors of influenza virus RNA polymerase gene expression. Here we investigate the modified capped RNA derivative containing phosphorothioate oligonucleotides (Cap decoy) as a potential influenza virus RNA polymerase inhibitor. The modified 5'-capped short phosphorothioate RNA fragments (Gms 12-15 nt) with the 5'-capped structure (m7GpppGm) were synthesized by T7 RNA polymerase. The 5'-capped short RNA fragments (Gms 12-15 nt) were encapsulated in liposome particulates and tested for their inhibitory effects on influenza virus RNA polymerase gene expression in the clone 76 cells. The 12-15 nt long Gms RNA fragments showed highly inhibitory effects. By contrast, the inhibitory effects of the 13 nt long short RNA fragments (Gm 13 nt) were considerably less in comparison with the 5'-capped short phosphorothioate RNA fragments (Gms 12-15 nt). In particular, the various Gms RNA chain lengths showed no significant differences in the inhibition of influenza virus RNA polymerase gene expression. Furthermore, the capped RNA with a phosphorothioate backbone was resistant to nuclease activity. These phosphorothioate RNA fragments exhibited higher inhibitory activity than the 5'-capped short RNA fragments (Gm 12 nt). These decoys may prove to be useful in anti-influenza virus therapeutics. PMID:12018680

  18. Unmasking Upstream Gene Expression Regulators with miRNA-corrected mRNA Data.

    PubMed

    Bollmann, Stephanie; Bu, Dengpan; Wang, Jiaqi; Bionaz, Massimo

    2015-01-01

    Expressed micro-RNA (miRNA) affects messenger RNA (mRNA) abundance, hindering the accuracy of upstream regulator analysis. Our objective was to provide an algorithm to correct such bias. Large mRNA and miRNA analyses were performed on RNA extracted from bovine liver and mammary tissue. Using four levels of target scores from TargetScan (all miRNA:mRNA target gene pairs or only the top 25%, 50%, or 75%). Using four levels of target scores from TargetScan (all miRNA:mRNA target gene pairs or only the top 25%, 50%, or 75%) and four levels of the magnitude of miRNA effect (ME) on mRNA expression (30%, 50%, 75%, and 83% mRNA reduction), we generated 17 different datasets (including the original dataset). For each dataset, we performed upstream regulator analysis using two bioinformatics tools. We detected an increased effect on the upstream regulator analysis with larger miRNA:mRNA pair bins and higher ME. The miRNA correction allowed identification of several upstream regulators not present in the analysis of the original dataset. Thus, the proposed algorithm improved the prediction of upstream regulators.

  19. Unmasking Upstream Gene Expression Regulators with miRNA-corrected mRNA Data

    PubMed Central

    Bollmann, Stephanie; Bu, Dengpan; Wang, Jiaqi; Bionaz, Massimo

    2015-01-01

    Expressed micro-RNA (miRNA) affects messenger RNA (mRNA) abundance, hindering the accuracy of upstream regulator analysis. Our objective was to provide an algorithm to correct such bias. Large mRNA and miRNA analyses were performed on RNA extracted from bovine liver and mammary tissue. Using four levels of target scores from TargetScan (all miRNA:mRNA target gene pairs or only the top 25%, 50%, or 75%). Using four levels of target scores from TargetScan (all miRNA:mRNA target gene pairs or only the top 25%, 50%, or 75%) and four levels of the magnitude of miRNA effect (ME) on mRNA expression (30%, 50%, 75%, and 83% mRNA reduction), we generated 17 different datasets (including the original dataset). For each dataset, we performed upstream regulator analysis using two bioinformatics tools. We detected an increased effect on the upstream regulator analysis with larger miRNA:mRNA pair bins and higher ME. The miRNA correction allowed identification of several upstream regulators not present in the analysis of the original dataset. Thus, the proposed algorithm improved the prediction of upstream regulators. PMID:27279737

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

    PubMed

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

    2016-01-01

    RNA-mediated knockdowns are widely used to control gene expression. This versatile family of techniques makes use of short RNA (sRNA) that can be synthesized with any sequence and designed to complement any gene targeted for silencing. Because sRNA constructs can be introduced to many cell types directly or using a variety of vectors, gene expression can be repressed in living cells without laborious genetic modification. The most common RNA knockdown technology, RNA interference (RNAi), makes use of the endogenous RNA-induced silencing complex (RISC) to mediate sequence recognition and cleavage of the target mRNA. Applications of this technique are therefore limited to RISC-expressing organisms, primarily eukaryotes. Recently, a new generation of RNA biotechnologists have developed alternative mechanisms for controlling gene expression through RNA, and so made possible RNA-mediated gene knockdowns in bacteria. Here we describe a method for silencing gene expression in E. coli that functionally resembles RNAi. In this system a synthetic phagemid is designed to express sRNA, which may designed to target any sequence. The expression construct is delivered to a population of E. coli cells with non-lytic M13 phage, after which it is able to stably replicate as a plasmid. Antisense recognition and silencing of the target mRNA is mediated by the Hfq protein, endogenous to E. coli. This protocol includes methods for designing the antisense sRNA, constructing the phagemid vector, packaging the phagemid into M13 bacteriophage, preparing a live cell population for infection, and performing the infection itself. The fluorescent protein mKate2 and the antibiotic resistance gene chloramphenicol acetyltransferase (CAT) are targeted to generate representative data and to quantify knockdown effectiveness. PMID:27023729

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

    PubMed Central

    Lindner, Ariel B.; Wintermute, Edwin H.

    2016-01-01

    RNA-mediated knockdowns are widely used to control gene expression. This versatile family of techniques makes use of short RNA (sRNA) that can be synthesized with any sequence and designed to complement any gene targeted for silencing. Because sRNA constructs can be introduced to many cell types directly or using a variety of vectors, gene expression can be repressed in living cells without laborious genetic modification. The most common RNA knockdown technology, RNA interference (RNAi), makes use of the endogenous RNA-induced silencing complex (RISC) to mediate sequence recognition and cleavage of the target mRNA. Applications of this technique are therefore limited to RISC-expressing organisms, primarily eukaryotes. Recently, a new generation of RNA biotechnologists have developed alternative mechanisms for controlling gene expression through RNA, and so made possible RNA-mediated gene knockdowns in bacteria. Here we describe a method for silencing gene expression in E. coli that functionally resembles RNAi. In this system a synthetic phagemid is designed to express sRNA, which may designed to target any sequence. The expression construct is delivered to a population of E. coli cells with non-lytic M13 phage, after which it is able to stably replicate as a plasmid. Antisense recognition and silencing of the target mRNA is mediated by the Hfq protein, endogenous to E. coli. This protocol includes methods for designing the antisense sRNA, constructing the phagemid vector, packaging the phagemid into M13 bacteriophage, preparing a live cell population for infection, and performing the infection itself. The fluorescent protein mKate2 and the antibiotic resistance gene chloramphenicol acetyltransferase (CAT) are targeted to generate representative data and to quantify knockdown effectiveness. PMID:27023729

  2. The baculovirus expression vector system: A commercial manufacturing platform for viral vaccines and gene therapy vectors.

    PubMed

    Felberbaum, Rachael S

    2015-05-01

    The baculovirus expression vector system (BEVS) platform has become an established manufacturing platform for the production of viral vaccines and gene therapy vectors. Nine BEVS-derived products have been approved - four for human use (Cervarix(®), Provenge(®), Glybera(®) and Flublok(®)) and five for veterinary use (Porcilis(®) Pesti, BAYOVAC CSF E2(®), Circumvent(®) PCV, Ingelvac CircoFLEX(®) and Porcilis(®) PCV). The BEVS platform offers many advantages, including manufacturing speed, flexible product design, inherent safety and scalability. This combination of features and product approvals has previously attracted interest from academic researchers, and more recently from industry leaders, to utilize BEVS to develop next generation vaccines, vectors for gene therapy, and other biopharmaceutical complex proteins. In this review, we explore the BEVS platform, detailing how it works, platform features and limitations and important considerations for manufacturing and regulatory approval. To underscore the growth in opportunities for BEVS-derived products, we discuss the latest product developments in the gene therapy and influenza vaccine fields that follow in the wake of the recent product approvals of Glybera(®) and Flublok(®), respectively. We anticipate that the utility of the platform will expand even further as new BEVS-derived products attain licensure. Finally, we touch on some of the areas where new BEVS-derived products are likely to emerge.

  3. Deciphering the impact of parameters influencing transgene expression kinetics after repeated cell transduction with integration-deficient retroviral vectors.

    PubMed

    Schott, Juliane W; Jaeschke, Nico M; Hoffmann, Dirk; Maetzig, Tobias; Ballmaier, Matthias; Godinho, Tamaryin; Cathomen, Toni; Schambach, Axel

    2015-05-01

    Lentiviral and gammaretroviral vectors are state-of-the-art tools for transgene expression within target cells. The integration of these vectors can be deliberately suppressed to derive a transient gene expression system based on extrachromosomal circular episomes with intact coding regions. These episomes can be used to deliver DNA templates and to express RNA or protein. Importantly, transient gene transfer avoids the genotoxic side effects of integrating vectors. Restricting their applicability, episomes are rapidly lost upon dilution in dividing target cells. Addressing this limitation, we could establish comparably stable percentages of transgene-positive cells over prolonged time periods in proliferating cells by repeated transductions. Flow cytometry was applied for kinetic analyses to decipher the impact of individual parameters on the kinetics of fluoroprotein expression after episomal retransduction and to visualize sequential and simultaneous transfer of heterologous fluoroproteins. Expression windows could be exactly timed by the number of transduction steps. The kinetics of signal loss was affected by the cell proliferation rate. The transfer of genes encoding fluoroproteins with different half-lives revealed a major impact of protein stability on temporal signal distribution and accumulation, determining optimal retransduction intervals. In addition, sequential transductions proved broad applicability in different cell types and using different envelope pseudotypes without receptor overload. Stable percentages of cells coexpressing multiple transgenes could be generated upon repeated coadministration of different episomal vectors. Alternatively, defined patterns of transgene expression could be recapitulated by sequential transductions. Altogether, we established a methodology to control and adjust a temporally defined window of transgene expression using retroviral episomal vectors. Combined with the highly efficient cell entry of these vectors while

  4. Telomere Length, TERT, and miRNA Expression

    PubMed Central

    Slattery, Martha L.; Herrick, Jennifer S.; Pellatt, Andrew J.; Wolff, Roger K.; Mullany, Lila E.

    2016-01-01

    It has been proposed that miRNAs are involved in the control of telomeres. We test that hypothesis by examining the association between miRNAs and telomere length (TL). Additionally, we evaluate if genetic variation in telomerase reverse transcriptase (TERT) is associated with miRNA expression levels. We use data from a population-based study of colorectal cancer (CRC), where we have previously shown associations between TL and TERT and CRC, to test associations between TL and miRNA expression and TERT and miRNA expression. To gain insight into functions of miRNAs associated with TERT we tested linear associations between miRNAs and their targeted gene mRNAs. An Agilent platform that contained information on over 2000 miRNAs was used. TL was measured using a multiplexed quantitative PCR (qPCR). RNAseq was used to assess gene expression. Our sample consisted of 1152 individuals with SNP data and miRNA expression data; 363 individuals with both TL and miRNA; and 148 individuals with miRNA and mRNA data. Thirty-three miRNAs were directly associated with TL after adjusting for age and sex (false discovery rate (FDR) of 0.05). TERT rs2736118 was associated with differences in miRNA expression between carcinoma and normal colonic mucosa for 75 miRNAs (FDR <0.05). Genes regulated by these miRNAs, as indicated by mRNA/miRNA associations, were associated with major signaling pathways beyond their TL-related functions, including PTEN, and PI3K/AKT signaling. Our data support a direct association between miRNAs and TL; differences in miRNA expression levels by TERT genotype were observed. Based on miRNA and targeted mRNA associations our data suggest that TERT is involved in non-TL-related functions by acting through altered miRNA expression. PMID:27627813

  5. Telomere Length, TERT, and miRNA Expression.

    PubMed

    Slattery, Martha L; Herrick, Jennifer S; Pellatt, Andrew J; Wolff, Roger K; Mullany, Lila E

    2016-01-01

    It has been proposed that miRNAs are involved in the control of telomeres. We test that hypothesis by examining the association between miRNAs and telomere length (TL). Additionally, we evaluate if genetic variation in telomerase reverse transcriptase (TERT) is associated with miRNA expression levels. We use data from a population-based study of colorectal cancer (CRC), where we have previously shown associations between TL and TERT and CRC, to test associations between TL and miRNA expression and TERT and miRNA expression. To gain insight into functions of miRNAs associated with TERT we tested linear associations between miRNAs and their targeted gene mRNAs. An Agilent platform that contained information on over 2000 miRNAs was used. TL was measured using a multiplexed quantitative PCR (qPCR). RNAseq was used to assess gene expression. Our sample consisted of 1152 individuals with SNP data and miRNA expression data; 363 individuals with both TL and miRNA; and 148 individuals with miRNA and mRNA data. Thirty-three miRNAs were directly associated with TL after adjusting for age and sex (false discovery rate (FDR) of 0.05). TERT rs2736118 was associated with differences in miRNA expression between carcinoma and normal colonic mucosa for 75 miRNAs (FDR <0.05). Genes regulated by these miRNAs, as indicated by mRNA/miRNA associations, were associated with major signaling pathways beyond their TL-related functions, including PTEN, and PI3K/AKT signaling. Our data support a direct association between miRNAs and TL; differences in miRNA expression levels by TERT genotype were observed. Based on miRNA and targeted mRNA associations our data suggest that TERT is involved in non-TL-related functions by acting through altered miRNA expression. PMID:27627813

  6. Novel Strategy to Control Transgene Expression Mediated by a Sendai Virus-Based Vector Using a Nonstructural C Protein and Endogenous MicroRNAs

    PubMed Central

    Ohtaka, Manami; Nakanishi, Mahito

    2016-01-01

    Tissue-specific control of gene expression is an invaluable tool for studying various biological processes and medical applications. Efficient regulatory systems have been utilized to control transgene expression in various types of DNA viral or integrating viral vectors. However, existing regulatory systems are difficult to transfer into negative-strand RNA virus vector platforms because of significant differences in their transcriptional machineries. In this study, we developed a novel strategy for regulating transgene expression mediated by a cytoplasmic RNA vector based on a replication-defective and persistent Sendai virus (SeVdp). Because of the capacity of Sendai virus (SeV) nonstructural C proteins to specifically inhibit viral RNA synthesis, overexpression of C protein significantly reduced transgene expression mediated by SeVdp vectors. We found that SeV C overexpression concomitantly reduced SeVdp mRNA levels and genomic RNA synthesis. To control C expression, target sequences for an endogenous microRNA were incorporated into the 3′ untranslated region of the C genes. Incorporation of target sequences for miR-21 into the SeVdp vector restored transgene expression in HeLa cells by decreasing C expression. Furthermore, the SeVdp vector containing target sequences for let-7a enabled cell-specific control of transgene expression in human fibroblasts and induced pluripotent stem cells. Our findings demonstrate that SeV C can be used as an effective regulator for controlling transgene expression. This strategy will contribute to efficient and less toxic SeVdp-mediated gene transfer in various biological applications. PMID:27764162

  7. [Silencing HSV1 gD expression in cultured cells by RNA interference].

    PubMed

    Zhu, Qin-Chang; Ren, Zhe; Zhang, Chun-Long; Zhang, Mei-Ying; Liao, Hong-Juan; Liu, Qiu-Ying; Zhang, Pei-Zhuo; Li, Jiu-Xiang; Hu, Chao-Feng; Wang, Hua-Dong; Wang, Yi-Fei

    2007-01-01

    To explore the anti-HSV-1 effect of silencing gD gene expression by RNA interference, five 21-nucleotide duplex small interfering RNAs(siRNAs) targeting the HSV1 gD sequence were designed and the gD-EGFP fusion gene expression vector was constructed, then co-transfected into Vero cell, and screened the effective siRNA through analyzing the intensity of the EGFP fluorescence. Finally, the anti-HSV1 effect was confirmed by plaque reduction assay, real-time PCR and daughter virus titration of HSV1 infected Vero cells transfected with siRNAs. The study demonstrated that siRNAs could effectively and specifically inhibit gD gene expression in HSV1-infected cells, but only had a little effect on HSV1 infection, so taking gD as the target of siRNA against HSV1 needs further study.

  8. MicroRNA expression during demosponge dissociation, reaggregation, and differentiation and a evolutionarily conserved demosponge miRNA expression profile.

    PubMed

    Robinson, Jeffrey M

    2015-11-01

    Demosponges share eight orthologous microRNAs (miRNAs), with none in common with Bilateria. Biological functions of these demosponge miRNAs are unknown. Bilaterian miRNAs are key regulators of cellular processes including cell cycle, differentiation, and metabolism. Resolving if demosponge miRNAs participate in such biological functions will provide clues whether these functions are convergent, evidence on the mode of evolution of cellular developmental processes. Here, a quantitative PCR (qPCR) assay was developed and used to test for differential miRNA expression during dissociation and reaggregation in Spongosorites, compare expression profiles between choanosome and cortex in Spongosorites, and compare undifferentiated gemmules to differentiated juveniles in Ephydatia. During Spongosorites dissociation and reaggregation, miRNA expression showed a global decrease in expression across a range of reaggregating cell densities. miRNA differential response could be related to various general cellular responses, potentially related to nutrient-poor conditions of the minimal artificial seawater media, stress response from tissue dissociation, or loss of cell-cell or cell-matrix contact. In Ephydatia, overall increase in miRNA expression in gemmule-hatched stage 4/5 juveniles relative to gemmules is observed, indicating that increased miRNA expression may be related to increased cellular activity such as migration, cell cycle, and/or differentiation. Observed differential miRNA expression of miRNA during dissociation in Spongosorites (lowered global expression), and during activation, and differentiation of Ephydatia gemmules (increased global expression) could indicate that miRNA expression is associated with cell cycle, differentiation, or metabolism pathways. Interspecies comparison was performed, results indicating that orthologous miRNAs share similar relative expression pattern between the four species tested (Spongosorites, Cinachyrella, Haliclona, and Ephydatia

  9. Effect of dual Bt-expression transformation vectors on transgene expression in tobacco.

    PubMed

    Xu, L N; Dong, Y; Zhang, J; Wang, R X; Liu, H M; Yang, Q; Yang, M S

    2016-01-01

    This study aimed to determine the influence of vector structure on dual Bt gene expression and establish an efficient expression vector using Cry1Ac and Cry3A genes. Four vectors (N4, N5, N10, and S23) were developed and used for genetic transformation of tobacco to obtain insect-resistant transgenic lines. The vectors were constructed using the MAR structure, applying different promoter and enhancer sequences, and changing the transgene open-reading frame sequence. The average Cry1Ac toxalbumin expression quantity was 67 times higher in N5 than in N4 transgenic lines (8.77 and 0.13 μg/g, respectively). In contrast, the average Cry3A toxalbumin expression quantity was 1.5 times higher in N4 than in N5 lines (12.70 and 8.21 μg/g, respectively). The sequences of both Bt genes significantly influenced toxalbumin expression, although upstream Bt genes presented lower expression levels. The average Cry1Ac toxalbumin content was 13 times higher in the transgenic lines of AtADH 5'-non-translated sequence N5 (8.77 mg/g) than in the omega N10 lines (0.67 mg/g). Furthermore, the average Cry1Ac toxalbumin content was 5 times higher in MAR N5 than in non-MAR S23 lines (8.77 and 1.63 mg/g, respectively). The average Cry3A toxalbumin content was 1.3 times higher in N5 than in S23 lines (8.21 and 6.48 mg/g, respectively). Moreover, toxalbumin expression levels differed significantly among the S23-transformed lines. The MAR structure applied on both ends of the genes increased both the level and stability of exogenous gene expression. In conclusion, N5 was the most optimal of the four tested vectors. PMID:27525887

  10. Novel redox nanomedicine improves gene expression of polyion complex vector

    NASA Astrophysics Data System (ADS)

    Toh, Kazuko; Yoshitomi, Toru; Ikeda, Yutaka; Nagasaki, Yukio

    2011-12-01

    Gene therapy has generated worldwide attention as a new medical technology. While non-viral gene vectors are promising candidates as gene carriers, they have several issues such as toxicity and low transfection efficiency. We have hypothesized that the generation of reactive oxygen species (ROS) affects gene expression in polyplex supported gene delivery systems. The effect of ROS on the gene expression of polyplex was evaluated using a nitroxide radical-containing nanoparticle (RNP) as an ROS scavenger. When polyethyleneimine (PEI)/pGL3 or PEI alone was added to the HeLa cells, ROS levels increased significantly. In contrast, when (PEI)/pGL3 or PEI was added with RNP, the ROS levels were suppressed. The luciferase expression was increased by the treatment with RNP in a dose-dependent manner and the cellular uptake of pDNA was also increased. Inflammatory cytokines play an important role in ROS generation in vivo. In particular, tumor necrosis factor (TNF)-α caused intracellular ROS generation in HeLa cells and decreased gene expression. RNP treatment suppressed ROS production even in the presence of TNF-α and increased gene expression. This anti-inflammatory property of RNP suggests that it may be used as an effective adjuvant for non-viral gene delivery systems.

  11. [Immune efficacy of rabies virus glycoprotein expressed by baculovirus vector].

    PubMed

    Chen, Qi; Zhang, Shou-Feng; Liu, Ye; Fu, Yun-Hong; Sun, Cheng-Long; Yang, Yang; Gong, Ting; Song, Fei-Fei; Hu, Rong-Liang

    2012-09-01

    To construct a recombinant baculovirus expressing glycoprotein (GP) of RV SRV9 strain and test the immunological efficacy in mice, open reading frame of rabies virus GP gene of SRV9 strain was cloned into the shuttle vector Bacmid to construct the recombinant shuttle plasmid Bacmid-G and transfection was performed into S f9 cells with the recombinant shuttle plasmid. CPE appeared in cell cultures was identified by electronmicroscopy. Western-blot, IFA and immunity tests in mice were performed to identify the immunoreactivity and immunogenicity of the expression products. Our results showed a recombinant baculovirus expressing GP protein of rabies virus SRV9 was obtained. The expression products possessed a favorable immunogenicity and fall immunized mice could develop 100% protective level of anti-rabies neutralizing antibody. In conclusion, The SRV9 glycoprotein expressed by the recombinant baculovirus in this study had good immunogenicity and could induce anti-rabies neutralizing antibody, which laid the foundation of further development of rabies subunit vaccine.

  12. Intramammary expression and therapeutic effect of a human lysozyme-expressing vector for treating bovine mastitis*

    PubMed Central

    Sun, Huai-Chang; Xue, Fang-Ming; Qian, Ke; Fang, Hao-Xia; Qiu, Hua-Lei; Zhang, Xin-Yu; Yin, Zhao-Hua

    2006-01-01

    To develop a gene therapy strategy for treating bovine mastitis, a new mammary-specific vector containing human lysozyme (hLYZ) cDNA and kanamycin resistance gene was constructed for intramammary expression and clinical studies. After one time acupuncture or intracisternal infusion of healthy cows with 400 μg of the p215C3LYZ vector, over 2.0 μg/ml of rhLYZ could be detected by enzymatic assay for about 3 weeks in the milk samples. Western blotting showed that rhLYZ secreted into milk samples from the vector-injected cows had molecular weight similar to that of the natural hLYZ in human colostrums. Twenty days after the primary injection, the quarters were re-injected with the same vector by quarter acupuncture and even higher concentrations of rhLYZ could be detected. Indirect competitive ELISA of milk samples showed that the vector injection did not induce detectable humoral immune response against hLYZ. Clinical studies showed that twice acupuncture of quarters with the p215C3LYZ vector had overt therapeutic effect on clinical and subclinical mastitis previously treated with antibiotics, including disappearance of clinical symptoms and relatively high microbiological cure rates. These data provide a solid rationale for using the vector to develop gene therapy for treating bovine mastitis. PMID:16532537

  13. Decreasing lncRNA HOTAIR expression inhibits human colorectal cancer stem cells

    PubMed Central

    Dou, Jun; Ni, Yaoyao; He, Xiangfeng; Wu, Di; Li, Miao; Wu, Songyan; Zhang, Rong; Guo, Mei; Zhao, Fengsu

    2016-01-01

    Research on the relationship between aberrant long non-coding RNA (lncRNA) and cancer stem cell (CSC) biology in cancer patients has been recently gaining attention. The goal of this study was to investigate whether the decreasing lncRNA HOTAIR expression would inhibit human colorectal cancer (CRC) stem cells. CD133+CSCs were isolated from human CRC LoVo cell line by using a magnetic-activated cell sorting system, and were transfected with the expression vector-based small hairpin RNA targeting HOTAIR (shHOTAIR). The ability of cellular proliferation, migration, invasion, colony-forming, and the epithelial-mesenchymal transition (EMT)-associated molecule expression as well as the tumorigenicity of CD133+-shHOTAIR were evaluated by the MTT, wound-healing, cellular invasion, colony formation and Western blot assays, respectively. This study found that, when compared with control cells in vitro, CD133+-shHOTAIR exhibited the decreased HOTAIR expression, suppressed cellular proliferation, migration, invasion, colony-forming, and inhibited the Vimentin expression with increased E-cadherin expression. In particular, the down-regulation of the HOTAIR expression in CD133+CSCs markedly attenuated the tumor growth and lung metastasis in xenograft nude mice. Taken together, this study found that down-regulating the HOTAIR expression in CD133+CSCs could serve as a potential anti-cancer regimen to inhibit the invasiveness and metastasis of CRC CSCs. PMID:27069543

  14. Right ventricular long noncoding RNA expression in human heart failure.

    PubMed

    Di Salvo, Thomas G; Guo, Yan; Su, Yan Ru; Clark, Travis; Brittain, Evan; Absi, Tarek; Maltais, Simon; Hemnes, Anna

    2015-03-01

    The expression of long noncoding RNAs (lncRNAs) in human heart failure (HF) has not been widely studied. Using RNA sequencing (RNA-Seq), we compared lncRNA expression in 22 explanted human HF hearts with lncRNA expression in 5 unused donor human hearts. We used Cufflinks to identify isoforms and DESeq to identify differentially expressed genes. We identified the noncoding RNAs by cross-reference to Ensembl release 73 (Genome Reference Consortium human genome build 37) and explored possible functional roles using a variety of online tools. In HF hearts, RNA-Seq identified 84,793 total messenger RNA coding and noncoding different transcripts, including 13,019 protein-coding genes, 2,085 total lncRNA genes, and 1,064 pseudogenes. By Ensembl noncoding RNA categories, there were 48 lncRNAs, 27 pseudogenes, and 30 antisense RNAs for a total of 105 differentially expressed lncRNAs in HF hearts. Compared with donor hearts, HF hearts exhibited differential expression of 7.7% of protein-coding genes, 3.7% of lncRNAs (including pseudogenes), and 2.5% of pseudogenes. There were not consistent correlations between antisense lncRNAs and parent genes and between pseudogenes and parent genes, implying differential regulation of expression. Exploratory in silico functional analyses using online tools suggested a variety of possible lncRNA regulatory roles. By providing a comprehensive profile of right ventricular polyadenylated messenger RNA transcriptome in HF, RNA-Seq provides an inventory of differentially expressed lncRNAs, including antisense transcripts and pseudogenes, for future mechanistic study.

  15. Biodegradable nanoparticles of mPEG-PLGA-PLL triblock copolymers as novel non-viral vectors for improving siRNA delivery and gene silencing.

    PubMed

    Du, Jing; Sun, Ying; Shi, Qiu-Sheng; Liu, Pei-Feng; Zhu, Ming-Jie; Wang, Chun-Hui; Du, Lian-Fang; Duan, You-Rong

    2012-01-01

    Degradation of mRNA by RNA interference is one of the most powerful and specific mechanisms for gene silencing. However, insufficient cellular uptake and poor stability have limited its usefulness. Here, we report efficient delivery of siRNA via the use of biodegradable nanoparticles (NPs) made from monomethoxypoly(ethylene glycol)-poly(lactic-co-glycolic acid)-poly-l-lysine (mPEG-PLGA-PLL) triblock copolymers. Various physicochemical properties of mPEG-PLGA-PLL NPs, including morphology, size, surface charge, siRNA encapsulation efficiency, and in vitro release profile of siRNA from NPs, were characterized by scanning electron microscope, particle size and zeta potential analyzer, and high performance liquid chromatography. The levels of siRNA uptake and targeted gene inhibition were detected in human lung cancer SPC-A1-GFP cells stably expressing green fluorescent protein. Examination of the cultured SPC-A1-GFP cells with fluorescent microscope and flow cytometry showed NPs loading Cy3-labeled siRNA had much higher intracellular siRNA delivery efficiencies than siRNA alone and Lipofectamine-siRNA complexes. The gene silencing efficiency of mPEG-PLGA-PLL NPs was higher than that of commercially available transfecting agent Lipofectamine while showing no cytotoxicity. Thus, the current study demonstrates that biodegradable NPs of mPEG-PLGA-PLL triblock copolymers can be potentially applied as novel non-viral vectors for improving siRNA delivery and gene silencing.

  16. Biodegradable Nanoparticles of mPEG-PLGA-PLL Triblock Copolymers as Novel Non-Viral Vectors for Improving siRNA Delivery and Gene Silencing

    PubMed Central

    Du, Jing; Sun, Ying; Shi, Qiu-Sheng; Liu, Pei-Feng; Zhu, Ming-Jie; Wang, Chun-Hui; Du, Lian-Fang; Duan, You-Rong

    2012-01-01

    Degradation of mRNA by RNA interference is one of the most powerful and specific mechanisms for gene silencing. However, insufficient cellular uptake and poor stability have limited its usefulness. Here, we report efficient delivery of siRNA via the use of biodegradable nanoparticles (NPs) made from monomethoxypoly(ethylene glycol)-poly(lactic-co-glycolic acid)-poly-l-lysine (mPEG-PLGA-PLL) triblock copolymers. Various physicochemical properties of mPEG-PLGA-PLL NPs, including morphology, size, surface charge, siRNA encapsulation efficiency, and in vitro release profile of siRNA from NPs, were characterized by scanning electron microscope, particle size and zeta potential analyzer, and high performance liquid chromatography. The levels of siRNA uptake and targeted gene inhibition were detected in human lung cancer SPC-A1-GFP cells stably expressing green fluorescent protein. Examination of the cultured SPC-A1-GFP cells with fluorescent microscope and flow cytometry showed NPs loading Cy3-labeled siRNA had much higher intracellular siRNA delivery efficiencies than siRNA alone and Lipofectamine-siRNA complexes. The gene silencing efficiency of mPEG-PLGA-PLL NPs was higher than that of commercially available transfecting agent Lipofectamine while showing no cytotoxicity. Thus, the current study demonstrates that biodegradable NPs of mPEG-PLGA-PLL triblock copolymers can be potentially applied as novel non-viral vectors for improving siRNA delivery and gene silencing. PMID:22312268

  17. siRNA-Mediated Silencing of doublesex during Female Development of the Dengue Vector Mosquito Aedes aegypti

    PubMed Central

    Mysore, Keshava; Sun, Longhua; Tomchaney, Michael; Sullivan, Gwyneth; Adams, Haley; Piscoya, Andres S.; Severson, David W.; Syed, Zainulabeuddin; Duman-Scheel, Molly

    2015-01-01

    The development of sex-specific traits, including the female-specific ability to bite humans and vector disease, is critical for vector mosquito reproduction and pathogen transmission. Doublesex (Dsx), a terminal transcription factor in the sex determination pathway, is known to regulate sex-specific gene expression during development of the dengue fever vector mosquito Aedes aegypti. Here, the effects of developmental siRNA-mediated dsx silencing were assessed in adult females. Targeting of dsx during A. aegypti development resulted in decreased female wing size, a correlate for body size, which is typically larger in females. siRNA-mediated targeting of dsx also resulted in decreased length of the adult female proboscis. Although dsx silencing did not impact female membrane blood feeding or mating behavior in the laboratory, decreased fecundity and fertility correlated with decreased ovary length, ovariole length, and ovariole number in dsx knockdown females. Dsx silencing also resulted in disruption of olfactory system development, as evidenced by reduced length of the female antenna and maxillary palp and the sensilla present on these structures, as well as disrupted odorant receptor expression. Female lifespan, a critical component of the ability of A. aegypti to transmit pathogens, was also significantly reduced in adult females following developmental targeting of dsx. The results of this investigation demonstrate that silencing of dsx during A. aegypti development disrupts multiple sex-specific morphological, physiological, and behavioral traits of adult females, a number of which are directly or indirectly linked to mosquito reproduction and pathogen transmission. Moreover, the olfactory phenotypes observed connect Dsx to development of the olfactory system, suggesting that A. aegypti will be an excellent system in which to further assess the developmental genetics of sex-specific chemosensation. PMID:26544686

  18. siRNA-Mediated Silencing of doublesex during Female Development of the Dengue Vector Mosquito Aedes aegypti.

    PubMed

    Mysore, Keshava; Sun, Longhua; Tomchaney, Michael; Sullivan, Gwyneth; Adams, Haley; Piscoya, Andres S; Severson, David W; Syed, Zainulabeuddin; Duman-Scheel, Molly

    2015-11-01

    The development of sex-specific traits, including the female-specific ability to bite humans and vector disease, is critical for vector mosquito reproduction and pathogen transmission. Doublesex (Dsx), a terminal transcription factor in the sex determination pathway, is known to regulate sex-specific gene expression during development of the dengue fever vector mosquito Aedes aegypti. Here, the effects of developmental siRNA-mediated dsx silencing were assessed in adult females. Targeting of dsx during A. aegypti development resulted in decreased female wing size, a correlate for body size, which is typically larger in females. siRNA-mediated targeting of dsx also resulted in decreased length of the adult female proboscis. Although dsx silencing did not impact female membrane blood feeding or mating behavior in the laboratory, decreased fecundity and fertility correlated with decreased ovary length, ovariole length, and ovariole number in dsx knockdown females. Dsx silencing also resulted in disruption of olfactory system development, as evidenced by reduced length of the female antenna and maxillary palp and the sensilla present on these structures, as well as disrupted odorant receptor expression. Female lifespan, a critical component of the ability of A. aegypti to transmit pathogens, was also significantly reduced in adult females following developmental targeting of dsx. The results of this investigation demonstrate that silencing of dsx during A. aegypti development disrupts multiple sex-specific morphological, physiological, and behavioral traits of adult females, a number of which are directly or indirectly linked to mosquito reproduction and pathogen transmission. Moreover, the olfactory phenotypes observed connect Dsx to development of the olfactory system, suggesting that A. aegypti will be an excellent system in which to further assess the developmental genetics of sex-specific chemosensation. PMID:26544686

  19. siRNA-Mediated Silencing of doublesex during Female Development of the Dengue Vector Mosquito Aedes aegypti.

    PubMed

    Mysore, Keshava; Sun, Longhua; Tomchaney, Michael; Sullivan, Gwyneth; Adams, Haley; Piscoya, Andres S; Severson, David W; Syed, Zainulabeuddin; Duman-Scheel, Molly

    2015-11-01

    The development of sex-specific traits, including the female-specific ability to bite humans and vector disease, is critical for vector mosquito reproduction and pathogen transmission. Doublesex (Dsx), a terminal transcription factor in the sex determination pathway, is known to regulate sex-specific gene expression during development of the dengue fever vector mosquito Aedes aegypti. Here, the effects of developmental siRNA-mediated dsx silencing were assessed in adult females. Targeting of dsx during A. aegypti development resulted in decreased female wing size, a correlate for body size, which is typically larger in females. siRNA-mediated targeting of dsx also resulted in decreased length of the adult female proboscis. Although dsx silencing did not impact female membrane blood feeding or mating behavior in the laboratory, decreased fecundity and fertility correlated with decreased ovary length, ovariole length, and ovariole number in dsx knockdown females. Dsx silencing also resulted in disruption of olfactory system development, as evidenced by reduced length of the female antenna and maxillary palp and the sensilla present on these structures, as well as disrupted odorant receptor expression. Female lifespan, a critical component of the ability of A. aegypti to transmit pathogens, was also significantly reduced in adult females following developmental targeting of dsx. The results of this investigation demonstrate that silencing of dsx during A. aegypti development disrupts multiple sex-specific morphological, physiological, and behavioral traits of adult females, a number of which are directly or indirectly linked to mosquito reproduction and pathogen transmission. Moreover, the olfactory phenotypes observed connect Dsx to development of the olfactory system, suggesting that A. aegypti will be an excellent system in which to further assess the developmental genetics of sex-specific chemosensation.

  20. A primer vector system that allows temperature dependent gene amplification and expression in mammalian cells: regulation of the influenza virus NS1 gene expression.

    PubMed

    Portela, A; Melero, J A; Martínez, C; Domingo, E; Ortín, J

    1985-11-25

    Influenza virus RNA segment 8 has been cloned into primer-vector pSLts1. This vector was designed to replicate in simian cells in a temperature dependent fashion by use of the SV40 tsA209 T-antigen gene. The oriented synthesis of cDNA on dT-tailed pSLts1 was performed on in vitro synthesized mRNA, and the second DNA strand was primed with an influenza-specific terminal oligodeoxynucleotide. Recombinant pSLVa232 contained the RNA segment 8 sequence directly fused to the SV40 late promoter contained in pSLts1, and followed by the SV40 polyadenylation signal. Expression of NS1 gene in transfected COS cells took place at a level comparable to that found in infected cells. When VERO cell cultures were transfected with recombinant pSLVa232, expression of the NS1 gene was temperature dependent. Close to one hundred fold increase in the amplification and expression of the cloned gene was observed after shift down of the transfected cells to permissive temperature. Vector pSLts1 and the cloning strategy described may be useful for the specific cloning and regulated expression of mRNAs of known 5'-terminal sequence.

  1. Combining miRNA and mRNA Expression Profiles in Wilms Tumor Subtypes

    PubMed Central

    Ludwig, Nicole; Werner, Tamara V.; Backes, Christina; Trampert, Patrick; Gessler, Manfred; Keller, Andreas; Lenhof, Hans-Peter; Graf, Norbert; Meese, Eckart

    2016-01-01

    Wilms tumor (WT) is the most common childhood renal cancer. Recent findings of mutations in microRNA (miRNA) processing proteins suggest a pivotal role of miRNAs in WT genesis. We performed miRNA expression profiling of 36 WTs of different subtypes and four normal kidney tissues using microarrays. Additionally, we determined the gene expression profile of 28 of these tumors to identify potentially correlated target genes and affected pathways. We identified 85 miRNAs and 2107 messenger RNAs (mRNA) differentially expressed in blastemal WT, and 266 miRNAs and 1267 mRNAs differentially expressed in regressive subtype. The hierarchical clustering of the samples, using either the miRNA or mRNA profile, showed the clear separation of WT from normal kidney samples, but the miRNA pattern yielded better separation of WT subtypes. A correlation analysis of the deregulated miRNA and mRNAs identified 13,026 miRNA/mRNA pairs with inversely correlated expression, of which 2844 are potential interactions of miRNA and their predicted mRNA targets. We found significant upregulation of miRNAs-183, -301a/b and -335 for the blastemal subtype, and miRNAs-181b, -223 and -630 for the regressive subtype. We found marked deregulation of miRNAs regulating epithelial to mesenchymal transition, especially in the blastemal subtype, and miRNAs influencing chemosensitivity, especially in regressive subtypes. Further research is needed to assess the influence of preoperative chemotherapy and tumor infiltrating lymphocytes on the miRNA and mRNA patterns in WT. PMID:27043538

  2. Expression of cellulase genes in Rhodobacter capsulatus by use of plasmid expression vectors.

    PubMed Central

    Johnson, J A; Wong, W K; Beatty, J T

    1986-01-01

    Broad-host-range plasmid vectors were constructed for expression of heterologous genes in the photosynthetic bacterium Rhodobacter capsulatus. These plasmids utilize an RK2-derived replicon for maintenance and conjugative transfer and the R. capsulatus rxcA promoter to obtain transcription of genes within appropriately positioned DNA fragments. The expression vectors were used to obtain synthesis of endoglucanase and exoglucanase in R. capsulatus from cellulase genes present on exogenously derived DNA fragments. The cellulase genes were expressed either by use of their native translation initiation signals or by in-frame fusion with the rxcA B870 beta gene translation initiation signals to form a hybrid protein. The level of cellulase gene expression was found to be modulated in response to the extent of aeration of plasmid host cultures. Images PMID:3090019

  3. Complete reduction of p53 expression by RNA interference following heterozygous knockout in porcine fibroblasts.

    PubMed

    Kim, Young June; Kim, Tae-Hyun; Kim, Minjeong; Kim, Min Ju; Kim, Hae-Won; Shim, Hosup

    2016-08-01

    Tumor suppressor p53 plays a critical role in the regulation of cell cycle and apoptosis in mammals. Mutations of p53 often cause various cancers. Murine models have improved our understanding on tumorigenesis associated with p53 mutations. However, mice and humans are different in many ways. For example, the short lifespans of mice limit the clinical application of the data obtained from this species. Porcine model could be an alternative as pigs share many anatomical and physiological similarities with humans. Here, we modified the expression levels of p53 messenger RNA (mRNA) and protein in porcine fetal fibroblasts using a combination of gene targeting and RNA interference. First, we disrupted the p53 gene to produce p53 knockout (KO) cells. Second, the p53 shRNA expression vector was introduced into fibroblasts to isolate p53 knockdown (KD) cells. We obtained p53 KO, KD, and KO + KD fibroblasts which involve p53 KO and KD either separately or simultaneously. The mRNA expression of p53 in p53 KO fibroblasts was similar to that in the wild-type control. However, the mRNA expression levels of p53 in KD and KO + KD cells were significantly decreased. The p53 protein level significant reduced in p53 KD. Interestingly, no p53 protein was detected in KO + KD, suggesting a complete reduction of the protein by synergistic effect of KO and KD. This study demonstrated that various expression levels of p53 in porcine fibroblasts could be achieved by gene targeting and RNA interference. Moreover, complete abolishment of protein expression is feasible using a combination of gene targeting and RNA interference.

  4. Complete reduction of p53 expression by RNA interference following heterozygous knockout in porcine fibroblasts.

    PubMed

    Kim, Young June; Kim, Tae-Hyun; Kim, Minjeong; Kim, Min Ju; Kim, Hae-Won; Shim, Hosup

    2016-08-01

    Tumor suppressor p53 plays a critical role in the regulation of cell cycle and apoptosis in mammals. Mutations of p53 often cause various cancers. Murine models have improved our understanding on tumorigenesis associated with p53 mutations. However, mice and humans are different in many ways. For example, the short lifespans of mice limit the clinical application of the data obtained from this species. Porcine model could be an alternative as pigs share many anatomical and physiological similarities with humans. Here, we modified the expression levels of p53 messenger RNA (mRNA) and protein in porcine fetal fibroblasts using a combination of gene targeting and RNA interference. First, we disrupted the p53 gene to produce p53 knockout (KO) cells. Second, the p53 shRNA expression vector was introduced into fibroblasts to isolate p53 knockdown (KD) cells. We obtained p53 KO, KD, and KO + KD fibroblasts which involve p53 KO and KD either separately or simultaneously. The mRNA expression of p53 in p53 KO fibroblasts was similar to that in the wild-type control. However, the mRNA expression levels of p53 in KD and KO + KD cells were significantly decreased. The p53 protein level significant reduced in p53 KD. Interestingly, no p53 protein was detected in KO + KD, suggesting a complete reduction of the protein by synergistic effect of KO and KD. This study demonstrated that various expression levels of p53 in porcine fibroblasts could be achieved by gene targeting and RNA interference. Moreover, complete abolishment of protein expression is feasible using a combination of gene targeting and RNA interference. PMID:27142766

  5. An animal model of PDH deficiency using AAV8-siRNA vector-mediated knockdown of pyruvate dehydrogenase E1α

    PubMed Central

    Ojano-Dirain, Carolyn; Glushakova, Lyudmyla G.; Zhong, Li; Zolotukhin, Sergei; Muzyczka, Nicholas; Srivastava, Arun; Stacpoole, Peter W.

    2010-01-01

    We evaluated the feasibility of self-complementary adeno-associated virus (scAAV) vector-mediated knockdown of the pyruvate dehydrogenase complex using small interfering RNAs directed against the E1α subunit gene (PDHA1). AAV serotype 8 was used to stereotaxically deliver scAAV8-si3-PDHA1-Enhanced Green Fluorescent Protein (knockdown) or scAAV8-EGFP (control) vectors into the right striatum and substantia nigra of rats. Rotational asymmetry was employed to quantify abnormal rotation following neurodegeneration in the nigrostriatal system. By 20 weeks after surgery, the siRNA-injected rats exhibited higher contralateral rotation during the first 10 min following amphetamine administration and lower 90-min total rotations (p≤0.05). Expression of PDC E1α, E1β and E2 subunits in striatum was decreased (p≤0.05) in the siRNA-injected striatum after 14 weeks. By week 25, both PDC activity and expression of E1α were lower (p≤0.05) in siRNA-injected striata compared to controls. E1α expression was associated with PDC activity (R2=0.48; p=0.006) and modestly associated with counterclockwise rotation (R2=0.51;p=0.07). The use of tyrosine-mutant scAAV8 vectors resulted in ~17-fold increase in transduction efficiency of rat striatal neurons in vivo. We conclude that scAAV8-siRNA vector-mediated knockdown of PDC E1α in brain regions typically affected in humans with PDC deficiency results in a reproducible biochemical and clinical phenotype in rats that may be further enhanced with the use of tyrosine-mutant vectors. PMID:20685142

  6. MicroRNA-339 and microRNA-556 regulate Klotho expression in vitro.

    PubMed

    Mehi, Stephen J; Maltare, Astha; Abraham, Carmela R; King, Gwendalyn D

    2014-02-01

    Klotho is an anti-aging protein with direct effects on life-span in mice. Klotho functions to regulate pathways classically associated with longevity including insulin/IGF1 and Wnt signaling. Decreased Klotho protein expression is observed throughout the body during the normal aging process. While increased methylation of the Klotho promoter is reported, other epigenetic mechanisms could contribute to age-related downregulation of Klotho expression, including microRNA-mediated regulation. Following in silico identification of potential microRNA binding sites within the Klotho 3' untranslated region, reporter assays reveal regulation by microRNA-339, microRNA-556, and, to a lesser extent, microRNA-10 and microRNA-199. MicroRNA-339 and microRNA-556 were further found to directly decrease Klotho protein expression indicating that, if upregulated in aging tissue, these microRNA could play a role in age-related downregulation of Klotho messenger RNA. These microRNAs are differentially regulated in cancer cells compared to normal cells and may imply a role for microRNA-mediated regulation of Klotho in cancer. PMID:23818104

  7. Self-assembled Messenger RNA Nanoparticles (mRNA-NPs) for Efficient Gene Expression

    PubMed Central

    Kim, Hyejin; Park, Yongkuk; Lee, Jong Bum

    2015-01-01

    Although mRNA has several advantages over plasmid DNA when delivered into cells for gene expression, mRNA transfection is a very rare occurrence in gene delivery. This is mainly because of the labile nature of RNA, resulting in a low expression level of the desired protein. In this study, self-assembled mRNA nanoparticles (mRNA-NPs) packed with multiple repeats of mRNA were synthesized to achieve efficient gene expression. This approach required only a one-step process to synthesize particles with a minimal amount of plasmid DNA to produce the RNA transcripts via rolling circle transcription. Moreover, there are no concerns for cytotoxicity which can be caused by chemical condensates because mRNA-NPs are made entirely of mRNA. An examination of the cells transfected with the mRNA-NPs encoding the green fluorescence protein (GFP) confirmed that the mRNA-NPs can be used as a novel platform for effective gene delivery. PMID:26235529

  8. A Lentiviral Vector Expressing Desired Gene Only in Transduced Cells: An Approach for Suicide Gene Therapy.

    PubMed

    Mohammadi, Zahra; Shariati, Laleh; Khanahmad, Hossein; Kolahdouz, Mahsa; Kianpoor, Fariborz; Ghanbari, Jahan Afrooz; Hejazi, Zahra; Salehi, Mansoor; Nikpour, Parvaneh; Tabatabaiefar, Mohammad Amin

    2015-09-01

    Suicide gene therapy is a therapeutic strategy, in which cell suicide inducing transgenes are introduced into target cells. Inserting a toxin-encoding gene into a lentiviral vector leads to decreased efficiency of virus production due to lethal effect of toxin on packaging cells. In this study, we designed and constructed a transfer vector to express the toxin in transduced cells but not in packaging cells. Plasmid pLenti-F/GFP was constructed by cutting out R 5'LTR-R 3'LTR fragment with the AflII restriction endonuclease from a plasmid pLenti4-GW/H1/TO-laminshRNA, followed by ligating R 5'LTR-R 3'LTR fragment, constructed by three PCR stages. The promoter and GFP CDS were inserted in opposite strand. For lentiviral production, the HEK293T cell line was co-transfected with the PMD2G, psPAX2, and pLenti-F/GFP plasmids (envelope, packaging, and transfer plasmids).Viral vector titers were assayed. The HEK293T cell line was transduced with this virus. PCR was performed to confirm the presence of the promoter fragment between the R and U5 in 3'LTR. The lentivirus titers were approximately 2 × 10(5). The GFP expression was seen in 51 % of the HEK293T cells transduced with lentivirus. The PCR product size was 1440 bp confirming the promoter fragment position between the R and U5 in 3'LTR. The strategy enables us to use a broad spectrum of toxin genes in gene therapy and helps avoid the death of the packaging cells with lentiviral vectors carrying a toxin-encoding gene, thereby increasing the efficiency of viral production in packaging cells.

  9. Inhibition of luciferase expression in transgenic Aedes aegypti mosquitoes by Sindbis virus expression of antisense luciferase RNA

    PubMed Central

    Johnson, Barbara W.; Olson, Ken E.; Allen-Miura, Tanya; Rayms-Keller, Alfredo; Carlson, Jonathan O.; Coates, Craig J.; Jasinskiene, Nijole; James, Anthony A.; Beaty, Barry J.; Higgs, Stephen

    1999-01-01

    A rapid and reproducible method of inhibiting the expression of specific genes in mosquitoes should further our understanding of gene function and may lead to the identification of mosquito genes that determine vector competence or are involved in pathogen transmission. We hypothesized that the virus expression system based on the mosquito-borne Alphavirus, Sindbis (Togaviridae), may efficiently transcribe effector RNAs that inhibit expression of a targeted mosquito gene. To test this hypothesis, germ-line-transformed Aedes aegypti that express luciferase (LUC) from the mosquito Apyrase promoter were intrathoracically inoculated with a double subgenomic Sindbis (dsSIN) virus TE/3′2J/anti-luc (Anti-luc) that transcribes RNA complementary to the 5′ end of the LUC mRNA. LUC activity was monitored in mosquitoes infected with either Anti-luc or control dsSIN viruses expressing unrelated antisense RNAs. Mosquitoes infected with Anti-luc virus exhibited 90% reduction in LUC compared with uninfected and control dsSIN-infected mosquitoes at 5 and 9 days postinoculation. We demonstrate that a gene expressed from the mosquito genome can be inhibited by using an antisense strategy. The dsSIN antisense RNA expression system is an important tool for studying gene function in vivo. PMID:10557332

  10. Inhibition of luciferase expression in transgenic Aedes aegypti mosquitoes by Sindbis virus expression of antisense luciferase RNA.

    PubMed

    Johnson, B W; Olson, K E; Allen-Miura, T; Rayms-Keller, A; Carlson, J O; Coates, C J; Jasinskiene, N; James, A A; Beaty, B J; Higgs, S

    1999-11-01

    A rapid and reproducible method of inhibiting the expression of specific genes in mosquitoes should further our understanding of gene function and may lead to the identification of mosquito genes that determine vector competence or are involved in pathogen transmission. We hypothesized that the virus expression system based on the mosquito-borne Alphavirus, Sindbis (Togaviridae), may efficiently transcribe effector RNAs that inhibit expression of a targeted mosquito gene. To test this hypothesis, germ-line-transformed Aedes aegypti that express luciferase (LUC) from the mosquito Apyrase promoter were intrathoracically inoculated with a double subgenomic Sindbis (dsSIN) virus TE/3'2J/anti-luc (Anti-luc) that transcribes RNA complementary to the 5' end of the LUC mRNA. LUC activity was monitored in mosquitoes infected with either Anti-luc or control dsSIN viruses expressing unrelated antisense RNAs. Mosquitoes infected with Anti-luc virus exhibited 90% reduction in LUC compared with uninfected and control dsSIN-infected mosquitoes at 5 and 9 days postinoculation. We demonstrate that a gene expressed from the mosquito genome can be inhibited by using an antisense strategy. The dsSIN antisense RNA expression system is an important tool for studying gene function in vivo. PMID:10557332

  11. Intratumoral Heterogeneity of MicroRNA Expression in Rectal Cancer

    PubMed Central

    Andersen, Rikke Fredslund; Nielsen, Boye Schnack; Sørensen, Flemming Brandt; Appelt, Ane Lindegaard; Jakobsen, Anders; Hansen, Torben Frøstrup

    2016-01-01

    Introduction An increasing number of studies have investigated microRNAs (miRNAs) as potential markers of diagnosis, treatment and prognosis. So far, agreement between studies has been minimal, which may in part be explained by intratumoral heterogeneity of miRNA expression. The aim of the present study was to assess the heterogeneity of a panel of selected miRNAs in rectal cancer, using two different technical approaches. Materials and Methods The expression of the investigated miRNAs was analysed by real-time quantitative polymerase chain reaction (RT-qPCR) and in situ hybridization (ISH) in tumour specimens from 27 patients with T3-4 rectal cancer. From each tumour, tissue from three different luminal localisations was examined. Inter- and intra-patient variability was assessed by calculating intraclass correlation coefficients (ICCs). Correlations between RT-qPCR and ISH were evaluated using Spearman’s correlation. Results ICCsingle (one sample from each patient) was higher than 50% for miRNA-21 and miRNA-31. For miRNA-125b, miRNA-145, and miRNA-630, ICCsingle was lower than 50%. The ICCmean (mean of three samples from each patient) was higher than 50% for miRNA-21(RT-qPCR and ISH), miRNA-125b (RT-qPCR and ISH), miRNA-145 (ISH), miRNA-630 (RT-qPCR), and miRNA-31 (RT-qPCR). For miRNA-145 (RT-qPCR) and miRNA-630 (ISH), ICCmean was lower than 50%. Spearman correlation coefficients, comparing results obtained by RT-qPCR and ISH, respectively, ranged from 0.084 to 0.325 for the mean value from each patient, and from -0.085 to 0.515 in the section including the deepest part of the tumour. Conclusion Intratumoral heterogeneity may influence the measurement of miRNA expression and consequently the number of samples needed for representative estimates. Our findings with two different methods suggest that one sample is sufficient for adequate assessment of miRNA-21 and miRNA-31, whereas more samples would improve the assessment of miRNA-125b, miRNA-145, and miRNA-630

  12. Increasing the efficiency of homologous recombination vector-mediated end joining repair by inhibition of Lig4 gene using siRNA in sheep embryo fibroblasts.

    PubMed

    Wei, Wang; Yushuang, Wang; Lanlan, Huang; Zijian, Jian; Xinhua, Wang; Shouren, Liu; Wenhui, Pi

    2016-09-01

    In animal cells, inhibition of non-homologous end joining (NHEJ) pathway improves the efficiency of homologous recombination (HR)-mediated double-strand brakes (DSBs) repair. To improve the efficiency of HR in sheep embryo fibroblasts, the NHEJ key molecule DNA ligase 4 (Lig4) was suppressed by siRNA interference. Four pairs of siRNA targeting Lig4 were designed and chemically synthesized. These siRNA were electro-transferred into sheep embryo fibroblasts respectively. Compared with the control groups, two pairs of siRNA were identified to effectively inhibit the expression of sheep Lig4 gene by qRT-PCR and Western blotting. The plasmid rejoining assay was adopted for examining the efficiency of HR-mediated DSB repair. I-SceⅠ endonuclease linearized vector and siRNA were co-transfected into sheep embryo fibroblasts. Flow cytometry analysis of cells after transfection for 72 h showed that suppression of Lig4 using siRNAs increased the rejoining efficiency of HR vector by 3-4 times compared with the control groups. Therefore, enhanced HR vector rejoining frequency by instant inhabition of Lig4 gene provides theoretical basis for improving gene targeting efficiency of sheep embryo fibroblasts. PMID:27644744

  13. RNA-based, transient modulation of gene expression in human haematopoietic stem and progenitor cells

    PubMed Central

    Diener, Yvonne; Jurk, Marion; Kandil, Britta; Choi, Yeong-Hoon; Wild, Stefan; Bissels, Ute; Bosio, Andreas

    2015-01-01

    Modulation of gene expression is a useful tool to study the biology of haematopoietic stem and progenitor cells (HSPCs) and might also be instrumental to expand these cells for therapeutic approaches. Most of the studies so far have employed stable gene modification by viral vectors that are burdensome when translating protocols into clinical settings. Our study aimed at exploring new ways to transiently modify HSPC gene expression using non-integrating, RNA-based molecules. First, we tested different methods to deliver these molecules into HSPCs. The delivery of siRNAs with chemical transfection methods such as lipofection or cationic polymers did not lead to target knockdown, although we observed more than 90% fluorescent cells using a fluorochrome-coupled siRNA. Confocal microscopic analysis revealed that despite extensive washing, siRNA stuck to or in the cell surface, thereby mimicking a transfection event. In contrast, electroporation resulted in efficient, siRNA-mediated protein knockdown. For transient overexpression of proteins, we used optimised mRNA molecules with modified 5′- and 3′-UTRs. Electroporation of mRNA encoding GFP resulted in fast, efficient and persistent protein expression for at least seven days. Our data provide a broad-ranging comparison of transfection methods for hard-to-transfect cells and offer new opportunities for DNA-free, non-integrating gene modulation in HSPCs. PMID:26599627

  14. Enhanced antitumor effect and reduced vector dissemination with fiber-modified adenovirus vectors expressing herpes simplex virus thymidine kinase.

    PubMed

    Mizuguchi, Hiroyuki; Hayakawa, Takao

    2002-03-01

    There are at least two hurdles confronting the use of the adenovirus (Ad)-mediated herpes simplex virus thymidine kinase (HSVtk)/ganciclovir (GCV) system for the treatment of cancer. One is inefficient Ad vector-mediated gene transfer into tumor cells lacking the primary receptor, i.e., the coxsackievirus and adenovirus receptor (CAR). The other is hepatotoxicity due to unwanted vector spread into the liver, even when Ad vectors are injected intratumorally. Herein, we present an attractive strategy for overcoming such limitations based on use of a fiber-modified Ad vector containing an RGD peptide motif in the fiber knob. HSVtk-expressing Ad vectors containing mutant fiber (AdRGD-tk) or wild-type fiber (Ad-tk) were injected intratumorally into CAR-negative B16 melanoma cells inoculated into mice, after which GCV was injected intraperitoneally for 10 days. AdRGD-tk showed approximately 25 times more antitumor activity than Ad-tk. Histopathological studies suggested that liver damage in mice injected with AdRGD-tk was significantly lower than that in mice injected with Ad-tk. Intratumoral administration of luciferase-expressing Ad vectors containing the mutant fiber (AdRGD-L2) resulted in nearly 40 times more luciferase production in the tumor, but 8 times less production in the liver than the conventional Ad vectors (Ad-L2). These results indicate that combination of fiber-modified vectors and a HSVtk/GCV system is a potentially useful and safe approach for the treatment of tumors lacking CAR expression, and that fiber-modified vectors could be of great utility for gene therapy and gene transfer experiments. PMID:11896439

  15. Intratumoral heterogeneity of microRNA expression in breast cancer.

    PubMed

    Raychaudhuri, Mithu; Schuster, Tibor; Buchner, Theresa; Malinowsky, Katharina; Bronger, Holger; Schwarz-Boeger, Ulrike; Höfler, Heinz; Avril, Stefanie

    2012-07-01

    Profiling studies have identified specific microRNA (miRNA) signatures in malignant tumors including breast cancer. Our aim was to assess intratumoral heterogeneity in miRNA expression levels within primary breast cancers and between axillary lymph node metastases from the same patient. Specimens of 16 primary breast cancers were sampled in 8-10 distinct locations including the peripheral, intermediate, and central tumor zones, as well as two to five axillary lymph node metastases (n = 9). Total RNA was extracted from 132 paraffin-embedded samples, and the expression of miR-10b, miR-210, miR-31, and miR-335 was assessed as well as the reproducibility of RNA extraction and miRNA analysis by quantitative RT-PCR. Considerable intratumoral heterogeneity existed for all four miRNAs within primary breast cancers (CV 40%). No significant differences within (CV 34%) or between different tumor zones (CV 33%) were found. A similar variation in miRNA expression was observed between corresponding lymph node metastases (mean CV 40%). In comparison, the variation among different patients showed a CV of 80% for primary tumors and 103% for lymph node metastases. Both miRNA extraction procedures and quantitative RT-PCR showed high reproducibility (CV ≤ 2%). Thus, the intratumoral heterogeneity of miRNA expression in breast cancers can lead to significant sampling bias. Assessment of breast cancer miRNA profiles may require sampling at several different tumor locations and of several tumor-involved lymph nodes when deriving miRNA expression profiles of metastases.

  16. Application of a Fas Ligand Encoding a Recombinant Adenovirus Vector for Prolongation of Transgene Expression

    PubMed Central

    Zhang, Huang-Ge; Bilbao, Guadalupe; Zhou, Tong; Contreras, Juan Luis; Gómez-Navarro, Jesús; Feng, Meizhen; Saito, Izumu; Mountz, John D.; Curiel, David T.

    1998-01-01

    An adenovirus vector encoding murine Fas ligand (mFasL) under an inducible control was derived. In vivo ectopic expression of mFasL in murine livers induced an inflammatory cellular infiltration. Furthermore, ectopic expression of mFasL by myocytes did not allow prolonged vector-mediated transgene expression. Thus, ectopic expression of functional mFasL in vector-transduced cells does not appear to confer, by itself, an immunoprivileged site sufficient to mitigate adenovirus vector immunogenicity. PMID:9499110

  17. Inhibition of infectious bursal disease virus by vector delivered SiRNA in cell culture.

    PubMed

    Sahare, Amol Ashok; Bedekar, Megha Kadam; Jain, Sudhir Kumar; Singh, Azad; Singh, Sanjeev; Sarkhel, Bikas Chandra

    2015-01-01

    Infectious Bursal Disease (IBD) is major threat to poultry industry. It causes severe immunosuppression and mortality in chicken generally at 3 to 6 weeks of age. RNA intereference (RNAi) emerges as a potent gene regulatory tool in last few years. The present study was conducted to evaluate the efficiency of RNAi to inhibit the IBD virus (IDBV) replication in-vitro. VP2 gene of virus encodes protein involved in capsid formation, cell entry and induction of protective immune responses against it. Thus, VP2 gene of IBDV is the candidate target for the molecular techniques applied for IBDV detection and inhibition assay. In this study, IBDV was isolated from field cases and confirmed by RT-PCR. The virus was then adapted on chicken embryo fibroblast cells (CEF) in which it showed severe cytopathic effects (CPE). The short hairpin RNA (shRNAs) constructs homologous to the VP2 gene were designed and one, having maximum score and fulfilling maximum Reynolds criteria, was selected for evaluation of effective inhibition. Selected shRNA construct (i.e., VP2-shRNA) was observed to be the most effective for inhibiting VP2 gene expression. Real time PCR analysis was performed to measure the relative expression of VP2 gene in different experimental groups. The VP2 gene was less expressed in virus infected cells co-transfected with VP2-shRNA as compared to mock transfected cells and IBDV+ cells (control) at dose 1.6 µ g. The result showed ∼95% efficient down regulation of VP2 gene mRNA in VP2-shRNA treated cells. These findings suggested that designed shRNA construct achieved high level of inhibition of VP2 gene expression in-vitro. PMID:25153457

  18. microRNA Expression Profiling: Technologies, Insights, and Prospects.

    PubMed

    Roden, Christine; Mastriano, Stephen; Wang, Nayi; Lu, Jun

    2015-01-01

    Since the early days of microRNA (miRNA) research, miRNA expression profiling technologies have provided important tools toward both better understanding of the biological functions of miRNAs and using miRNA expression as potential diagnostics. Multiple technologies, such as microarrays, next-generation sequencing, bead-based detection system, single-molecule measurements, and quantitative RT-PCR, have enabled accurate quantification of miRNAs and the subsequent derivation of key insights into diverse biological processes. As a class of ~22 nt long small noncoding RNAs, miRNAs present unique challenges in expression profiling that require careful experimental design and data analyses. We will particularly discuss how normalization and the presence of miRNA isoforms can impact data interpretation. We will present one example in which the consideration in data normalization has provided insights that helped to establish the global miRNA expression as a tumor suppressor. Finally, we discuss two future prospects of using miRNA profiling technologies to understand single cell variability and derive new rules for the functions of miRNA isoforms. PMID:26663195

  19. Gene gymnastics: Synthetic biology for baculovirus expression vector system engineering.

    PubMed

    Vijayachandran, Lakshmi S; Thimiri Govinda Raj, Deepak B; Edelweiss, Evelina; Gupta, Kapil; Maier, Josef; Gordeliy, Valentin; Fitzgerald, Daniel J; Berger, Imre

    2013-01-01

    Most essential activities in eukaryotic cells are catalyzed by large multiprotein assemblies containing up to ten or more interlocking subunits. The vast majority of these protein complexes are not easily accessible for high resolution studies aimed at unlocking their mechanisms, due to their low cellular abundance and high heterogeneity. Recombinant overproduction can resolve this bottleneck and baculovirus expression vector systems (BEVS) have emerged as particularly powerful tools for the provision of eukaryotic multiprotein complexes in high quality and quantity. Recently, synthetic biology approaches have begun to make their mark in improving existing BEVS reagents by de novo design of streamlined transfer plasmids and by engineering the baculovirus genome. Here we present OmniBac, comprising new custom designed reagents that further facilitate the integration of heterologous genes into the baculovirus genome for multiprotein expression. Based on comparative genome analysis and data mining, we herein present a blueprint to custom design and engineer the entire baculovirus genome for optimized production properties using a bottom-up synthetic biology approach. PMID:23328086

  20. Prediction of miRNA-disease associations with a vector space model.

    PubMed

    Pasquier, Claude; Gardès, Julien

    2016-01-01

    MicroRNAs play critical roles in many physiological processes. Their dysregulations are also closely related to the development and progression of various human diseases, including cancer. Therefore, identifying new microRNAs that are associated with diseases contributes to a better understanding of pathogenicity mechanisms. MicroRNAs also represent a tremendous opportunity in biotechnology for early diagnosis. To date, several in silico methods have been developed to address the issue of microRNA-disease association prediction. However, these methods have various limitations. In this study, we investigate the hypothesis that information attached to miRNAs and diseases can be revealed by distributional semantics. Our basic approach is to represent distributional information on miRNAs and diseases in a high-dimensional vector space and to define associations between miRNAs and diseases in terms of their vector similarity. Cross validations performed on a dataset of known miRNA-disease associations demonstrate the excellent performance of our method. Moreover, the case study focused on breast cancer confirms the ability of our method to discover new disease-miRNA associations and to identify putative false associations reported in databases. PMID:27246786

  1. Plasmid vector with temperature-controlled gene expression

    SciTech Connect

    Kravchenko, V.V.; Yamshchikov, V.F.; Pletnev, A.G.

    1986-02-01

    In plasmid pBR327, a fragment 169 b.p. long including promotor p/sub 3/ of the bla gene has been deleted. The deletional derivative so obtained (pSP2) has been used to construct a recombinant plasmid bearing a fragment of phage lambda DNA with the p/sub R/ promotor and the gene of the temperature-sensitive repressor cI. It has been shown that the plasmid vector so constructed (pCE119) with promotor cR performs repressor-cI-controlled transcription of the bla gene, as a result of which induction for an hour at 42/sup 0/C leads to an almost 100-fold increase in the amount of product of the bla gene as compared with that at 32/sup 0/C. The possibility of the use of plasmid cPE119 for the expression of other genes has been demonstrated for the case of the semisynthetic ..beta..-galactosidase gene of E. coli. In this case, on induction of the cells with recombinant plasmid pCEZ12 for 3 hours at 42/sup 0/C, a 300-fold increase in the amount of active ..beta..-galactosidase, as compared with that at 32/sup 0/C, was observed. It is important to point out that under these conditions (at 42/sup 0/C), at least 99% of the cells containing the plasmid retain the phenotype lacZ/sup +/, which indicates the stability of the proposed vector system

  2. [Construction of venus vector carrying IGFBP7 gene and its expression in K562 cells].

    PubMed

    Wu, Shui-Yan; Hu, Shao-Yan; Cen, Jian-Nong; Chen, Zi-Xing

    2012-02-01

    The aim of this study was to construct venus vector carrying the gene encoding insulin-like growth factor binding protein 7 (IGFBP7), which provides an effective platform for exploring the function of this gene in leukemia. After digestion by restriction endonuclease, the IGFBP7 gene was recombined with the transfer plasmid. The venus particles were packaged using 293T cells to transfect K562 cells, and identification was performed by means of flow cytometry, RT-PCR and Western blot. The results showed that the sequence of cloned IGFBP7 gene was the same as that in GenBank. The size of product restricted by BamHI was same as the predicted one. GFP expression was observed in 293T and K562 cells with the fluorescent microscopy and flow cytometry. The expression level of mRNA and protein of IGFBP7 was confirmed by RT-PCR and Western blotting in K562 cells. It is concluded that venus vector carrying IGFBP7 gene has been successfully constructed and provides basis for exploring function of IGFBP7 in K562 cells.

  3. Methods for analyzing microRNA expression and function during osteogenic differentiation of human adipose tissue-derived mesenchymal stem cells.

    PubMed

    Kim, Yeon Jeong; Jung, Jin Sup

    2011-01-01

    MicroRNAs (miRNA) are single-stranded RNA molecules of 21-23 nucleotides in length that regulate gene expression at the posttranscriptional level. They may play important roles during osteogenic differentiation of adipose tissue-derived mesenchymal stem cells (hASC). In this chapter, we focus on the methods and strategies for elucidating miRNA function during osteogenic differentiation. We describe a miRNA expression analysis protocol, and a lentiviral vector strategy for the ectopic expression of miRNA in hASC to determine the role of miRNA during osteogenic differentiation. We also describe miRNA inhibition to further determine the role of miRNA during osteogenic differentiation, and a luciferase assay to demonstrate direct binding between a specific miRNA and its putative target.

  4. Helper virus-mediated downregulation of transgene expression permits production of recalcitrant helper-dependent adenoviral vector

    PubMed Central

    Palmer, Donna J; Grove, Nathan C; Ng, Philip

    2016-01-01

    Helper-dependent adenoviral vectors (HDAd) that express certain transgene products are impossible to produce because the transgene product is toxic to the producer cells, especially when made in large amounts during vector production. Downregulating transgene expression from the HDAd during vector production is a way to solve this problem. In this report, we show that this can be accomplished by inserting the target sequence for the adenoviral VA RNAI into the 3’ untranslated region of the expression cassette in the HDAd. Thus during vector production, when the producer cells are coinfected with both the helper virus (HV) and the HDAd, the VA RNAI produced by the HV will target the transgene mRNA from the HDAd via the endogenous cellular RNAi pathway. Once the HDAd is produced and purified, transduction of the target cells results in unimpeded transgene expression because of the absence of HV. This simple and universal strategy permits for the robust production of otherwise recalcitrant HDAds. PMID:27331077

  5. A high-throughput microRNA expression profiling system.

    PubMed

    Guo, Yanwen; Mastriano, Stephen; Lu, Jun

    2014-01-01

    As small noncoding RNAs, microRNAs (miRNAs) regulate diverse biological functions, including physiological and pathological processes. The expression and deregulation of miRNA levels contain rich information with diagnostic and prognostic relevance and can reflect pharmacological responses. The increasing interest in miRNA-related research demands global miRNA expression profiling on large numbers of samples. We describe here a robust protocol that supports high-throughput sample labeling and detection on hundreds of samples simultaneously. This method employs 96-well-based miRNA capturing from total RNA samples and on-site biochemical reactions, coupled with bead-based detection in 96-well format for hundreds of miRNAs per sample. With low-cost, high-throughput, high detection specificity, and flexibility to profile both small and large numbers of samples, this protocol can be adapted in a wide range of laboratory settings. PMID:25030917

  6. A Large U3 Deletion Causes Increased In Vivo Expression from a Nonintegrating Lentiviral Vector

    PubMed Central

    Bayer, Matthew; Kantor, Boris; Cockrell, Adam; Ma, Hong; Zeithaml, Brian; Li, Xiangping; McCown, Thomas; Kafri, Tal

    2008-01-01

    The feasibility of employing nonintegrating lentiviral vectors has been demonstrated by recent studies showing the ability of nonintegrating lentiviral vectors to maintain transgene expression in vitro and in vivo. Furthermore, HIV-1 vectors packaged with a mutated integrase were able to correct retinal disease in a mouse model. Interestingly, these results differ from earlier studies in which first-generation nonintegrating lentiviral vectors yielded insignificant levels of transduction. However, to date a rigorous characterization of transgene expression from the currently used self-inactivating (SIN) nonintegrating lentiviral vectors has not been published. Here we characterize transgene expression from SIN nonintegrating lentiviral vectors. Overall, we found that nonintegrating vectors express transgenes at a significantly lower level than their integrating counterparts. Expression from nonintegrating vectors was improved upon introducing a longer deletion in the vector’s U3 region. A unique shuttle-vector assay indicated that the relative abundance of the different episomal forms was not altered by the longer U3 deletion. Interestingly, the longer U3 deletion did not enhance expression in the corpus callosum of the rat brain, suggesting that the extent of silencing of episomal transcription is influenced by tissue-specific factors. Finally, and for the first time, episomal expression in the mouse liver was potent and sustained. PMID:18797449

  7. A large U3 deletion causes increased in vivo expression from a nonintegrating lentiviral vector.

    PubMed

    Bayer, Matthew; Kantor, Boris; Cockrell, Adam; Ma, Hong; Zeithaml, Brian; Li, Xiangping; McCown, Thomas; Kafri, Tal

    2008-12-01

    The feasibility of using nonintegrating lentiviral vectors has been demonstrated by recent studies showing their ability to maintain transgene expression both in vitro and in vivo. Furthermore, human immunodeficiency virus-1 (HIV-1) vectors packaged with a mutated integrase were able to correct retinal disease in a mouse model. Interestingly, these results differ from earlier studies in which first-generation nonintegrating lentiviral vectors yielded insignificant levels of transduction. However, to date, a rigorous characterization of transgene expression from the currently used self-inactivating (SIN) nonintegrating lentiviral vectors has not been published. In this study, we characterize transgene expression from SIN nonintegrating lentiviral vectors. Overall, we found that nonintegrating vectors express transgenes at a significantly lower level than their integrating counterparts. Expression from nonintegrating vectors was improved upon introducing a longer deletion in the vector's U3 region. A unique shuttle-vector assay indicated that the relative abundance of the different episomal forms was not altered by the longer U3 deletion. Interestingly, the longer U3 deletion did not enhance expression in the corpus callosum of the rat brain, suggesting that the extent of silencing of episomal transcription is influenced by tissue-specific factors. Finally, and for the first time, episomal expression in the mouse liver was potent and sustained.

  8. Patterns of miRNA expression in Arctic charr development.

    PubMed

    Kapralova, Kalina H; Franzdóttir, Sigrídur Rut; Jónsson, Hákon; Snorrason, Sigurður S; Jónsson, Zophonías O

    2014-01-01

    Micro-RNAs (miRNAs) are now recognized as a major class of developmental regulators. Sequences of many miRNAs are highly conserved, yet they often exhibit temporal and spatial heterogeneity in expression among species and have been proposed as an important reservoir for adaptive evolution and divergence. With this in mind we studied miRNA expression during embryonic development of offspring from two contrasting morphs of the highly polymorphic salmonid Arctic charr (Salvelinus alpinus), a small benthic morph from Lake Thingvallavatn (SB) and an aquaculture stock (AC). These morphs differ extensively in morphology and adult body size. We established offspring groups of the two morphs and sampled at several time points during development. Four time points (3 embryonic and one just before first feeding) were selected for high-throughput small-RNA sequencing. We identified a total of 326 conserved and 427 novel miRNA candidates in Arctic charr, of which 51 conserved and 6 novel miRNA candidates were differentially expressed among developmental stages. Furthermore, 53 known and 19 novel miRNAs showed significantly different levels of expression in the two contrasting morphs. Hierarchical clustering of the 53 conserved miRNAs revealed that the expression differences are confined to the embryonic stages, where miRNAs such as sal-miR-130, 30, 451, 133, 26 and 199a were highly expressed in AC, whereas sal-miR-146, 183, 206 and 196a were highly expressed in SB embryos. The majority of these miRNAs have previously been found to be involved in key developmental processes in other species such as development of brain and sensory epithelia, skeletogenesis and myogenesis. Four of the novel miRNA candidates were only detected in either AC or SB. miRNA candidates identified in this study will be combined with available mRNA expression data to identify potential targets and involvement in developmental regulation. PMID:25170615

  9. Patterns of MiRNA Expression in Arctic Charr Development

    PubMed Central

    Kapralova, Kalina H.; Franzdóttir, Sigrídur Rut; Jónsson, Hákon; Snorrason, Sigurður S.; Jónsson, Zophonías O.

    2014-01-01

    Micro-RNAs (miRNAs) are now recognized as a major class of developmental regulators. Sequences of many miRNAs are highly conserved, yet they often exhibit temporal and spatial heterogeneity in expression among species and have been proposed as an important reservoir for adaptive evolution and divergence. With this in mind we studied miRNA expression during embryonic development of offspring from two contrasting morphs of the highly polymorphic salmonid Arctic charr (Salvelinus alpinus), a small benthic morph from Lake Thingvallavatn (SB) and an aquaculture stock (AC). These morphs differ extensively in morphology and adult body size. We established offspring groups of the two morphs and sampled at several time points during development. Four time points (3 embryonic and one just before first feeding) were selected for high-throughput small-RNA sequencing. We identified a total of 326 conserved and 427 novel miRNA candidates in Arctic charr, of which 51 conserved and 6 novel miRNA candidates were differentially expressed among developmental stages. Furthermore, 53 known and 19 novel miRNAs showed significantly different levels of expression in the two contrasting morphs. Hierarchical clustering of the 53 conserved miRNAs revealed that the expression differences are confined to the embryonic stages, where miRNAs such as sal-miR-130, 30, 451, 133, 26 and 199a were highly expressed in AC, whereas sal-miR-146, 183, 206 and 196a were highly expressed in SB embryos. The majority of these miRNAs have previously been found to be involved in key developmental processes in other species such as development of brain and sensory epithelia, skeletogenesis and myogenesis. Four of the novel miRNA candidates were only detected in either AC or SB. miRNA candidates identified in this study will be combined with available mRNA expression data to identify potential targets and involvement in developmental regulation. PMID:25170615

  10. Stable Delivery of CCR5-Directed shRNA into Human Primary Peripheral Blood Mononuclear Cells and Hematopoietic Stem/Progenitor Cells via a Lentiviral Vector

    PubMed Central

    Shimizu, Saki; Yadav, Swati Seth; An, Dong Sung

    2016-01-01

    RNAi is a powerful tool to achieve suppression of a specific gene expression and therefore it has tremendous potential for gene therapy applications. A number of vector systems have been developed to express short-hairpin RNAs (shRNAs) to produce siRNAs within mammalian T-cells, primary hematopoietic stem/progenitor cells (HSPC), human peripheral blood mononuclear cells, and in animal model systems. Among these, vectors based on lentivirus backbones have significantly transformed our ability to transfer shRNAs into nondividing cells, such as HSPC, resulting in high transduction efficiencies. However, delivery and long-term expression of shRNAs should be carefully optimized for efficient knock down of target gene without causing cytotoxicity in mammalian cells. Here, we describe our protocols for the development of shRNA against a major HIV co-receptor/chemokine receptor CCR5 and the use of lentiviral vectors for stable shRNA delivery and expression in primary human PBMC and HSPC. PMID:26472455

  11. Stable Delivery of CCR5-Directed shRNA into Human Primary Peripheral Blood Mononuclear Cells and Hematopoietic Stem/Progenitor Cells via a Lentiviral Vector.

    PubMed

    Shimizu, Saki; Yadav, Swati Seth; An, Dong Sung

    2016-01-01

    RNAi is a powerful tool to achieve suppression of a specific gene expression and therefore it has tremendous potential for gene therapy applications. A number of vector systems have been developed to express short-hairpin RNAs (shRNAs) to produce siRNAs within mammalian T-cells, primary hematopoietic stem/progenitor cells (HSPC), human peripheral blood mononuclear cells, and in animal model systems. Among these, vectors based on lentivirus backbones have significantly transformed our ability to transfer shRNAs into nondividing cells, such as HSPC, resulting in high transduction efficiencies. However, delivery and long-term expression of shRNAs should be carefully optimized for efficient knock down of target gene without causing cytotoxicity in mammalian cells. Here, we describe our protocols for the development of shRNA against a major HIV co-receptor/chemokine receptor CCR5 and the use of lentiviral vectors for stable shRNA delivery and expression in primary human PBMC and HSPC. PMID:26472455

  12. Construction of novel shuttle expression vectors for gene expression in Bacillus subtilis and Bacillus pumilus.

    PubMed

    Shao, Huanhuan; Cao, Qinghua; Zhao, Hongyan; Tan, Xuemei; Feng, Hong

    2015-01-01

    A native plasmid (pSU01) was detected by genome sequencing of Bacillus subtilis strain S1-4. Two pSU01-based shuttle expression vectors pSU02-AP and pSU03-AP were constructed enabling stable replication in B. subtilis WB600. These vectors contained the reporter gene aprE, encoding an alkaline protease from Bacillus pumilus BA06. The expression vector pSU03-AP only possessed the minimal replication elements (rep, SSO, DSO) and exhibited more stability on structure, suggesting that the rest of the genes in pSU01 (ORF1, ORF2, mob, hsp) were unessential for the structural stability of plasmid in B. subtilis. In addition, recombinant production of the alkaline protease was achieved more efficiently with pSU03-AP whose copy number was estimated to be more than 100 per chromosome. Furthermore, pSU03-AP could also be used to transform and replicate in B. pumilus BA06 under selective pressure. In conclusion, pSU03-AP is expected to be a useful tool for gene expression in Bacillus subtilis and B. pumilus. PMID:26377132

  13. Impact of Host Genes and Strand Selection on miRNA and miRNA* Expression

    PubMed Central

    Biasiolo, Marta; Sales, Gabriele; Lionetti, Marta; Agnelli, Luca; Todoerti, Katia; Bisognin, Andrea; Coppe, Alessandro; Romualdi, Chiara; Neri, Antonino; Bortoluzzi, Stefania

    2011-01-01

    Dysregulation of miRNAs expression plays a critical role in the pathogenesis of genetic, multifactorial disorders and in human cancers. We exploited sequence, genomic and expression information to investigate two main aspects of post-transcriptional regulation in miRNA biogenesis, namely strand selection regulation and expression relationships between intragenic miRNAs and host genes. We considered miRNAs expression profiles, measured in five sizeable microarray datasets, including samples from different normal cell types and tissues, as well as different tumours and disease states. First, the study of expression profiles of “sister” miRNA pairs (miRNA/miRNA*, 5′ and 3′ strands of the same hairpin precursor) showed that the strand selection is highly regulated since it shows tissue-/cell-/condition-specific modulation. We used information about the direction and the strength of the strand selection bias to perform an unsupervised cluster analysis for the sample classification evidencing that is able to distinguish among different tissues, and sometimes between normal and malignant cells. Then, considering a minimum expression threshold, in few miRNA pairs only one mature miRNA is always present in all considered cell types, whereas the majority of pairs were concurrently expressed in some cell types and alternatively in others. In a significant fraction of concurrently expressed pairs, the major and the minor forms found at comparable levels may contribute to post-transcriptional gene silencing, possibly in a coordinate way. In the second part of the study, the behaved tendency to co-expression of intragenic miRNAs and their “host” mRNA genes was confuted by expression profiles examination, suggesting that the expression profile of a given host gene can hardly be a good estimator of co-transcribed miRNA(s) for post-transcriptional regulatory networks inference. Our results point out the regulatory importance of post-transcriptional phases of mi

  14. Rapid construction of multiple sgRNA vectors and knockout of the Arabidopsis IAA2 gene using the CRISPR/Cas9 genomic editing technology.

    PubMed

    Dingyuan, Liu; Ting, Qiu; Xiaohui, Ding; Miaomiao, Li; Muyuan, Zhu; Junhui, Wang

    2016-08-01

    IAA2 is a member of the Aux/IAA auxin responsive gene family in Arabidopsis thaliana. No iaa2 mutant has been reported until now, thus hindering its further mechanistic investigations. The normal genomic editing technology of CRISPR/Cas9 (Clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9) uses only a single guide RNA (sgRNA) to target one site in a specific gene, and the gene knockout efficiency is not high. Instead, multiple sgRNAs can target multiple sites; therefore, the efficiency may be improved. In the present investigation, we used the golden-gate cloning strategy and two rounds of PCR reactions to combine three sgRNAs in the same entry vector. The final expression vector was obtained by LR reactions with the destination vector containing the Cas9 expression cassette. Four out of the six sgRNAs were effective, and we also obtained a lot of insertion and deletion mutations. Compared with one sgRNA approach, multiple sgRNAs displayed higher gene-knockout efficiency and produced more germ-line mutants. Thus, we established a more rapid and efficient method and generated five mutants for further studies of IAA2 functions.

  15. Rapid construction of multiple sgRNA vectors and knockout of the Arabidopsis IAA2 gene using the CRISPR/Cas9 genomic editing technology.

    PubMed

    Dingyuan, Liu; Ting, Qiu; Xiaohui, Ding; Miaomiao, Li; Muyuan, Zhu; Junhui, Wang

    2016-08-01

    IAA2 is a member of the Aux/IAA auxin responsive gene family in Arabidopsis thaliana. No iaa2 mutant has been reported until now, thus hindering its further mechanistic investigations. The normal genomic editing technology of CRISPR/Cas9 (Clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9) uses only a single guide RNA (sgRNA) to target one site in a specific gene, and the gene knockout efficiency is not high. Instead, multiple sgRNAs can target multiple sites; therefore, the efficiency may be improved. In the present investigation, we used the golden-gate cloning strategy and two rounds of PCR reactions to combine three sgRNAs in the same entry vector. The final expression vector was obtained by LR reactions with the destination vector containing the Cas9 expression cassette. Four out of the six sgRNAs were effective, and we also obtained a lot of insertion and deletion mutations. Compared with one sgRNA approach, multiple sgRNAs displayed higher gene-knockout efficiency and produced more germ-line mutants. Thus, we established a more rapid and efficient method and generated five mutants for further studies of IAA2 functions. PMID:27531614

  16. Expression of neurotensin messenger RNA in a human carcinoid tumor.

    PubMed Central

    Evers, B M; Ishizuka, J; Townsend, C M; Rajaraman, S; Thompson, J C

    1991-01-01

    Neurotensin (NT), a distal gut peptide, has important regulatory and trophic effects throughout the gut; however the intracellular mechanisms that regulate the gene expression and release of human NT are not known. The purpose of this endeavor was to study a functioning human pancreatic carcinoid cell line (called BON) in vitro that expresses the NT gene, and to study the effect of the cyclic adenosine monophosphate (cAMP) signal-transduction pathway on the expression and release of human NT. RNA was prepared from BON cell line (which has been established in this laboratory); the RNA was analyzed for NT mRNA expression by Northern hybridization with a complementary DNA probe. RNA blot analysis demonstrated that the NT gene is expressed in BON and is transcribed to two mRNAs of 1.0- and 1.5-kb sizes. In the second part of this study, BON cells were treated with either forskolin (FSK), which increases intracellular levels of cAMP, or with serotonin (5-HT), which reduces cAMP in BON cells. Forskolin produced a dose-dependent increase in NT peptide release and, furthermore, FSK (10(-6) mol/L) rapidly increased NT mRNA abundance 1 hour after addition; conversely, 5-HT (10(-5) mol/L) decreased NT mRNA at 1 hour. Neurotensin mRNA levels returned to control values by 3 hours after either FSK or 5-HT, which suggests that the transcript half-life for NT is relatively short. These findings show that the expression and peptide release of human NT is mediated, in part, by the cAMP signal-transduction pathway. Our human carcinoid cell line will provide a useful model to study the in vitro regulation of NT gene expression and peptide release. Images Fig. 1. Fig. 2. Fig. 3. Fig. 5. Fig. 6. PMID:1659338

  17. Optimized lentiviral vectors for HIV gene therapy: multiplexed expression of small RNAs and inclusion of MGMT(P140K) drug resistance gene.

    PubMed

    Chung, Janet; Scherer, Lisa J; Gu, Angel; Gardner, Agnes M; Torres-Coronado, Monica; Epps, Elizabeth W; Digiusto, David L; Rossi, John J

    2014-05-01

    Gene therapy with hematopoietic stem and progenitor cells is a promising approach to engineering immunity to human immunodeficiency virus (HIV) that may lead to a functional cure for acquired immunodeficiency syndrome (AIDS). In support of this approach, we created lentiviral vectors with an engineered polycistronic platform derived from the endogenous MCM7 gene to express a diverse set of small antiviral RNAs and a drug resistance MGMT(P140K) marker. Multiple strategies for simultaneous expression of up to five RNA transgenes were tested. The placement and orientation of each transgene and its promoter were important determinants for optimal gene expression. Antiviral RNA expression from the MCM7 platform with a U1 promoter was sufficient to provide protection from R5-tropic HIV in macrophages and resulted in reduced hematopoietic toxicity compared with constructs expressing RNA from independent RNA polymerase III promoters. The addition of an HIV entry inhibitor and nucleolar TAR RNA decoy did not enhance antiviral potency over constructs that targeted only viral RNA transcripts. We also demonstrated selective enrichment of gene-modified cells in vivo using a humanized mouse model. The use of these less toxic, potent anti-HIV vectors expressing a drug selection marker is likely to enhance the in vivo efficacy of our stem cell gene therapy approach in treating HIV/AIDS.

  18. Analysis of microRNA expression by in situ hybridization with RNA oligonucleotide probes

    PubMed Central

    Thompson, Robert C.; Deo, Monika; Turner, David L.

    2007-01-01

    In situ hybridization is an important tool for analyzing gene expression and developing hypotheses about gene functions. The discovery of hundreds of microRNA (miRNA) genes in animals has provided new challenges for analyzing gene expression and functions. The small size of the mature miRNAs (∼20-24 nucleotides in length) presents difficulties for conventional in situ hybridization methods. However, we have developed a modified in situ hybridization method for detection of mammalian miRNAs in tissue sections, based upon the use of RNA oligonucleotide probes in combination with highly specific wash conditions. Here we present detailed procedures for detection of miRNAs in tissue sections or cultured cells. The methods described can utilize either nonradioactive hapten-conjugated probes that are detected by enzyme-coupled antibodies, or radioactively labeled probes that are detected by autoradiography. The ability to visualize miRNA expression patterns in tissue sections provides an additional tool for the analyses of miRNA expression and function. In addition, the use of radioactively labeled probes should facilitate quantitative analyses of changes in miRNA gene expression. PMID:17889803

  19. Ultrasensitive DNA chip: gene expression profile analysis without RNA amplification.

    PubMed

    Nagino, Kunihisa; Nomura, Osamu; Takii, Yuki; Myomoto, Akira; Ichikawa, Makiko; Nakamura, Fumio; Higasa, Masashi; Akiyama, Hideo; Nobumasa, Hitoshi; Shiojima, Satoshi; Tsujimoto, Gozoh

    2006-04-01

    We have developed a new DNA chip whose substrate has a unique minute columnar array structure made of plastic. The DNA chip exhibits ultrahigh sensitivity, up to 100-fold higher than that of reference DNA chips, which makes it possible to monitor gene expression profiles even with very small amounts of RNA (0.1-0.01 microg of total RNA) without amplification. Differential expression ratios obtained with the new DNA chip were validated against those obtained with quantitative real-time PCR assays. This novel microarray technology would be a powerful tool for monitoring gene expression profiles, especially for clinical diagnosis.

  20. mRNA modifications: Dynamic regulators of gene expression?

    PubMed Central

    Hoernes, Thomas Philipp; Hüttenhofer, Alexander; Erlacher, Matthias David

    2016-01-01

    ABSTRACT The expression of a gene is a tightly regulated process and is exerted by a myriad of different mechanisms. Recently, RNA modifications located in coding sequences of mRNAs, have been identified as potential regulators of gene expression. N6-methyladenosine (m6A), 5-methylcytosine (m5C), pseudouridine (Ψ) and N1-methyladenosine (m1A) have been found within open reading frames of mRNAs. The presence of these mRNA modifications has been implicated to modulate the fate of an mRNA, ranging from maturation to its translation and even degradation. However, many aspects concerning the biological functions of mRNA modifications remain elusive. Recently, systematic in vitro studies allowed a first glimpse of the direct interplay of mRNA modifications and the efficiency and fidelity of ribosomal translation. It thereby became evident that the effects of mRNA modifications were, astonishingly versatile, depending on the type, position or sequence context. The incorporation of a single modification could either prematurely terminate protein synthesis, reduce the peptide yield or alter the amino acid sequence identity. These results implicate that mRNA modifications are a powerful mechanism to post-transcriptionally regulate gene expression. PMID:27351916

  1. MicroRNA expression profiling of male breast cancer

    PubMed Central

    Fassan, Matteo; Baffa, Raffaele; Palazzo, Juan P; Lloyd, Joshua; Crosariol, Marco; Liu, Chang-Gong; Volinia, Stefano; Alder, Hannes; Rugge, Massimo; Croce, Carlo M; Rosenberg, Anne

    2009-01-01

    Introduction MicroRNAs (miRNAs) are a class of small noncoding RNAs that control gene expression by targeting mRNAs and triggering either translation repression or RNA degradation. Their aberrant expression may be involved in human diseases, including cancer. To test the hypothesis that there is a specific miRNA expression signature which characterizes male breast cancers, we performed miRNA microarray analysis in a series of male breast cancers and compared them with cases of male gynecomastia and female breast cancers. Methods Paraffin blocks were obtained at the Department of Pathology of Thomas Jefferson University from 28 male patients including 23 breast cancers and five cases of male gynecomastia, and from 10 female ductal breast carcinomas. The RNA harvested was hybridized to miRNA microarrays (~1,100 miRNA probes, including 326 human and 249 mouse miRNA genes, spotted in duplicate). To further support the microarray data, an immunohistochemical analysis for two specific miRNA gene targets (HOXD10 and VEGF) was performed in a small series of male breast carcinoma and gynecomastia samples. Results We identified a male breast cancer miRNA signature composed of a large portion of underexpressed miRNAs. In particular, 17 miRNAs with increased expression and 26 miRNAs with decreased expression were identified in male breast cancer compared with gynecomastia. Among these miRNAs, some had well-characterized cancer development association and some showed a deregulation in cancer specimens similar to the one previously observed in the published signatures of female breast cancer. Comparing male with female breast cancer miRNA expression signatures, 17 significantly deregulated miRNAs were observed (four overexpressed and 13 underexpressed in male breast cancers). The HOXD10 and VEGF gene immunohistochemical expression significantly follows the corresponding miRNA deregulation. Conclusions Our results suggest that specific miRNAs may be directly involved in male

  2. Heterologous protein production using euchromatin-containing expression vectors in mammalian cells.

    PubMed

    Zboray, Katalin; Sommeregger, Wolfgang; Bogner, Edith; Gili, Andreas; Sterovsky, Thomas; Fauland, Katharina; Grabner, Beatrice; Stiedl, Patricia; Moll, Herwig P; Bauer, Anton; Kunert, Renate; Casanova, Emilio

    2015-09-18

    Upon stable cell line generation, chromosomal integration site of the vector DNA has a major impact on transgene expression. Here we apply an active gene environment, rather than specified genetic elements, in expression vectors used for random integration. We generated a set of Bacterial Artificial Chromosome (BAC) vectors with different open chromatin regions, promoters and gene regulatory elements and tested their impact on recombinant protein expression in CHO cells. We identified the Rosa26 BAC as the most efficient vector backbone showing a nine-fold increase in both polyclonal and clonal production of the human IgG-Fc. Clonal protein production was directly proportional to integrated vector copy numbers and remained stable during 10 weeks without selection pressure. Finally, we demonstrated the advantages of BAC-based vectors by producing two additional proteins, HIV-1 glycoprotein CN54gp140 and HIV-1 neutralizing PG9 antibody, in bioreactors and shake flasks reaching a production yield of 1 g/l.

  3. P and M gene junction is the optimal insertion site in Newcastle disease virus vaccine vector for foreign gene expression.

    PubMed

    Zhao, Wei; Zhang, Zhenyu; Zsak, Laszlo; Yu, Qingzhong

    2015-01-01

    Newcastle disease virus (NDV) has been developed as a vector for vaccine and gene therapy purposes. However, the optimal insertion site for foreign gene expression remained to be determined. In the present study, we inserted the green fluorescence protein (GFP) gene into five different intergenic regions of the enterotropic NDV VG/GA vaccine strain using reverse genetics technology. The rescued recombinant viruses retained lentogenic pathotype and displayed delayed growth dynamics, particularly when the GFP gene was inserted between the NP and P genes of the virus. The GFP mRNA level was most abundant when the gene was inserted closer to the 3' end and gradually decreased as the gene was inserted closer to the 5' end. Measurement of the GFP fluorescence intensity in recombinant virus-infected cells demonstrated that the non-coding region between the P and M genes is the optimal insertion site for foreign gene expression in the VG/GA vaccine vector.

  4. Polycistronic Expression of the Influenza A Virus RNA-Dependent RNA Polymerase by Using the Thosea asigna Virus 2A-Like Self-Processing Sequence

    PubMed Central

    Momose, Fumitaka; Morikawa, Yuko

    2016-01-01

    The RNA-dependent RNA polymerase (RdRp) of influenza A virus consists of three subunits, PB2, PB1, and PA, and catalyses both viral RNA genome replication and transcription. Cotransfection of four monocistronic expression vectors for these subunits and nucleoprotein with an expression vector for viral RNA reconstitutes functional viral ribonucleoprotein complex (vRNP). However, the specific activity of reconstituted RdRp is usually very low since the expression level and the ratio of the three subunits by transfection are uncontrollable at single-cell levels. For efficient reconstitution of RdRp and vRNP, their levels need to be at least comparable. We constructed polycistronic expression vectors in which the coding sequences of the three subunits were joined with the 2A-like self-processing sequence of Thosea asigna virus (TaV2A) in various orders. The level of PB1 protein, even when it was placed at the most downstream, was comparable with that expressed from the monocistronic PB1 vector. In contrast, the levels of PB2 and PA were very low, the latter of which was most likely due to proteasomal degradation caused by the TaV2A-derived sequences attached to the amino- and/or carboxyl-terminal ends in this expression system. Interestingly, two of the constructs, in which the PB1 coding sequence was placed at the most upstream, showed much higher reporter activity in a luciferase-based mini-genome assay than that observed by cotransfection of the monocistronic vectors. When the coding sequence of selective antibiotic marker was further placed at the most downstream of the PB1-PA-PB2 open reading frame, stable cells expressing RdRp were easily established, indicating that acquisition of antibiotic resistance assured the expression of upstream RdRp. The addition of an affinity tag to the carboxyl-terminal end of PB2 allowed us to isolate reconstituted vRNP. Taken together, the polycistronic expression system for influenza virus RdRp may be available for functional and

  5. Production of a recombinant Fab in Pichia pastoris from a Monocistronic expression vector.

    PubMed

    Burtet, Rafael Trindade; Santos-Silva, Marcos Antônio; Buss, Guilherme Antônio Marques; Moraes, Lidia Maria Pepe; Maranhão, Andrea Queiroz; Brigido, Marcelo Macedo

    2007-12-01

    Recombinant Fab is usually expressed using dicistronic vectors producing the heavy and light chains separately. We developed an improved vector for Fab fragment expression in Pichia pastoris, which allows a stoichiometric expression of both chains based on a monocistronic arrangement. The protein is produced as a unique polypeptide harbouring a KEX2 processing site between both chains. After KEX cleavage, a correctly folded mature Fab is formed. The produced recombinant protein is characterized as a heterodimeric functional Fab. The vector described is a new tool for the proper expression of antibody fragments or any heterodimeric polypeptides.

  6. Joint analysis of miRNA and mRNA expression data.

    PubMed

    Muniategui, Ander; Pey, Jon; Planes, Francisco J; Rubio, Angel

    2013-05-01

    miRNAs are small RNA molecules ('22 nt) that interact with their target mRNAs inhibiting translation or/and cleavaging the target mRNA. This interaction is guided by sequence complentarity and results in the reduction of mRNA and/or protein levels. miRNAs are involved in key biological processes and different diseases. Therefore, deciphering miRNA targets is crucial for diagnostics and therapeutics. However, miRNA regulatory mechanisms are complex and there is still no high-throughput and low-cost miRNA target screening technique. In recent years, several computational methods based on sequence complementarity of the miRNA and the mRNAs have been developed. However, the predicted interactions using these computational methods are inconsistent and the expected false positive rates are still large. Recently, it has been proposed to use the expression values of miRNAs and mRNAs (and/or proteins) to refine the results of sequence-based putative targets for a particular experiment. These methods have shown to be effective identifying the most prominent interactions from the databases of putative targets. Here, we review these methods that combine both expression and sequence-based putative targets to predict miRNA targets. PMID:22692086

  7. Development of Lentiviral Vectors Simultaneously Expressing Multiple siRNAs Against CCR5, vif and tat/rev Genes for an HIV-1 Gene Therapy Approach.

    PubMed

    Spanevello, Francesca; Calistri, Arianna; Del Vecchio, Claudia; Mantelli, Barbara; Frasson, Chiara; Basso, Giuseppe; Palù, Giorgio; Cavazzana, Marina; Parolin, Cristina

    2016-04-19

    Gene therapy holds considerable promise for the functional cure of HIV-1 infection and, in this context, RNA interference (RNAi)-based approaches represent powerful strategies. Stable expression of small interfering RNAs (siRNAs) targeting HIV genes or cellular cofactors has the potential to render HIV-1 susceptible cells resistant to infection. To inhibit different steps of virus life cycle, self-inactivating lentiviral vectors expressing multiple siRNAs targeting the CCR5 cellular gene as well as vif and tat/rev viral transcripts, under the control of different RNA polymerase III promoters (U6, 7SK, H1) were developed. The use of a single RNA polymerase III promoter driving the expression of a sequence giving rise to three siRNAs directed against the selected targets (e-shRNA) was also investigated. Luciferase assay and inhibition of HIV-1 replication in human Jurkat T-cell line were adopted to select the best combination of promoter/siRNA. The efficacy of selected developed combinatorial vectors in interfering with viral replication was evaluated in human primary CD4(+) T lymphocytes. We identified two effective anti-HIV combinatorial vectors that conferred protection against R5- and X4- tropic viruses. Overall, our results showed that the antiviral effect is influenced by different factors, including the promoter used to express the RNAi molecules and the selected cassette combination. These findings contribute to gain further insights in the design of RNAi-based gene therapy approaches against HIV-1 for clinical application.

  8. Selective MicroRNA-Offset RNA Expression in Human Embryonic Stem Cells

    PubMed Central

    Juhila, Juuso; Holm, Frida; Weltner, Jere; Trokovic, Ras; Mikkola, Milla; Toivonen, Sanna; Balboa, Diego; Lampela, Riina; Icay, Katherine; Tuuri, Timo; Otonkoski, Timo; Wong, Garry; Hovatta, Outi

    2015-01-01

    Small RNA molecules, including microRNAs (miRNAs), play critical roles in regulating pluripotency, proliferation and differentiation of embryonic stem cells. miRNA-offset RNAs (moRNAs) are similar in length to miRNAs, align to miRNA precursor (pre-miRNA) loci and are therefore believed to derive from processing of the pre-miRNA hairpin sequence. Recent next generation sequencing (NGS) studies have reported the presence of moRNAs in human neurons and cancer cells and in several tissues in mouse, including pluripotent stem cells. In order to gain additional knowledge about human moRNAs and their putative development-related expression, we applied NGS of small RNAs in human embryonic stem cells (hESCs) and fibroblasts. We found that certain moRNA isoforms are notably expressed in hESCs from loci coding for stem cell-selective or cancer-related miRNA clusters. In contrast, we observed only sparse moRNAs in fibroblasts. Consistent with earlier findings, most of the observed moRNAs derived from conserved loci and their expression did not appear to correlate with the expression of the adjacent miRNAs. We provide here the first report of moRNAs in hESCs, and their expression profile in comparison to fibroblasts. Moreover, we expand the repertoire of hESC miRNAs. These findings provide an expansion on the known repertoire of small non-coding RNA contents in hESCs. PMID:25822230

  9. MicroRNA expression in the aging mouse thymus.

    PubMed

    Ye, Yaqiong; Li, Daotong; Ouyang, Dan; Deng, Li; Zhang, Yuan; Ma, Yongjiang; Li, Yugu

    2014-09-01

    MicroRNAs (miRNAs) have been implicated in the process of aging in many model organisms, such as Caenorhabditis elegans, and in many organs, such as the mouse lung and human epididymis. However, the role of miRNAs in the thymus tissues of the aging mouse remains unclear. To address this question, we investigated the miRNA expression profiles in the thymuses of 1-, 10- and 19-month-old mice using miRNA array and qRT-PCR assays. A total of 223 mouse miRNAs were screened, and the expression levels of those miRNAs exhibited gradual increases and decreases over the course of thymus aging. Fifty miRNAs in the 10-month-old thymus and 81 miRNAs in the 19-month-old thymus were defined as differentially expressed miRNAs (p<0.05) in comparison with their levels in the 1-month-old mouse, and approximately one-third of these miRNAs were grouped within 11 miRNA clusters. Each miRNA cluster contained 2 to 5 miRNA genes, and most of the cluster members displayed similar expression patterns, being either increased or decreased. In addition, Ingenuity Pathway Analysis (IPA) software and the IPA database were used to analyze the 12 miRNAs that exhibited significant expression changes, revealing that as many as 15 pathways may be involved. Thus, our current study determined the expression profiles of miRNAs in the mouse thymus during the process of aging. The results suggested that these miRNAs could become meaningful biomarkers for studying thymus aging and that the aging-related alternations in miRNA expression may be involved in the regulation of cell proliferation, apoptosis, development and carcinogenesis/tumorigenesis.

  10. Construction and verification of the targeted uPA-shRNA lentiviral vector and evaluation of the transfection and silencing rate

    PubMed Central

    WANG, WEI-SHAN; GUO, FENG-JING; LI, CHANG-JUN; ZHANG, ZHEN-DONG; SHI, CHEN-HUI

    2014-01-01

    Urokinase-type plasminogen activator (uPA) receptors, which are released by the synovial tissue, are responsible for the activation of cartilage-breakdown proteases and play critical roles in cartilage degradation during the progression of osteoarthritis (OA). RNA interference (RNAi) technology has emerged as a potent tool to generate cellular knockdown phenotypes of a desired gene. The aims of the present study were to investigate the effect of siRNA specific to the uPA gene on chondrocytes and to investigate the possible mechanisms of OA. Firstly, four types of small hairpin RNA (shRNA) sequence (P1, P2, P3 and P4) were obtained from the targeted uPA gene of the New Zealand rabbit, based on siRNA theory. The sequences were designed, constructed and subjected to restriction enzyme digestion, transformation, polymerase chain reaction (PCR) identification, positive clone sequencing and lentivirus packaging. Secondly, primary culturing cartilage cells from the New Zealand rabbit were transfected with P1, P2, P3 or P4 to observe the transfection rate under a fluorescence microscope. The mRNA expression levels of uPA were analyzed in cartilage cells using quantitative PCR, while protein expression levels were analyzed in the cartilage cells using western blot technology. Four types of uPA-shRNA lentiviral vectors were constructed successfully, which were all able to be transfected into the primary culturing cartilage cells. The transfection rate was as high as 85% when the multiplicity of infection was 100, which demonstrated that P1, P2, P3 and P4 were all capable of inhibiting the mRNA and protein expression of uPA in cartilage cells. In addition, among the four sequences, the P2 sequence exhibited the highest silencing rate of 70%. Statistical significance (P<0.05) was observed when analyzing the silencing rate of P2 compared to the other three groups. The most efficient targeted uPA-shRNA sequence was identified following screening. The results strongly verified

  11. Principles of microRNA Regulation Revealed Through Modeling microRNA Expression Quantitative Trait Loci.

    PubMed

    Budach, Stefan; Heinig, Matthias; Marsico, Annalisa

    2016-08-01

    Extensive work has been dedicated to study mechanisms of microRNA-mediated gene regulation. However, the transcriptional regulation of microRNAs themselves is far less well understood, due to difficulties determining the transcription start sites of transient primary transcripts. This challenge can be addressed using expression quantitative trait loci (eQTLs) whose regulatory effects represent a natural source of perturbation of cis-regulatory elements. Here we used previously published cis-microRNA-eQTL data for the human GM12878 cell line, promoter predictions, and other functional annotations to determine the relationship between functional elements and microRNA regulation. We built a logistic regression model that classifies microRNA/SNP pairs into eQTLs or non-eQTLs with 85% accuracy; shows microRNA-eQTL enrichment for microRNA precursors, promoters, enhancers, and transcription factor binding sites; and depletion for repressed chromatin. Interestingly, although there is a large overlap between microRNA eQTLs and messenger RNA eQTLs of host genes, 74% of these shared eQTLs affect microRNA and host expression independently. Considering microRNA-only eQTLs we find a significant enrichment for intronic promoters, validating the existence of alternative promoters for intragenic microRNAs. Finally, in line with the GM12878 cell line derived from B cells, we find genome-wide association (GWA) variants associated to blood-related traits more likely to be microRNA eQTLs than random GWA and non-GWA variants, aiding the interpretation of GWA results. PMID:27260304

  12. Principles of microRNA Regulation Revealed Through Modeling microRNA Expression Quantitative Trait Loci.

    PubMed

    Budach, Stefan; Heinig, Matthias; Marsico, Annalisa

    2016-08-01

    Extensive work has been dedicated to study mechanisms of microRNA-mediated gene regulation. However, the transcriptional regulation of microRNAs themselves is far less well understood, due to difficulties determining the transcription start sites of transient primary transcripts. This challenge can be addressed using expression quantitative trait loci (eQTLs) whose regulatory effects represent a natural source of perturbation of cis-regulatory elements. Here we used previously published cis-microRNA-eQTL data for the human GM12878 cell line, promoter predictions, and other functional annotations to determine the relationship between functional elements and microRNA regulation. We built a logistic regression model that classifies microRNA/SNP pairs into eQTLs or non-eQTLs with 85% accuracy; shows microRNA-eQTL enrichment for microRNA precursors, promoters, enhancers, and transcription factor binding sites; and depletion for repressed chromatin. Interestingly, although there is a large overlap between microRNA eQTLs and messenger RNA eQTLs of host genes, 74% of these shared eQTLs affect microRNA and host expression independently. Considering microRNA-only eQTLs we find a significant enrichment for intronic promoters, validating the existence of alternative promoters for intragenic microRNAs. Finally, in line with the GM12878 cell line derived from B cells, we find genome-wide association (GWA) variants associated to blood-related traits more likely to be microRNA eQTLs than random GWA and non-GWA variants, aiding the interpretation of GWA results.

  13. Principles of microRNA Regulation Revealed Through Modeling microRNA Expression Quantitative Trait Loci

    PubMed Central

    Budach, Stefan; Heinig, Matthias; Marsico, Annalisa

    2016-01-01

    Extensive work has been dedicated to study mechanisms of microRNA-mediated gene regulation. However, the transcriptional regulation of microRNAs themselves is far less well understood, due to difficulties determining the transcription start sites of transient primary transcripts. This challenge can be addressed using expression quantitative trait loci (eQTLs) whose regulatory effects represent a natural source of perturbation of cis-regulatory elements. Here we used previously published cis-microRNA-eQTL data for the human GM12878 cell line, promoter predictions, and other functional annotations to determine the relationship between functional elements and microRNA regulation. We built a logistic regression model that classifies microRNA/SNP pairs into eQTLs or non-eQTLs with 85% accuracy; shows microRNA-eQTL enrichment for microRNA precursors, promoters, enhancers, and transcription factor binding sites; and depletion for repressed chromatin. Interestingly, although there is a large overlap between microRNA eQTLs and messenger RNA eQTLs of host genes, 74% of these shared eQTLs affect microRNA and host expression independently. Considering microRNA-only eQTLs we find a significant enrichment for intronic promoters, validating the existence of alternative promoters for intragenic microRNAs. Finally, in line with the GM12878 cell line derived from B cells, we find genome-wide association (GWA) variants associated to blood-related traits more likely to be microRNA eQTLs than random GWA and non-GWA variants, aiding the interpretation of GWA results. PMID:27260304

  14. MicroRNA Expression during Bovine Oocyte Maturation and Fertilization.

    PubMed

    Gilchrist, Graham C; Tscherner, Allison; Nalpathamkalam, Thomas; Merico, Daniele; LaMarre, Jonathan

    2016-01-01

    Successful fertilization and subsequent embryo development rely on complex molecular processes starting with the development of oocyte competence through maturation. MicroRNAs (miRNAs) are small non-coding RNA molecules that function as gene regulators in many biological systems, including the oocyte and embryo. In order to further explore the roles of miRNAs in oocyte maturation, we employed small RNA sequencing as a screening tool to identify and characterize miRNA populations present in pools of bovine germinal vesicle (GV) oocytes, metaphase II (MII) oocytes, and presumptive zygotes (PZ). Each stage contained a defined miRNA population, some of which showed stable expression while others showed progressive changes between stages that were subsequently confirmed by quantitative reverse transcription polymerase chain reaction (RT-PCR). Bta-miR-155, bta-miR-222, bta-miR-21, bta-let-7d, bta-let-7i, and bta-miR-190a were among the statistically significant differentially expressed miRNAs (p < 0.05). To determine whether changes in specific primary miRNA (pri-miRNA) transcripts were responsible for the observed miRNA changes, we evaluated pri-miR-155, -222 and let-7d expression. Pri-miR-155 and -222 were not detected in GV oocytes but pri-miR-155 was present in MII oocytes, indicating transcription during maturation. In contrast, levels of pri-let-7d decreased during maturation, suggesting that the observed increase in let-7d expression was likely due to processing of the primary transcript. This study demonstrates that both dynamic and stable populations of miRNAs are present in bovine oocytes and zygotes and extend previous studies supporting the importance of the small RNA landscape in the maturing bovine oocyte and early embryo. PMID:26999121

  15. MicroRNA Expression during Bovine Oocyte Maturation and Fertilization

    PubMed Central

    Gilchrist, Graham C.; Tscherner, Allison; Nalpathamkalam, Thomas; Merico, Daniele; LaMarre, Jonathan

    2016-01-01

    Successful fertilization and subsequent embryo development rely on complex molecular processes starting with the development of oocyte competence through maturation. MicroRNAs (miRNAs) are small non-coding RNA molecules that function as gene regulators in many biological systems, including the oocyte and embryo. In order to further explore the roles of miRNAs in oocyte maturation, we employed small RNA sequencing as a screening tool to identify and characterize miRNA populations present in pools of bovine germinal vesicle (GV) oocytes, metaphase II (MII) oocytes, and presumptive zygotes (PZ). Each stage contained a defined miRNA population, some of which showed stable expression while others showed progressive changes between stages that were subsequently confirmed by quantitative reverse transcription polymerase chain reaction (RT-PCR). Bta-miR-155, bta-miR-222, bta-miR-21, bta-let-7d, bta-let-7i, and bta-miR-190a were among the statistically significant differentially expressed miRNAs (p < 0.05). To determine whether changes in specific primary miRNA (pri-miRNA) transcripts were responsible for the observed miRNA changes, we evaluated pri-miR-155, -222 and let-7d expression. Pri-miR-155 and -222 were not detected in GV oocytes but pri-miR-155 was present in MII oocytes, indicating transcription during maturation. In contrast, levels of pri-let-7d decreased during maturation, suggesting that the observed increase in let-7d expression was likely due to processing of the primary transcript. This study demonstrates that both dynamic and stable populations of miRNAs are present in bovine oocytes and zygotes and extend previous studies supporting the importance of the small RNA landscape in the maturing bovine oocyte and early embryo. PMID:26999121

  16. Expression of chimeric tRNA-driven antisense transcripts renders NIH 3T3 cells highly resistant to Moloney murine leukemia virus replication.

    PubMed Central

    Sullenger, B A; Lee, T C; Smith, C A; Ungers, G E; Gilboa, E

    1990-01-01

    NIH 3T3 cells infected with Moloney murine leukemia virus (MoMLV) express high levels of virus-specific RNA. To inhibit replication of the virus, we stably introduced chimeric tRNA genes encoding antisense templates into NIH 3T3 cells via a retroviral vector. Efficient expression of hybrid tRNA-MoMLV antisense transcripts and inhibition of MoMLV replication were dependent on the use of a particular type of retroviral vector, the double-copy vector, in which the chimeric tRNA gene was inserted in the 3' long terminal repeat. MoMLV replication was inhibited up to 97% in cells expressing antisense RNA corresponding to the gag gene and less than twofold in cells expressing antisense RNA corresponding to the pol gene. RNA and protein analyses suggest that inhibition was exerted at the level of translation. These results suggest that RNA polymerase III-based antisense inhibition systems can be used to inhibit highly expressed viral genes and render cells resistant to viral replication via intracellular immunization strategies. Images PMID:2247070

  17. Design and Generation of Drosophila Single Guide RNA Expression Constructs.

    PubMed

    Housden, Benjamin E; Hu, Yanhui; Perrimon, Norbert

    2016-01-01

    The recent advances in CRISPR-based genome engineering have enabled a plethora of new experiments to study a wide range of biological questions. The major attraction of this system over previous methods is its high efficiency and simplicity of use. For example, whereas previous genome engineering technologies required the generation of new proteins to target each new locus, CRISPR requires only the expression of a different single guide RNA (sgRNA). This sgRNA binds to the Cas9 endonuclease protein and directs the generation of a double-strand break to a highly specific genomic site determined by the sgRNA sequence. In addition, the relative simplicity of the Drosophila genome is a particular advantage, as possible sgRNA off-target sites can easily be avoided. Here, we provide a step-by-step protocol for designing sgRNA target sites using the Drosophila RNAi Screening Center (DRSC) Find CRISPRs tool (version 2). We also describe the generation of sgRNA expression plasmids for the use in cultured Drosophila cells or in vivo. Finally, we discuss specific design requirements for various genome engineering applications. PMID:27587779

  18. The use of chromatin insulators to improve the expression and safety of integrating gene transfer vectors.

    PubMed

    Emery, David W

    2011-06-01

    The therapeutic application of recombinant retroviruses and other integrating gene transfer vectors has been limited by problems of vector expression and vector-mediated genotoxicity. These problems arise in large part from the interactions between vector sequences and the genomic environment surrounding sites of integration. Strides have been made in overcoming both of these problems through the modification of deleterious vector sequences, the inclusion of better enhancers and promoters, and the use of alternative virus systems. However, these modifications often add other restrictions on vector design, which in turn can further limit therapeutic applications. As an alternative, several groups have been investigating a class of DNA regulatory elements known as chromatin insulators. These elements provide a means of blocking the interaction between an integrating vector and the target cell genome in a manner that is independent of the vector transgene, regulatory elements, or virus of origin. This review outlines the background, rationale, and evidence for using chromatin insulators to improve the expression and safety of gene transfer vectors. Also reviewed are topological factors that constrain the use of insulators in integrating gene transfer vectors, alternative sources of insulators, and the role of chromatin insulators as one of several components for optimal vector design.

  19. Posttranscriptional regulation of retroviral gene expression: primary RNA transcripts play three roles as pre-mRNA, mRNA, and genomic RNA

    PubMed Central

    LeBlanc, Jason; Weil, Jason; Beemon, Karen

    2013-01-01

    After reverse transcription of the retroviral RNA genome and integration of the DNA provirus into the host genome, host machinery is used for viral gene expression along with viral proteins and RNA regulatory elements. Here, we discuss co-transcriptional and posttranscriptional regulation of retroviral gene expression, comparing simple and complex retroviruses. Cellular RNA polymerase II synthesizes full-length viral primary RNA transcripts that are capped and polyadenylated. All retroviruses generate a singly spliced env mRNA from this primary transcript, which encodes the viral glycoproteins. In addition, complex viral RNAs are alternatively spliced to generate accessory proteins, such as Rev, which is involved in posttranscriptional regulation of HIV-1 RNA. Importantly, the splicing of all retroviruses is incomplete; they must maintain and export a fraction of their primary RNA transcripts. This unspliced RNA functions both as the major mRNA for Gag and Pol proteins and as the packaged genomic RNA. Different retroviruses export their unspliced viral RNA from the nucleus to the cytoplasm by either Tap-dependent or Rev/CRM1-dependent routes. Translation of the unspliced mRNA involves frame-shifting or termination codon suppression so that the Gag proteins, which make up the capsid, are expressed more abundantly than the Pol proteins, which are the viral enzymes. After the viral polyproteins assemble into viral particles and bud from the cell membrane, a viral encoded protease cleaves them. Some retroviruses have evolved mechanisms to protect their unspliced RNA from decay by nonsense-mediated RNA decay and to prevent genome editing by the cellular APOBEC deaminases. PMID:23754689

  20. Rev-erb beta regulates the Srebp-1c promoter and mRNA expression in skeletal muscle cells

    SciTech Connect

    Ramakrishnan, Sathiya N.; Lau, Patrick; Crowther, Lisa M.; Cleasby, Mark E.; Millard, Susan; Leong, Gary M.; Cooney, Gregory J.; Muscat, George E.O.

    2009-10-30

    The nuclear hormone receptor, Rev-erb beta operates as a transcriptional silencer. We previously demonstrated that exogenous expression of Rev-erb{beta}{Delta}E in skeletal muscle cells increased Srebp-1c mRNA expression. We validated these in vitro observations by injection of an expression vector driving Rev-erb{beta}{Delta}E expression into mouse tibialis muscle that resulted in increased Srebp-1c mRNA expression. Paradoxically, Rev-erb{beta} siRNA expression in skeletal muscle cells repressed Srebp-1c expression, and indicated that Rev-erb{beta} expression was necessary for Srebp-1c expression. ChIP analysis demonstrated that Rev-erb{beta} was recruited to the Srebp-1c promoter. Moreover, Rev-erb{beta} trans-activated the Srebp-1c promoter, in contrast, Rev-erb{beta} efficiently repressed the Rev-erb{alpha} promoter, a previously characterized target gene. Finally, treatment with the Rev-erb agonist (hemin) (i) increased the trans-activation of the Srebp-1c promoter by Rev-erb{beta}; and (ii) increased Rev-erb{beta} and Srebp-1c mRNA expression. These data suggest that Rev-erb{beta} has the potential to activate gene expression, and is a positive regulator of Srebp-1c, a regulator of lipogenesis.

  1. Enhancement of heterologous gene expression in Flammulina velutipes using polycistronic vectors containing a viral 2A cleavage sequence.

    PubMed

    Lin, Yu-Ju; Huang, Li-Hsin; Huang, Ching-Tsan

    2013-01-01

    Agrobacterium tumefaciens-mediated transformation for edible mushrooms has been previously established. However, the enhancement of heterologous protein production and the expression of multi-target genes remains a challenge. In this study, heterologous protein expression in the enoki mushroom Flammulina velutipes was notably enhanced using 2A peptide-mediated cleavage to co-express multiple copies of single gene. The polycistronic expression vectors were constructed by connecting multi copies of the enhanced green fluorescent protein (egfp) gene using 2A peptides derived from porcine teschovirus-1. The P2A peptides properly self-cleaved as shown by the formation of the transformants with antibiotic resistant capacity and exciting green fluorescence levels after introducing the vectors into F. velutipes mycelia. The results of western blot analysis, epifluorescent microscopy and EGFP production showed that heterologous protein expression in F. velutipes using the polycistronic strategy increased proportionally as the gene copy number increased from one to three copies. In contrast, much lower EGFP levels were detected in the F. velutipes transformants harboring four copies of the egfp gene due to mRNA instability. The polycistronic strategy using 2A peptide-mediated cleavage developed in this study can not only be used to express single gene in multiple copies, but also to express multiple genes in a single reading frame. It is a promising strategy for the application of mushroom molecular pharming.

  2. Enhancement of heterologous gene expression in Flammulina velutipes using polycistronic vectors containing a viral 2A cleavage sequence.

    PubMed

    Lin, Yu-Ju; Huang, Li-Hsin; Huang, Ching-Tsan

    2013-01-01

    Agrobacterium tumefaciens-mediated transformation for edible mushrooms has been previously established. However, the enhancement of heterologous protein production and the expression of multi-target genes remains a challenge. In this study, heterologous protein expression in the enoki mushroom Flammulina velutipes was notably enhanced using 2A peptide-mediated cleavage to co-express multiple copies of single gene. The polycistronic expression vectors were constructed by connecting multi copies of the enhanced green fluorescent protein (egfp) gene using 2A peptides derived from porcine teschovirus-1. The P2A peptides properly self-cleaved as shown by the formation of the transformants with antibiotic resistant capacity and exciting green fluorescence levels after introducing the vectors into F. velutipes mycelia. The results of western blot analysis, epifluorescent microscopy and EGFP production showed that heterologous protein expression in F. velutipes using the polycistronic strategy increased proportionally as the gene copy number increased from one to three copies. In contrast, much lower EGFP levels were detected in the F. velutipes transformants harboring four copies of the egfp gene due to mRNA instability. The polycistronic strategy using 2A peptide-mediated cleavage developed in this study can not only be used to express single gene in multiple copies, but also to express multiple genes in a single reading frame. It is a promising strategy for the application of mushroom molecular pharming. PMID:23516605

  3. Micro-RNA Expression and Function in Lymphomas

    PubMed Central

    Sandhu, Sukhinder K.; Croce, Carlo M.; Garzon, Ramiro

    2011-01-01

    The recent discovery of microRNAs (miRNAs) has introduced a new layer of complexity to the process of gene regulation. MiRNAs are essential for cellular function, and their dysregulation often results in disease. Study of miRNA expression and function in animal models and human lymphomas has improved our knowledge of the pathogenesis of this heterogeneous disease. In this paper, we attempt to describe the expression of miRNAs and their function in lymphomas and discuss potential miRNA-based therapies in the diagnosis and treatment of lymphomas. PMID:21461378

  4. Assessment of microRNA differential expression and detection in multiplexed small RNA sequencing data.

    PubMed

    Campbell, Joshua D; Liu, Gang; Luo, Lingqi; Xiao, Ji; Gerrein, Joseph; Juan-Guardela, Brenda; Tedrow, John; Alekseyev, Yuriy O; Yang, Ivana V; Correll, Mick; Geraci, Mark; Quackenbush, John; Sciurba, Frank; Schwartz, David A; Kaminski, Naftali; Johnson, W Evan; Monti, Stefano; Spira, Avrum; Beane, Jennifer; Lenburg, Marc E

    2015-02-01

    Small RNA sequencing can be used to gain an unprecedented amount of detail into the microRNA transcriptome. The relatively high cost and low throughput of sequencing bases technologies can potentially be offset by the use of multiplexing. However, multiplexing involves a trade-off between increased number of sequenced samples and reduced number of reads per sample (i.e., lower depth of coverage). To assess the effect of different sequencing depths owing to multiplexing on microRNA differential expression and detection, we sequenced the small RNA of lung tissue samples collected in a clinical setting by multiplexing one, three, six, nine, or 12 samples per lane using the Illumina HiSeq 2000. As expected, the numbers of reads obtained per sample decreased as the number of samples in a multiplex increased. Furthermore, after normalization, replicate samples included in distinct multiplexes were highly correlated (R > 0.97). When detecting differential microRNA expression between groups of samples, microRNAs with average expression >1 reads per million (RPM) had reproducible fold change estimates (signal to noise) independent of the degree of multiplexing. The number of microRNAs detected was strongly correlated with the log2 number of reads aligning to microRNA loci (R = 0.96). However, most additional microRNAs detected in samples with greater sequencing depth were in the range of expression which had lower fold change reproducibility. These findings elucidate the trade-off between increasing the number of samples in a multiplex with decreasing sequencing depth and will aid in the design of large-scale clinical studies exploring microRNA expression and its role in disease.

  5. Inhibitory receptor expression on memory CD8 T cells following Ad vector immunization.

    PubMed

    Penaloza-MacMaster, Pablo; Alayo, Quazim A; Ra, Joshua; Provine, Nicholas M; Larocca, Rafael; Lee, Benjamin; Barouch, Dan H

    2016-09-22

    T cells are an important component of immune responses, and their function is influenced by their expression of inhibitory receptors. Immunization with alternative serotype adenovirus (Ad) vectors induces highly functional T cell responses with lower programmed cell death 1 (PD-1) expression and increased boostability relative to Ad5 vectors. However, a detailed phenotypic characterization of other inhibitory receptors is lacking, and it is unknown whether Ad5-induced CD8 T cells eventually recover function with time. In this report, we measure the expression of various inhibitory receptors and memory markers during early and late time points following vaccination with Ad5 and alternative serotype Ad vectors. CD8 T cells induced by Ad5 exhibited increased expression of the inhibitory receptor Tim-3 and showed decreased central memory differentiation as compared with alternative serotype Ad vectors, even a year following immunization. Moreover, relative to Ad5-primed mice, Ad26-primed mice exhibited substantially improved recall of SIV Gag-specific CD8 T cell responses following heterologous boosting with MVA or Ad35 vectors. We also demonstrate that low doses of Ad5 priming resulted in more boostable immune responses with lower PD-1 expression as compared to high Ad5 doses, suggesting a role for vector dose in influencing immune dysfunction following Ad5 vaccination. These data suggest that Ad5 vectors induce a long-term pattern of immune exhaustion that can be partly overcome by lowering vector dose and modulating inhibitory signals. PMID:27566899

  6. Small RNA cloning and sequencing strategy affects host and viral microRNA expression signatures.

    PubMed

    Stik, Grégoire; Muylkens, Benoît; Coupeau, Damien; Laurent, Sylvie; Dambrine, Ginette; Messmer, Mélanie; Chane-Woon-Ming, Béatrice; Pfeffer, Sébastien; Rasschaert, Denis

    2014-07-10

    The establishment of the microRNA (miRNA) expression signatures is the basic element to investigate the role played by these regulatory molecules in the biology of an organism. Marek's disease virus 1 (MDV-1) is an avian herpesvirus that naturally infects chicken and induces T cells lymphomas. During latency, MDV-1, like other herpesviruses, expresses a limited subset of transcripts. These include three miRNA clusters. Several studies identified the expression of virus and host encoded miRNAs from MDV-1 infected cell cultures and chickens. But a high discrepancy was observed when miRNA cloning frequencies obtained from different cloning and sequencing protocols were compared. Thus, we analyzed the effect of small RNA library preparation and sequencing on the miRNA frequencies obtained from the same RNA samples collected during MDV-1 infection of chicken at different steps of the oncoviral pathogenesis. Qualitative and quantitative variations were found in the data, depending on the strategy used. One of the mature miRNA derived from the latency-associated-transcript (LAT), mdv1-miR-M7-5p, showed the highest variation. Its cloning frequency was 50% of the viral miRNA counts when a small scale sequencing approach was used. Its frequency was 100 times less abundant when determined through the deep sequencing approach. Northern blot analysis showed a better correlation with the miRNA frequencies found by the small scale sequencing approach. By analyzing the cellular miRNA repertoire, we also found a gap between the two sequencing approaches. Collectively, our study indicates that next-generation sequencing data considered alone are limited for assessing the absolute copy number of transcripts. Thus, the quantification of small RNA should be addressed by compiling data obtained by using different techniques such as microarrays, qRT-PCR and NB analysis in support of high throughput sequencing data. These observations should be considered when miRNA variations are studied

  7. Profiling Pre-MicroRNA and Mature MicroRNA Expressions Using a Single Microarray and Avoiding Separate Sample Preparation

    PubMed Central

    Gan, Lin; Denecke, Bernd

    2013-01-01

    Mature microRNA is a crucial component in the gene expression regulation network. At the same time, microRNA gene expression and procession is regulated in a precise and collaborated way. Pre-microRNAs mediate products during the microRNA transcription process, they can provide hints of microRNA gene expression regulation or can serve as alternative biomarkers. To date, little effort has been devoted to pre-microRNA expression profiling. In this study, three human and three mouse microRNA profile data sets, based on the Affymetrix miRNA 2.0 array, have been re-analyzed for both mature and pre-microRNA signals as a primary test of parallel mature/pre-microRNA expression profiling on a single platform. The results not only demonstrated a glimpse of pre-microRNA expression in human and mouse, but also the relationship of microRNA expressions between pre- and mature forms. The study also showed a possible application of currently available microRNA microarrays in profiling pre-microRNA expression in a time and cost effective manner.

  8. miRTarVis: an interactive visual analysis tool for microRNA-mRNA expression profile data

    PubMed Central

    2015-01-01

    Background MicroRNAs (miRNA) are short nucleotides that down-regulate its target genes. Various miRNA target prediction algorithms have used sequence complementarity between miRNA and its targets. Recently, other algorithms tried to improve sequence-based miRNA target prediction by exploiting miRNA-mRNA expression profile data. Some web-based tools are also introduced to help researchers predict targets of miRNAs from miRNA-mRNA expression profile data. A demand for a miRNA-mRNA visual analysis tool that features novel miRNA prediction algorithms and more interactive visualization techniques exists. Results We designed and implemented miRTarVis, which is an interactive visual analysis tool that predicts targets of miRNAs from miRNA-mRNA expression profile data and visualizes the resulting miRNA-target interaction network. miRTarVis has intuitive interface design in accordance with the analysis procedure of load, filter, predict, and visualize. It predicts targets of miRNA by adopting Bayesian inference and MINE analyses, as well as conventional correlation and mutual information analyses. It visualizes a resulting miRNA-mRNA network in an interactive Treemap, as well as a conventional node-link diagram. miRTarVis is available at http://hcil.snu.ac.kr/~rati/miRTarVis/index.html. Conclusions We reported findings from miRNA-mRNA expression profile data of asthma patients using miRTarVis in a case study. miRTarVis helps to predict and understand targets of miRNA from miRNA-mRNA expression profile data. PMID:26361498

  9. Construction and evaluation of an adenoviral vector for the liver-specific expression of the serine/arginine-rich splicing factor, SRSF3.

    PubMed

    Suchanek, Amanda L; Salati, Lisa M

    2015-11-01

    Serine/arginine-rich splicing factor-3 (SRSF3), alternatively known as SRp20, is a member of the highly-conserved SR protein family of mRNA splicing factors. SRSF3 generally functions as an enhancer of mRNA splicing by binding to transcripts in a sequence-specific manner to both recruit and stabilize the binding of spliceosomal components to the mRNA. In liver, expression of SRSF3 is relatively low and its activity is increased in response to insulin and feeding a high carbohydrate diet. We sought to over-express SRSF3 in primary rat hepatocytes to identify regulatory targets. A standard adenoviral shuttle vector system containing an epitope-tagged SRSF3 under the transcriptional control of the CMV promoter could not be used to produce infectious adenoviral particles. SRSF3 over-expression in the packaging cell line prevented the production of infectious adenovirus particles by interfering with the viral splicing program. To circumvent this issue, SRSF3 expression from the shuttle vector was blocked by placing its expression under the control of the liver-specific albumin promoter. In this system, the FLAG-SRSF3 transgene is only expressed in the target cells (hepatocytes) but not in the packaging cell line. An additional benefit of the albumin promoter is that expression of the transgene does not require the addition of hormones or antibiotics to drive SRSF3 expression in the hepatocytes. Robust expression of FLAG-SRSF3 protein is detected in both HepG2 cells and primary rat hepatocytes infected with adenovirus prepared from this new shuttle vector. Furthermore, abundances of several known and suspected mRNA targets of SRSF3 action are increased in response to over-expression using this virus. This report details the construction of the albumin promoter-driven adenoviral shuttle vector, termed pmAlbAd5-FLAG.SRSF3, that can be used to generate functional adenovirus to express FLAG-SRSF3 specifically in liver. This vector would be suitable for over-expression of

  10. Boiler: lossy compression of RNA-seq alignments using coverage vectors.

    PubMed

    Pritt, Jacob; Langmead, Ben

    2016-09-19

    We describe Boiler, a new software tool for compressing and querying large collections of RNA-seq alignments. Boiler discards most per-read data, keeping only a genomic coverage vector plus a few empirical distributions summarizing the alignments. Since most per-read data is discarded, storage footprint is often much smaller than that achieved by other compression tools. Despite this, the most relevant per-read data can be recovered; we show that Boiler compression has only a slight negative impact on results given by downstream tools for isoform assembly and quantification. Boiler also allows the user to pose fast and useful queries without decompressing the entire file. Boiler is free open source software available from github.com/jpritt/boiler.

  11. Boiler: lossy compression of RNA-seq alignments using coverage vectors

    PubMed Central

    Pritt, Jacob; Langmead, Ben

    2016-01-01

    We describe Boiler, a new software tool for compressing and querying large collections of RNA-seq alignments. Boiler discards most per-read data, keeping only a genomic coverage vector plus a few empirical distributions summarizing the alignments. Since most per-read data is discarded, storage footprint is often much smaller than that achieved by other compression tools. Despite this, the most relevant per-read data can be recovered; we show that Boiler compression has only a slight negative impact on results given by downstream tools for isoform assembly and quantification. Boiler also allows the user to pose fast and useful queries without decompressing the entire file. Boiler is free open source software available from github.com/jpritt/boiler. PMID:27298258

  12. Boiler: lossy compression of RNA-seq alignments using coverage vectors.

    PubMed

    Pritt, Jacob; Langmead, Ben

    2016-09-19

    We describe Boiler, a new software tool for compressing and querying large collections of RNA-seq alignments. Boiler discards most per-read data, keeping only a genomic coverage vector plus a few empirical distributions summarizing the alignments. Since most per-read data is discarded, storage footprint is often much smaller than that achieved by other compression tools. Despite this, the most relevant per-read data can be recovered; we show that Boiler compression has only a slight negative impact on results given by downstream tools for isoform assembly and quantification. Boiler also allows the user to pose fast and useful queries without decompressing the entire file. Boiler is free open source software available from github.com/jpritt/boiler. PMID:27298258

  13. Inducible gene and shRNA expression in resident hematopoietic stem cells in vivo.

    PubMed

    Laurenti, Elisa; Barde, Isabelle; Verp, Sonia; Offner, Sandra; Wilson, Anne; Quenneville, Simon; Wiznerowicz, Maciej; Macdonald, H Robson; Trono, Didier; Trumpp, Andreas

    2010-08-01

    Hematopoietic stem cells (HSC) are probably the best understood somatic stem cells and often serve as a paradigm for other stem cells. Nevertheless, most current techniques to genetically manipulate them in vivo are either constitutive and/or induced in settings of hematopoietic stress such as after irradiation. Here, we present a conditional expression system that allows for externally controllable transgenesis and knockdown in resident HSCs, based on a lentiviral vector containing a tet-O sequence and a transgenic mouse line expressing a doxycyclin-regulated tTR-KRAB repressor protein. HSCs harvested from tTR-KRAB mice are transduced with the lentiviral vector containing a cDNA (i.e., Green Fluorescent Protein (GFP)) and/or shRNA (i.e., p53) of interest and then transplanted into lethally irradiated recipients. While the vector is effectively repressed by tTR-KRAB during homing and engraftment, robust GFP/shp53 expression is induced on doxycyclin treatment in HSCs and their progeny. Doxycylin-controllable transcription is maintained on serial transplantation, indicating that repopulating HSCs are stably modified by this approach. In summary, this easy to implement conditional system provides inducible and reversible overexpression or knock down of genes in resident HSCs in vivo using a drug devoid of toxic or activating effects. PMID:20641037

  14. MicroRNA expression profiling in human Barrett's carcinogenesis

    PubMed Central

    Fassan, Matteo; Volinia, Stefano; Palatini, Jeff; Pizzi, Marco; Baffa, Raffaele; De Bernard, Marina; Battaglia, Giorgio; Parente, Paola; Croce, Carlo M.; Zaninotto, Giovanni; Ancona, Ermanno; Rugge, Massimo

    2015-01-01

    Barrett's esophagus (BE) is characterized by the native stratified squamous epithelium (N) lining the esophagus being replaced by a columnar epithelium with intestinal differentiation (Barrett's mucosa; BM). BM is considered as the main risk factor for esophageal adenocarcinoma (Barrett's adenocarcinoma; BAc). MicroRNAs (miRNAs) are a class of small noncoding RNAs that control gene expression by targeting messenger RNAs and they are reportedly dysregulated in BM. To test the hypothesis that a specific miRNA expression signature characterizes BM development and progression, we performed miRNA microarray analysis comparing native esophageal mucosa with all the phenotypic lesions seen in the Barrett's carcinogenic process. Specimens were collected from 14 BE patients who had undergone esophagectomy, including: 14 with N, 14 with BM, 7 with low-grade intraepithelial neoplasia, 5 with high-grade intra-epithelial neoplasia and 11 with BAc. Microarray findings were further validated by quantitive real-time polymerase chain reaction and in situ hybridization analyses using a different series of consecutive cases (162 biopsy samples and 5 esophagectomies) of histologically proven, long-segment BE. We identified a miRNA signature of Barrett's carcinogenesis consisting of an increased expression of 6 miRNAs and a reduced expression of 7 miRNAs. To further support these results, we investigated target gene expression using the Oncomine database and/or immunohistochemical analysis. We found that target gene expression correlated significantly with miRNA dysregulation. Specific miRNAs are directly involved in BE progression to cancer. miRNA profiling significantly expands current knowledge on the molecular history of Barrett's carcinogenesis, also identifying molecular markers of cancer progression. PMID:21128279

  15. Insect cell transformation vectors that support high level expression and promoter assessment in insect cell culture

    Technology Transfer Automated Retrieval System (TEKTRAN)

    A somatic transformation vector, pDP9, was constructed that provides a simplified means of producing permanently transformed cultured insect cells that support high levels of protein expression of foreign genes. The pDP9 plasmid vector incorporates DNA sequences from the Junonia coenia densovirus th...

  16. Simultaneous cloning of open reading frames into several different expression vectors.

    PubMed

    Stanyon, Clement A; Limjindaporn, Thawornchai; Finley, Russell L

    2003-09-01

    Genomic sequencing has enabled the prediction of thousands of genes, most of which either cannot be assigned a function or can be only broadly categorized on the basis of sequence alone. High-throughput strategies for elucidating protein function are of high priority, and numerous approaches are being developed. Many of these approaches require the cloning of open reading frames (ORFs) into expression vectors that enable the encoded proteins to be tested for biological and biochemical activities. Typically, more than one type of vector must be employed, as different experiments require different conditions of protein production. Here we show that it is possible to simultaneously transfer a single ORF from a source vector to four target vectors using a commercially available in vitro recombination system. To test the approach, we constructed new vectors for expression of fusion proteins in yeast, including vectors for the LexA two-hybrid system. We show that individual ORFs can be efficiently transferred to four different vectors in a single in vitro reaction. The resulting expression plasmids can be separated using prototrophic markers specific to each vector. Using this system to produce multiple expression constructs simultaneously could greatly facilitate high-throughput subcloning and proteomic studies.

  17. miRNA expression during prickly pear cactus fruit development.

    PubMed

    Rosas-Cárdenas, Flor de Fátima; Caballero-Pérez, Juan; Gutiérrez-Ramos, Ximena; Marsch-Martínez, Nayelli; Cruz-Hernández, Andrés; de Folter, Stefan

    2015-02-01

    miRNAs are a class of small non-coding RNAs that regulate gene expression. They are involved in the control of many developmental processes, including fruit development. The increasing amount of information on miRNAs, on their expression, abundance, and conservation between various species, provides a new opportunity to study the role of miRNAs in non-model plant species. In this work, we used a combination of Northern blot and tissue print hybridization analysis to identify conserved miRNAs expressed during prickly pear cactus (Opuntia ficus indica) fruit development. Comparative profiling detected the expression of 34 miRNAs, which were clustered in three different groups that were associated with the different phases of fruit development. Variation in the level of miRNA expression was observed. Gradual expression increase of several miRNAs was observed during fruit development, including miR164. miR164 was selected for stem-loop RT-PCR and for a detailed spatial-temporal expression analysis. At early floral stages, miR164 was mainly localized in meristematic tissues, boundaries and fusion zones, while it was more homogenously expressed in fruit tissues. Our results provide the first evidence of miRNA expression in the prickly pear cactus and provide the basis for future research on miRNAs in Opuntia. Moreover, our analyses suggest that miR164 plays different roles during prickly pear cactus fruit development.

  18. miRNA expression during prickly pear cactus fruit development.

    PubMed

    Rosas-Cárdenas, Flor de Fátima; Caballero-Pérez, Juan; Gutiérrez-Ramos, Ximena; Marsch-Martínez, Nayelli; Cruz-Hernández, Andrés; de Folter, Stefan

    2015-02-01

    miRNAs are a class of small non-coding RNAs that regulate gene expression. They are involved in the control of many developmental processes, including fruit development. The increasing amount of information on miRNAs, on their expression, abundance, and conservation between various species, provides a new opportunity to study the role of miRNAs in non-model plant species. In this work, we used a combination of Northern blot and tissue print hybridization analysis to identify conserved miRNAs expressed during prickly pear cactus (Opuntia ficus indica) fruit development. Comparative profiling detected the expression of 34 miRNAs, which were clustered in three different groups that were associated with the different phases of fruit development. Variation in the level of miRNA expression was observed. Gradual expression increase of several miRNAs was observed during fruit development, including miR164. miR164 was selected for stem-loop RT-PCR and for a detailed spatial-temporal expression analysis. At early floral stages, miR164 was mainly localized in meristematic tissues, boundaries and fusion zones, while it was more homogenously expressed in fruit tissues. Our results provide the first evidence of miRNA expression in the prickly pear cactus and provide the basis for future research on miRNAs in Opuntia. Moreover, our analyses suggest that miR164 plays different roles during prickly pear cactus fruit development. PMID:25366556

  19. MicroRNA Expression and Identification of Putative miRNA Targets in Ovarian Cancer

    PubMed Central

    Dahiya, Neetu; Sherman-Baust, Cheryl A.; Wang, Tian-Li; Davidson, Ben; Shih, Ie-Ming; Zhang, Yongqing; Wood, William; Becker, Kevin G.; Morin, Patrice J.

    2008-01-01

    Background MicroRNAs (miRNAs) represent a class of small non-coding RNAs that control gene expression by targeting mRNAs and triggering either translation repression or RNA degradation. Emerging evidence suggests the potential involvement of altered regulation of miRNA in the pathogenesis of cancers, and these genes are thought to function as both tumor suppressors and oncogenes. Methodology/Principal Findings Using microRNA microarrays, we identify several miRNAs aberrantly expressed in human ovarian cancer tissues and cell lines. miR-221 stands out as a highly elevated miRNA in ovarian cancer, while miR-21 and several members of the let-7 family are found downregulated. Public databases were used to reveal potential targets for the highly differentially expressed miRNAs. In order to experimentally identify transcripts whose stability may be affected by the differentially expressed miRNAs, we transfected precursor miRNAs into human cancer cell lines and used oligonucleotide microarrays to examine changes in the mRNA levels. Interestingly, there was little overlap between the predicted and the experimental targets or pathways, or between experimental targets/pathways obtained using different cell lines, highlighting the complexity of miRNA target selection. Conclusion/Significance Our results identify several differentially expressed miRNAs in ovarian cancer and identify potential target transcripts that may be regulated by these miRNAs. These miRNAs and their targets may have important roles in the initiation and development of ovarian cancer. PMID:18560586

  20. Understanding mechanisms underlying human gene expression variation with RNA sequencing

    PubMed Central

    Pickrell, Joseph K.; Marioni, John C.; Pai, Athma A.; Degner, Jacob F.; Engelhardt, Barbara E.; Nkadori, Everlyne; Veyrieras, Jean-Baptiste; Stephens, Matthew; Gilad, Yoav; Pritchard, Jonathan K.

    2011-01-01

    Understanding the genetic mechanisms underlying natural variation in gene expression is a central goal of both medical and evolutionary genetics, and studies of expression quantitative trait loci (eQTLs) have become an important tool for achieving this goal1. Although all eQTL studies so far have assayed messenger RNA levels using expression microarrays, recent advances in RNA sequencing enable the analysis of transcript variation at unprecedented resolution. We sequenced RNA from 69 lymphoblastoid cell lines derived from unrelated Nigerian individuals that have been extensively genotyped by the International HapMap Project2. By pooling data from all individuals, we generated a map of the transcriptional landscape of these cells, identifying extensive use of unannotated untranslated regions and more than 100 new putative protein-coding exons. Using the genotypes from the HapMap project, we identified more than a thousand genes at which genetic variation influences overall expression levels or splicing. We demonstrate that eQTLs near genes generally act by a mechanism involving allele-specific expression, and that variation that influences the inclusion of an exon is enriched within and near the consensus splice sites. Our results illustrate the power of high-throughput sequencing for the joint analysis of variation in transcription, splicing and allele-specific expression across individuals. PMID:20220758

  1. Placental microRNA expression in pregnancies complicated by preeclampsia

    PubMed Central

    Enquobahrie, Daniel A.; Abetew, Dejene F.; Sorensen, Tanya K.; Willoughby, David; Chidambaram, Kumaravel; Williams, Michelle A.

    2010-01-01

    Objective The role of post-transcription regulation in preeclampsia is largely unknown. We investigated preeclampsia related placental microRNA (miRNA) expression using microarray and confirmatory qRT-PCR experiments. Study design Placental expressions of characterized and novel miRNAs (1,295 probes) were measured in samples collected from 20 preeclampsia cases and 20 controls. Differential expression was evaluated using Students T-test and fold change analyses. In pathway analysis, we examined functions/functional relationships of targets of differentially expressed miRNAs. Results Eight miRNAs were differentially expressed (1 up- and 7 down-regulated) among preeclampsia cases compared with controls. These included previously identified candidates (miR-210, miR-1 and a miRNA in the 14q32.31 cluster region) and others that are novel (miR- 584 and miR-34c-5p). These miRNAs target genes that participate in organ/system development (cardiovascular and reproductive system), immunologic dysfunction, cell adhesion, cell cycle and signaling. Conclusion Expression of microRNAs that target genes in diverse pathophysiological processes is altered in the setting of preeclampsia. PMID:21093846

  2. Identification and Expression Profiles of microRNA in Dolphin.

    PubMed

    Segawa, Takao; Kobayashi, Yuki; Inamoto, Satoko; Suzuki, Miwa; Endoh, Tomoko; Itou, Takuya

    2016-02-01

    Recently, microRNAs (miRNAs) are focused on the role of biomarker because they are stable in serum and plasma, and some of them express in the specific organs and increase with the organ injury. Thus miRNAs may be very useful as biomarkers for monitoring the health and condition of dolphins and for detecting disorders in aquariums. Here, a small RNA library was made from dolphin lung, liver and spleen, and miRNA expression patterns were then determined for 15 different tissues. We identified 62 conserved miRNA homologs in the dolphin small RNA library and found high expression miRNAs in specific tissues: miR-125b and miR-221 were highly expressed in brain, miR-23b in heart, miR-199a and miR-223 in lung, and miR-122-5p in liver. Some of these tissue-enriched miRNAs may be useful as specific and sensitive diagnostic blood biomarkers for organ injury in dolphins.

  3. Identification and Expression Profiles of microRNA in Dolphin.

    PubMed

    Segawa, Takao; Kobayashi, Yuki; Inamoto, Satoko; Suzuki, Miwa; Endoh, Tomoko; Itou, Takuya

    2016-02-01

    Recently, microRNAs (miRNAs) are focused on the role of biomarker because they are stable in serum and plasma, and some of them express in the specific organs and increase with the organ injury. Thus miRNAs may be very useful as biomarkers for monitoring the health and condition of dolphins and for detecting disorders in aquariums. Here, a small RNA library was made from dolphin lung, liver and spleen, and miRNA expression patterns were then determined for 15 different tissues. We identified 62 conserved miRNA homologs in the dolphin small RNA library and found high expression miRNAs in specific tissues: miR-125b and miR-221 were highly expressed in brain, miR-23b in heart, miR-199a and miR-223 in lung, and miR-122-5p in liver. Some of these tissue-enriched miRNAs may be useful as specific and sensitive diagnostic blood biomarkers for organ injury in dolphins. PMID:26853874

  4. Lipoplexes Strengthened by Anionic Polymers: Easy Preparation of Highly Effective siRNA Vectors Based on Cationic Lipids and Anionic Polymers.

    PubMed

    Arruda, Danielle Campiol; Schlegel, Anne; Bigey, Pascal; Escriou, Virginie

    2016-01-01

    RNA interference is an invaluable tool in biology to specifically silence a given gene. Synthetic duplexes of RNA oligonucleotides are widely used to induce mRNA degradation in cultured cells or in whole organisms. They have to be vectorized to reach their target site. Here, we describe the preparation of highly efficient siRNA vectors based on cationic liposomes and polyanionic polymers and their application in cultured cells to silence reporter and/or endogenous genes. PMID:27436316

  5. An intronic RNA structure modulates expression of the mRNA biogenesis factor Sus1.

    PubMed

    AbuQattam, Ali; Gallego, José; Rodríguez-Navarro, Susana

    2016-01-01

    Sus1 is a conserved protein involved in chromatin remodeling and mRNA biogenesis. Unlike most yeast genes, the SUS1 pre-mRNA of Saccharomyces cerevisiae contains two introns and is alternatively spliced, retaining one or both introns in response to changes in environmental conditions. SUS1 splicing may allow the cell to control Sus1 expression, but the mechanisms that regulate this process remain unknown. Using in silico analyses together with NMR spectroscopy, gel electrophoresis, and UV thermal denaturation experiments, we show that the downstream intron (I2) of SUS1 forms a weakly stable, 37-nucleotide stem-loop structure containing the branch site near its apical loop and the 3' splice site after the stem terminus. A cellular assay revealed that two of four mutants containing altered I2 structures had significantly impaired SUS1 expression. Semiquantitative RT-PCR experiments indicated that all mutants accumulated unspliced SUS1 pre-mRNA and/or induced distorted levels of fully spliced mRNA relative to wild type. Concomitantly, Sus1 cellular functions in histone H2B deubiquitination and mRNA export were affected in I2 hairpin mutants that inhibited splicing. This work demonstrates that I2 structure is relevant for SUS1 expression, and that this effect is likely exerted through modulation of splicing.

  6. An intronic RNA structure modulates expression of the mRNA biogenesis factor Sus1

    PubMed Central

    AbuQattam, Ali; Gallego, José; Rodríguez-Navarro, Susana

    2016-01-01

    Sus1 is a conserved protein involved in chromatin remodeling and mRNA biogenesis. Unlike most yeast genes, the SUS1 pre-mRNA of Saccharomyces cerevisiae contains two introns and is alternatively spliced, retaining one or both introns in response to changes in environmental conditions. SUS1 splicing may allow the cell to control Sus1 expression, but the mechanisms that regulate this process remain unknown. Using in silico analyses together with NMR spectroscopy, gel electrophoresis, and UV thermal denaturation experiments, we show that the downstream intron (I2) of SUS1 forms a weakly stable, 37-nucleotide stem–loop structure containing the branch site near its apical loop and the 3′ splice site after the stem terminus. A cellular assay revealed that two of four mutants containing altered I2 structures had significantly impaired SUS1 expression. Semiquantitative RT-PCR experiments indicated that all mutants accumulated unspliced SUS1 pre-mRNA and/or induced distorted levels of fully spliced mRNA relative to wild type. Concomitantly, Sus1 cellular functions in histone H2B deubiquitination and mRNA export were affected in I2 hairpin mutants that inhibited splicing. This work demonstrates that I2 structure is relevant for SUS1 expression, and that this effect is likely exerted through modulation of splicing. PMID:26546116

  7. Interaction between Brome mosaic virus proteins and RNAs: effects on RNA replication, protein expression, and RNA stability.

    PubMed

    Gopinath, K; Dragnea, B; Kao, C

    2005-11-01

    Brome mosaic virus (BMV) RNA replication has been examined in a number of systems, including Saccharomyces cerevisiae. We developed an efficient T-DNA-based gene delivery system using Agrobacterium tumefaciens to transiently express BMV RNAs in Nicotiana benthamiana. The expressed RNAs can systemically infect plants and provide material to extract BMV replicase that can perform template-dependent RNA-dependent RNA synthesis in vitro. We also expressed the four BMV-encoded proteins from nonreplicating RNAs and analyzed their effects on BMV RNA accumulation. The capsid protein that coinfiltrated with constructs expressing RNA1 and RNA2 suppressed minus-strand levels but increased plus-strand RNA accumulation. The replication proteins 1a and 2a could function in trans to replicate and transcribe the BMV RNAs. None of the BMV proteins or RNA could efficiently suppress posttranscriptional silencing. However, 1a expressed in trans will suppress the production of a recombinant green fluorescent protein expressed from the nontranslated portions of BMV RNA1 and RNA2, suggesting that 1a may regulate translation from BMV RNAs. BMV replicase proteins 1a did not affect the accumulation of the BMV RNAs in the absence of RNA replication, unlike the situation reported for S. cerevisiae. This work demonstrates that the Agrobacterium-mediated gene delivery system can be used to study the cis- and trans-acting requirements for BMV RNA replication in plants and that significant differences can exist for BMV RNA replication in different hosts.

  8. Elucidating the Potential of Plant Rhabdoviruses as Vector Expressions Systems

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Maize fine streak virus (MFSV) is a member of the genus Nucleorhabdovirus that is transmitted by the leafhopper Graminella nigrifons. The virus replicates in both its maize host and its insect vector. To determine whether Drosophila S2 cells support the production of full-length MFSV proteins, we ...

  9. Evaluation of inhibition of miRNA expression induced by anti-miRNA oligonucleotides.

    PubMed

    Chae, Dong-Kyu; Ban, Eunmi; Yoo, Young Sook; Baik, Ja-Hyun; Song, Eun Joo

    2016-07-01

    MicroRNAs (miRNAs) are short RNA molecules that control the expression of mRNAs associated with various biological processes. Therefore, deregulated miRNAs play an important role in the pathogenesis of diseases. Numerous studies aimed at developing novel miRNA-based drugs or determining miRNA functions have been conducted by inhibiting miRNAs using anti-miRNA oligonucleotides (AMOs), which inhibit the function by hybridizing with miRNA. To increase the binding affinity and specificity to target miRNA, AMOs with various chemical modifications have been developed. Evaluating the potency of these various types of AMOs is an essential step in their development. In this study, we developed a capillary electrophoresis with laser-induced fluorescence (CE-LIF) method to evaluate the potency of AMOs by measuring changes in miRNA levels with fluorescence-labeled ssDNA probes using AMO-miR-23a, which inhibits miR-23a related to lung cancer. In order to eliminate interference by excess AMOs during hybridization of the ssDNA probe with the miR-23a, the concentration of the ssDNA probe was optimized. This newly developed method was used to compare the potency of two different modified AMOs. The data were supported by the results of a luciferase assay. This study demonstrated that CE-LIF analysis could be used to accurately evaluate AMO potency in biological samples. PMID:27178549

  10. Chimeric adenoviral vector Ad5/F35-mediated APE1 siRNA enhances sensitivity of human colorectal cancer cells to radiotherapy in vitro and in vivo.

    PubMed

    Xiang, D-B; Chen, Z-T; Wang, D; Li, M-X; Xie, J-Y; Zhang, Y-S; Qing, Y; Li, Z-P; Xie, J

    2008-10-01

    Apurinic/apyrimidinic endonuclease (APE1), a bifunctional AP endonuclease/redox factor, is important in DNA repair and redox signaling, may be associated with radioresistance. Here we investigate whether targeted inhibition of APE1 can sensitize tumor cells to irradiation in vitro and in vivo. We first constructed chimeric adenoviral vector Ad5/F35 carrying human APE1 siRNA (Ad5/F35-APE1 siRNA). The infectivity of chimeric Ad5/F35 to LOVO colon cancer cells was greater than that of Ad5. APE1 was strongly expressed and nuclear factor kappaB (NF-kappaB), a downstream molecule of APE1, known as a radioresistance factor, was constitutively active in LOVO cells. Infection of LOVO cells with Ad5/F35-APE1 siRNA resulted in a dose-dependent decrease of APE1 protein and AP endonuclease activity in vitro. Ad5/F35-APE1 siRNA significantly enhanced sensitivity of LOVO cells to irradiation in clonogenic survival assays, associated with increased cell apoptosis. The APE1 expression in LOVO cells was induced by irradiation in a dose-dependent manner, accompanied with the enhancement of DNA-binding activity of NF-kappaB and Ad5/F35-APE1 siRNA effectively inhibited constitutive and irradiation-induced APE1 expression and NF-kappaB activation. In a subcutaneous nude mouse colon cancer model, Ad5/F35-APE1 siRNA (5 x 10(8) IU, intratumoral injection) inhibited the expression of APE1 protein in LOVO xenografts, and significantly enhanced inhibition of tumor growth by irradiation. In conclusion, APE1 may be involved as one of the radioresistance factors, and targeted inhibition of APE1 shows an effective means of enhancing tumor sensitivity to radiotherapy.

  11. AAVPG: A vigilant vector where transgene expression is induced by p53

    SciTech Connect

    Bajgelman, Marcio C.; Medrano, Ruan F.V.; Carvalho, Anna Carolina P.V.; Strauss, Bryan E.

    2013-12-15

    Using p53 to drive transgene expression from viral vectors may provide on demand expression in response to physiologic stress, such as hypoxia or DNA damage. Here we introduce AAVPG, an adeno-associated viral (AAV) vector where a p53-responsive promoter, termed PG, is used to control transgene expression. In vitro assays show that expression from the AAVPG-luc vector was induced specifically in the presence of functional p53 (1038±202 fold increase, p<0.001). The AAVPG-luc vector was an effective biosensor of p53 activation in response to hypoxia (4.48±0.6 fold increase in the presence of 250 µM CoCl{sub 2}, p<0.001) and biomechanical stress (2.53±0.4 fold increase with stretching, p<0.05). In vivo, the vigilant nature of the AAVPG-luc vector was revealed after treatment of tumor-bearing mice with doxorubicin (pre-treatment, 3.4×10{sup 5}±0.43×10{sup 5} photons/s; post-treatment, 6.6×10{sup 5}±2.1×10{sup 5} photons/s, p<0.05). These results indicate that the AAVPG vector is an interesting option for detecting p53 activity both in vitro and in vivo. - Highlights: • AAV vector where transgene expression is controlled by the tumor suppressor p53. • The new vector, AAVPG, shown to function as a biosensor of p53 activity, in vitro and in vivo. • The p53 activity monitored by the AAVPG vector is relevant to cancer and other diseases. • AAVPG reporter gene expression was activated upon DNA damage, hypoxia and mechanical stress.

  12. MicroRNA Expression Characterizes Oligometastasis(es)

    PubMed Central

    Lussier, Yves A.; Darga, Thomas E.; Malik, Renuka; Fan, Hanli; Perakis, Samantha; Filippo, Matthew; Corbin, Kimberly; Lee, Younghee; Posner, Mitchell C.; Chmura, Steven J.; Hellman, Samuel; Weichselbaum, Ralph R.

    2011-01-01

    Background Cancer staging and treatment presumes a division into localized or metastatic disease. We proposed an intermediate state defined by ≤5 cumulative metastasis(es), termed oligometastases. In contrast to widespread polymetastases, oligometastatic patients may benefit from metastasis-directed local treatments. However, many patients who initially present with oligometastases progress to polymetastases. Predictors of progression could improve patient selection for metastasis-directed therapy. Methods Here, we identified patterns of microRNA expression of tumor samples from oligometastatic patients treated with high-dose radiotherapy. Results Patients who failed to develop polymetastases are characterized by unique prioritized features of a microRNA classifier that includes the microRNA-200 family. We created an oligometastatic-polymetastatic xenograft model in which the patient-derived microRNAs discriminated between the two metastatic outcomes. MicroRNA-200c enhancement in an oligometastatic cell line resulted in polymetastatic progression. Conclusions These results demonstrate a biological basis for oligometastases and a potential for using microRNA expression to identify patients most likely to remain oligometastatic after metastasis-directed treatment. PMID:22174856

  13. MiRNA expression patterns predict survival in glioblastoma

    PubMed Central

    2011-01-01

    Background In order to define new prognostic subgroups in patients with glioblastoma a miRNA screen (> 1000 miRNAs) from paraffin tissues followed by a bio-mathematical analysis was performed. Methods 35 glioblastoma patients treated between 7/2005 - 8/2008 at a single institution with surgery and postoperative radio(chemo)therapy were included in this retrospective analysis. For microarray analysis the febit biochip "Geniom® Biochip MPEA homo-sapiens" was used. Total RNA was isolated from FFPE tissue sections and 1100 different miRNAs were analyzed. Results It was possible to define a distinct miRNA expression pattern allowing for a separation of distinct prognostic subgroups. The defined miRNA pattern was significantly associated with early death versus long-term survival (split at 450 days) (p = 0.01). The pattern and the prognostic power were both independent of the MGMT status. Conclusions At present, this is the first dataset defining a prognostic role of miRNA expression patterns in patients with glioblastoma. Having defined such a pattern, a prospective validation of this observation is required. PMID:22074483

  14. Effects of Chemically Modified Messenger RNA on Protein Expression.

    PubMed

    Li, Bin; Luo, Xiao; Dong, Yizhou

    2016-03-16

    Chemically modified nucleotides play significant roles in the effectiveness of mRNA translation. Here, we describe the synthesis of two sets of chemically modified mRNAs [encoding firefly Luciferase (FLuc) and enhanced green fluorescent protein (eGFP), respectively], evaluation of protein expression, and correlation analysis of expression level under various conditions. The results indicate that chemical modifications of mRNAs are able to significantly improve protein expression, which is dependent on cell types and coding sequences. Moreover, eGFP mRNAs with N1-methylpseudouridine (me(1)ψ), 5-methoxyuridine (5moU), and pseudouridine (ψ) modifications ranked top three in cell lines tested. Interestingly, 5moU-modified eGFP mRNA was more stable than other eGFP mRNAs. Consequently, me(1)ψ, 5moU, and ψ are promising nucleotides for chemical modification of mRNAs. PMID:26906521

  15. Wolbachia uses host microRNAs to manipulate host gene expression and facilitate colonization of the dengue vector Aedes aegypti.

    PubMed

    Hussain, Mazhar; Frentiu, Francesca D; Moreira, Luciano A; O'Neill, Scott L; Asgari, Sassan

    2011-05-31

    The obligate endosymbiont Wolbachia pipientis is found in a wide range of invertebrates where they are best known for manipulating host reproduction. Recent studies have shown that Wolbachia also can modulate the lifespan of host insects and interfere with the development of human pathogens in mosquito vectors. Despite considerable study, very little is known about the molecular interactions between Wolbachia and its hosts that might mediate these effects. Using microarrays, we show that the microRNA (miRNA) profile of the mosquito, Aedes aegypti, is significantly altered by the wMelPop-CLA strain of W. pipientis. We found that a host miRNA (aae-miR-2940) is induced after Wolbachia infection in both mosquitoes and cell lines. One target of aae-miR-2940 is the Ae. aegypti metalloprotease gene. Interestingly, expression of the target gene was induced after Wolbachia infection, ectopic expression of the miRNA independent of Wolbachia, or transfection of an artificial mimic of the miRNA into mosquito cells. We also confirmed the interaction of aae-miR-2940 with the target sequences using GFP as a reporter gene. Silencing of the metalloprotease gene in both Wolbachia-infected cells and adult mosquitoes led to a significant reduction in Wolbachia density, as did inhibition of the miRNA in cells. These results indicate that manipulation of the mosquito metalloprotease gene via aae-miR-2940 is crucial for efficient maintenance of the endosymbiont. This report shows how Wolbachia alters the host miRNA profile and provides insight into the mechanisms of host manipulation used by this widespread endosymbiont.

  16. Leptin mRNA expresses in the bull reproductive organ.

    PubMed

    Abavisani, A; Baghbanzadeh, A; Shayan, P; Tajik, P; Dehghani, H; Mirtorabi, M

    2009-12-01

    Leptin, a 167-amino acid hormone, is secreted mainly by fat tissue. It has some powerful effects on the regulation of metabolism and reproductive function through endocrine and probably paracrine mechanisms. The contribution rate of leptin function on the male reproductive system is not still clear. Characterization of leptin expression in reproductive organs will suggest that in addition to its endocrine action, leptin has also paracrine/autocrine effects on reproduction. The expression of functional leptin receptor mRNA has been already recognized in testis of rodents, human and cattle. Thus, the aim of the present study was to investigate the presence of leptin mRNA in the bovine testis, because it will be the first step for understanding of its paracrine/autocrine effects on the male reproductive organs in cattle. The present study was the first to showed leptin mRNA expression in the testis of Holstein cattle using reverse transcription and polymerase chain reaction (RT-PCR) analysis. RT-PCR products were amplified with nested PCR using inner leptin primer pairs to emphasis the first results. Besides, bovine beta actin gene was acted as an internal positive control as well as RNA purification marker. Our findings suggest that in addition to its endocrine actions at the hypothalamic-pituitary axis, leptin can has an autocrine and/or paracrine role in bull testicular function.

  17. miRNA Expression Analyses in Prostate Cancer Clinical Tissues

    PubMed Central

    Bucay, Nathan; Shahryari, Varahram; Majid, Shahana; Yamamura, Soichiro; Mitsui, Yozo; Tabatabai, Z. Laura; Greene, Kirsten; Deng, Guoren; Dahiya, Rajvir; Tanaka, Yuichiro; Saini, Sharanjot

    2015-01-01

    A critical challenge in prostate cancer (PCa) clinical management is posed by the inadequacy of currently used biomarkers for disease screening, diagnosis, prognosis and treatment. In recent years, microRNAs (miRNAs) have emerged as promising alternate biomarkers for prostate cancer diagnosis and prognosis. However, the development of miRNAs as effective biomarkers for prostate cancer heavily relies on their accurate detection in clinical tissues. miRNA analyses in prostate cancer clinical specimens is often challenging owing to tumor heterogeneity, sampling errors, stromal contamination etc. The goal of this article is to describe a simplified workflow for miRNA analyses in archived FFPE or fresh frozen prostate cancer clinical specimens using a combination of quantitative real-time PCR (RT-PCR) and in situ hybridization (ISH). Within this workflow, we optimize the existing methodologies for miRNA extraction from FFPE and frozen prostate tissues and expression analyses by Taqman-probe based miRNA RT-PCR. In addition, we describe an optimized method for ISH analyses formiRNA detection in prostate tissues using locked nucleic acid (LNA)- based probes. Our optimized miRNA ISH protocol can be applied to prostate cancer tissue slides or prostate cancer tissue microarrays (TMA). PMID:26382040

  18. miRNA Expression Analyses in Prostate Cancer Clinical Tissues.

    PubMed

    Bucay, Nathan; Shahryari, Varahram; Majid, Shahana; Yamamura, Soichiro; Mitsui, Yozo; Tabatabai, Z Laura; Greene, Kirsten; Deng, Guoren; Dahiya, Rajvir; Tanaka, Yuichiro; Saini, Sharanjot

    2015-01-01

    A critical challenge in prostate cancer (PCa) clinical management is posed by the inadequacy of currently used biomarkers for disease screening, diagnosis, prognosis and treatment. In recent years, microRNAs (miRNAs) have emerged as promising alternate biomarkers for prostate cancer diagnosis and prognosis. However, the development of miRNAs as effective biomarkers for prostate cancer heavily relies on their accurate detection in clinical tissues. miRNA analyses in prostate cancer clinical specimens is often challenging owing to tumor heterogeneity, sampling errors, stromal contamination etc. The goal of this article is to describe a simplified workflow for miRNA analyses in archived FFPE or fresh frozen prostate cancer clinical specimens using a combination of quantitative real-time PCR (RT-PCR) and in situ hybridization (ISH). Within this workflow, we optimize the existing methodologies for miRNA extraction from FFPE and frozen prostate tissues and expression analyses by Taqman-probe based miRNA RT-PCR. In addition, we describe an optimized method for ISH analyses formiRNA detection in prostate tissues using locked nucleic acid (LNA)- based probes. Our optimized miRNA ISH protocol can be applied to prostate cancer tissue slides or prostate cancer tissue microarrays (TMA). PMID:26382040

  19. miRNA Expression Analyses in Prostate Cancer Clinical Tissues.

    PubMed

    Bucay, Nathan; Shahryari, Varahram; Majid, Shahana; Yamamura, Soichiro; Mitsui, Yozo; Tabatabai, Z Laura; Greene, Kirsten; Deng, Guoren; Dahiya, Rajvir; Tanaka, Yuichiro; Saini, Sharanjot

    2015-09-08

    A critical challenge in prostate cancer (PCa) clinical management is posed by the inadequacy of currently used biomarkers for disease screening, diagnosis, prognosis and treatment. In recent years, microRNAs (miRNAs) have emerged as promising alternate biomarkers for prostate cancer diagnosis and prognosis. However, the development of miRNAs as effective biomarkers for prostate cancer heavily relies on their accurate detection in clinical tissues. miRNA analyses in prostate cancer clinical specimens is often challenging owing to tumor heterogeneity, sampling errors, stromal contamination etc. The goal of this article is to describe a simplified workflow for miRNA analyses in archived FFPE or fresh frozen prostate cancer clinical specimens using a combination of quantitative real-time PCR (RT-PCR) and in situ hybridization (ISH). Within this workflow, we optimize the existing methodologies for miRNA extraction from FFPE and frozen prostate tissues and expression analyses by Taqman-probe based miRNA RT-PCR. In addition, we describe an optimized method for ISH analyses formiRNA detection in prostate tissues using locked nucleic acid (LNA)- based probes. Our optimized miRNA ISH protocol can be applied to prostate cancer tissue slides or prostate cancer tissue microarrays (TMA).

  20. Construction of rice site-specific chloroplast transformation vector and transient expression of EGFP gene in Dunaliella salina.

    PubMed

    Li, Ding; Han, Xiaoxia; Zuo, Jia; Xie, Lingling; He, Ronghua; Gao, Jing; Chang, Lan; Yuan, Longping; Cao, Mengliang

    2011-12-01

    Chloroplast is a new hotspot in the field of plant transformation system of plant genetic engineering. Plastid transformation has several advantages: high expression, multiple expressed genes in a single transformation event, absence of gene silencing, et al. A series of elements for construction of dicistronic site-specific integration expression vector of rice chloroplast have been cloned, including trnl-trnA (rice chloroplast homologous recombination fragments), Prrn (16S rRNA operon promotor), PpsbA (the 3' untranslated region of the chloroplastpsbA gene), hptll gene (encoding hygromycin phosphotransferase) and EGFP (encoding enhanced green fluorescence protein). All the elements were constructed into a rice chloroplast dicistronic expression vector pCTE04 (-trnl-Prrn-RBS-hptlI-RBS-EGFP-PpsbA- trnA-). Then pCTE04 was introduced into chloroplasts of Dunaliella salina through particle bombardment. Strong green fluorescence was observed in chloroplasts of some bombarded Dunaliella salina cells under a stereo fluorescence microscope, indicating that pCTE04 could be expressed in Dunaliella salina chloroplasts transiently. It provides a solid foundation for further genetic engineering in rice chloroplast transformation.

  1. Small RNA viruses of insects: expression in plants and RNA silencing.

    PubMed

    Gordon, Karl H J; Waterhouse, Peter M

    2006-01-01

    Interest in insect small RNA viruses (SRVs) has grown slowly but steadily. A number of new viruses have been analyzed at the sequence level, adding to our knowledge of their diversity at the level of both individual virus species and families. In particular, a number of possible new virus families have emerged. This research has largely been driven by interest in their potential for pest control, as well as in their importance as the causal agents of disease in beneficial arthropods. At the same time, research into known viruses has made valuable contributions to our understanding of an emerging new field of central importance to molecular biology-the existence of RNA-based gene silencing, developmental control, and adaptive immune systems in eukaryotes. Subject to RNA-based adaptive immune responses in their hosts, viruses have evolved a variety of genes encoding proteins capable of suppressing the immune response. Such genes were first identified in plant viruses, but the first examples known from animal viruses were identified in insect RNA viruses. This chapter will address the diversity of insect SRVs, and attempts to harness their simplicity in the engineering of transgenic plants expressing viruses for resistance to insect pests. We also describe RNA interference and antiviral pathways identified in plants and animals, how they have led viruses to evolve genes capable of suppressing such adaptive immunity, and the problems presented by these pathways for the strategy of expressing viruses in transgenic plants. Approaches for countering these problems are also discussed.

  2. MicroRNA expression profiling of cat and dog kidneys.

    PubMed

    Ichii, Osamu; Otsuka, Saori; Ohta, Hiroshi; Yabuki, Akira; Horino, Taro; Kon, Yasuhiro

    2014-04-01

    MicroRNAs (miRNAs) play a role in the pathogenesis of certain diseases and may serve as biomarkers. Here, we present the first analysis of miRNA expression in the kidneys of healthy cats and dogs. Kidneys were divided into renal cortex (CO) and medulla (MD), and RNA sequence analysis was performed using the mouse genome as a reference. A total of 277, 276, 295, and 297 miRNAs were detected in cat CO, cat MD, dog CO, and dog MD, respectively. By comparing the expression ratio of CO to MD, we identified highly expressed miRNAs in each tissue as follows: 41 miRNAs including miR-192-5p in cat CO; 45 miRNAs including miR-323-3p in dog CO; 78 miRNAs including miR-20a-5p in cat MD; and 11 miRNAs including miR-132-5p in dog MD. Further, the target mRNAs of these miRNAs were identified. These data provide veterinary medicine critical information regarding renal miRNA expression.

  3. Label-free high-throughput microRNA expression profiling from total RNA

    PubMed Central

    Duan, Demin; Zheng, Ke-xiao; Shen, Ye; Cao, Rong; Jiang, Li; Lu, Zhuoxuan; Yan, Xiyun; Li, Jiong

    2011-01-01

    MicroRNAs (miRNAs) are key biological regulators and promising disease markers whose detection technologies hold great potentials in advancing fundamental research and medical diagnostics. Currently, miRNAs in biological samples have to be labeled before being applied to most high-throughput assays. Although effective, these labeling-based approaches are usually labor-intensive, time-consuming and liable to bias. Besides, the cross-hybridization of co-existing miRNA precursors (pre-miRNAs) is not adequately addressed in most assays that use total RNA as input. Here, we present a hybridization-triggered fluorescence strategy for label-free, microarray-based high-throughput miRNA expression profiling. The total RNA is directly applied to the microarray with a short fluorophore-linked oligonucleotide Universal Tag which can be selectively captured by the target-bound probes via base-stacking effects. This Stacking-Hybridized Universal Tag (SHUT) assay has been successfully used to analyze as little as 100 ng total RNA from human tissues, and found to be highly specific to homogenous miRNAs. Superb discrimination toward single-base mismatch at the 5′ or 3′ end has been demonstrated. Importantly, the pre-miRNAs generated negligible signals, validating the direct use of total RNA. PMID:21976734

  4. Vibrational force alters mRNA expression in osteoblasts

    NASA Technical Reports Server (NTRS)

    Tjandrawinata, R. R.; Vincent, V. L.; Hughes-Fulford, M.

    1997-01-01

    Serum-deprived mouse osteoblastic (MC3T3E1) cells were subjected to a vibrational force modeled by NASA to simulate a space shuttle launch (7.83 G rms). The mRNA levels for eight genes were investigated to determine the effect of vibrational force on mRNA expression. The mRNA levels of two growth-related protooncogenes, c-fos and c-myc, were up-regulated significantly within 30 min after vibration, whereas those of osteocalcin as well as transforming growth factor-beta1 were decreased significantly within 3 h after vibration. No changes were detected in the levels of beta-actin, histone H4, or cytoplasmic phospholipase A2 after vibration. No basal levels of cyclooxygenase-2 expression were detected. In addition, the extracellular concentrations of prostaglandin E2 (PGE2), a potent autocrine/paracrine growth factor in bone, were not significantly altered after vibration most likely due to the serum deprivation state of the osteoblasts. In comparison with the gravitational launch profile, vibrational-induced changes in gene expression were greater both in magnitude and number of genes activated. Taken together, these data suggest that the changes in mRNA expression are due to a direct mechanical effect of the vibrational force on the osteoblast cells and not to changes in the local PGE2 concentrations. The finding that launch forces induce gene expression is of utmost importance since many of the biological experiments do not dampen vibrational loads on experimental samples. This lack of dampening of vibrational forces may partially explain why 1-G onboard controls sometimes do not reflect 1-G ground controls. These data may also suggest that scientists use extra ground controls that are exposed to launch forces, have these forces dampened on launched samples, or use facilities such as Biorack that provide an onboard 1-G centrufuge in order to control for space shuttle launch forces.

  5. Vibrational force alters mRNA expression in osteoblasts.

    PubMed

    Tjandrawinata, R R; Vincent, V L; Hughes-Fulford, M

    1997-05-01

    Serum-deprived mouse osteoblastic (MC3T3E1) cells were subjected to a vibrational force modeled by NASA to simulate a space shuttle launch (7.83 G rms). The mRNA levels for eight genes were investigated to determine the effect of vibrational force on mRNA expression. The mRNA levels of two growth-related protooncogenes, c-fos and c-myc, were up-regulated significantly within 30 min after vibration, whereas those of osteocalcin as well as transforming growth factor-beta1 were decreased significantly within 3 h after vibration. No changes were detected in the levels of beta-actin, histone H4, or cytoplasmic phospholipase A2 after vibration. No basal levels of cyclooxygenase-2 expression were detected. In addition, the extracellular concentrations of prostaglandin E2 (PGE2), a potent autocrine/paracrine growth factor in bone, were not significantly altered after vibration most likely due to the serum deprivation state of the osteoblasts. In comparison with the gravitational launch profile, vibrational-induced changes in gene expression were greater both in magnitude and number of genes activated. Taken together, these data suggest that the changes in mRNA expression are due to a direct mechanical effect of the vibrational force on the osteoblast cells and not to changes in the local PGE2 concentrations. The finding that launch forces induce gene expression is of utmost importance since many of the biological experiments do not dampen vibrational loads on experimental samples. This lack of dampening of vibrational forces may partially explain why 1-G onboard controls sometimes do not reflect 1-G ground controls. These data may also suggest that scientists use extra ground controls that are exposed to launch forces, have these forces dampened on launched samples, or use facilities such as Biorack that provide an onboard 1-G centrufuge in order to control for space shuttle launch forces.

  6. A correlation between dexamethasone inducibility and basal expression levels of retroviral vector proviruses.

    PubMed Central

    Duch, M; Paludan, K; Lovmand, J; Pedersen, L; Jørgensen, P; Pedersen, F S

    1993-01-01

    Identical transcription units inserted at different positions of mammalian chromosomes may vary widely in transcriptional activity. We have used a set of ten cell clones with random unselected single integrations of retroviral vectors to study such position effects. The vector used carries a neo gene driven by the Akv murine leukemia virus long terminal repeat that has only a weak promoter-enhancer activity in the target cell, the lymphoid cell line L691. Under transient expression conditions, the strength of the Akv promoter-enhancer in the L691 cells is increased by dexamethasone. In cell clones with single vector integrations, a correlation is observed between the non-induced expression levels and the degree of dexamethasone induction. The strongest relative induction is found for the integrated vectors with the lowest non-induced expression levels and approaches the inducibility under transient expression. These results indicate that expression levels are composed of distinct contributions from the integrated vector and from the site of integration and are best explained in terms of a model in which the sites of chromosomal integration exert variable positive enhancer effects upon vector transcription. Images PMID:8233826

  7. Construction of vectors for inducible and constitutive gene expression in Lactobacillus.

    PubMed

    Duong, Tri; Miller, Michael J; Barrangou, Rodolphe; Azcarate-Peril, M Andrea; Klaenhammer, Todd R

    2011-05-01

    Microarray analysis of the genome of Lactobacillus acidophilus identified a number of operons that were differentially expressed in response to carbohydrate source or constitutively expressed regardless of carbohydrate source. These included operons implicated in the transport and catabolism of fructooligosaccharides (FOS), lactose (lac), trehalose (tre) and genes directing glycolysis. Analysis of these operons identified a number of putative promoter and repressor elements, which were used to construct a series of expression vectors for use in lactobacilli, based on the broad host range pWV01 replicon. A β-glucuronidase (GusA3) reporter gene was cloned into each vector to characterize expression from each promoter. GUS reporter assays showed FOS, lac and tre based vectors to be highly inducible by their specific carbohydrate and repressed by glucose. Additionally, a construct based on the phosphoglycerate mutase (pgm) promoter was constitutively highly expressed. To demonstrate the potential utility of these vectors, we constructed a plasmid for the overexpression of the oxalate degradation pathway (Frc and Oxc) of L. acidophilus NCFM. This construct was able to improve oxalate degradation by L. gasseri ATCC 33323 and compliment a L. acidophilus oxalate-deficient mutant. Development of these expression vectors could support several novel applications, including the expression of enzymes, proteins, vaccines and biotherapeutics by intestinal lactobacilli. PMID:21375708

  8. Construction of vectors for inducible and constitutive gene expression in Lactobacillus

    PubMed Central

    Duong, Tri; Miller, Michael J.; Barrangou, Rodolphe; Azcarate‐Peril, M. Andrea; Klaenhammer, Todd R.

    2011-01-01

    Summary Microarray analysis of the genome of Lactobacillus acidophilus identified a number of operons that were differentially expressed in response to carbohydrate source or constitutively expressed regardless of carbohydrate source. These included operons implicated in the transport and catabolism of fructooligosaccharides (FOS), lactose (lac), trehalose (tre) and genes directing glycolysis. Analysis of these operons identified a number of putative promoter and repressor elements, which were used to construct a series of expression vectors for use in lactobacilli, based on the broad host range pWV01 replicon. A β‐glucuronidase (GusA3) reporter gene was cloned into each vector to characterize expression from each promoter. GUS reporter assays showed FOS, lac and tre based vectors to be highly inducible by their specific carbohydrate and repressed by glucose. Additionally, a construct based on the phosphoglycerate mutase (pgm) promoter was constitutively highly expressed. To demonstrate the potential utility of these vectors, we constructed a plasmid for the overexpression of the oxalate degradation pathway (Frc and Oxc) of L. acidophilus NCFM. This construct was able to improve oxalate degradation by L. gasseri ATCC 33323 and compliment a L. acidophilus oxalate‐deficient mutant. Development of these expression vectors could support several novel applications, including the expression of enzymes, proteins, vaccines and biotherapeutics by intestinal lactobacilli. PMID:21375708

  9. Improved Production Efficiency of Virus-Like Particles by the Baculovirus Expression Vector System.

    PubMed

    López-Vidal, Javier; Gómez-Sebastián, Silvia; Bárcena, Juan; Nuñez, Maria del Carmen; Martínez-Alonso, Diego; Dudognon, Benoit; Guijarro, Eva; Escribano, José M

    2015-01-01

    Vaccines based on virus-like particles (VLPs) have proven effective in humans and animals. In this regard, the baculovirus expression vector system (BEVS) is one of the technologies of choice to generate such highly immunogenic vaccines. The extended use of these vaccines for human and animal populations is constrained because of high production costs, therefore a significant improvement in productivity is crucial to ensure their commercial viability. Here we describe the use of the previously described baculovirus expression cassette, called TB, to model the production of two VLP-forming vaccine antigens in insect cells. Capsid proteins from porcine circovirus type 2 (PCV2 Cap) and from the calicivirus that causes rabbit hemorrhagic disease (RHDV VP60) were expressed in insect cells using baculoviruses genetically engineered with the TB expression cassette. Productivity was compared to that obtained using standard counterpart vectors expressing the same proteins under the control of the polyhedrin promoter. Our results demonstrate that the use of the TB expression cassette increased the production yields of these vaccine antigens by around 300% with respect to the standard vectors. The recombinant proteins produced by TB-modified vectors were fully functional, forming VLPs identical in size and shape to those generated by the standard baculoviruses, as determined by electron microscopy analysis. The use of the TB expression cassette implies a simple modification of the baculovirus vectors that significantly improves the cost efficiency of VLP-based vaccine production, thereby facilitating the commercial viability and broad application of these vaccines for human and animal health.

  10. Circular RNA is expressed across the eukaryotic tree of life.

    PubMed

    Wang, Peter L; Bao, Yun; Yee, Muh-Ching; Barrett, Steven P; Hogan, Gregory J; Olsen, Mari N; Dinneny, José R; Brown, Patrick O; Salzman, Julia

    2014-01-01

    An unexpectedly large fraction of genes in metazoans (human, mouse, zebrafish, worm, fruit fly) express high levels of circularized RNAs containing canonical exons. Here we report that circular RNA isoforms are found in diverse species whose most recent common ancestor existed more than one billion years ago: fungi (Schizosaccharomyces pombe and Saccharomyces cerevisiae), a plant (Arabidopsis thaliana), and protists (Plasmodium falciparum and Dictyostelium discoideum). For all species studied to date, including those in this report, only a small fraction of the theoretically possible circular RNA isoforms from a given gene are actually observed. Unlike metazoans, Arabidopsis, D. discoideum, P. falciparum, S. cerevisiae, and S. pombe have very short introns (∼ 100 nucleotides or shorter), yet they still produce circular RNAs. A minority of genes in S. pombe and P. falciparum have documented examples of canonical alternative splicing, making it unlikely that all circular RNAs are by-products of alternative splicing or 'piggyback' on signals used in alternative RNA processing. In S. pombe, the relative abundance of circular to linear transcript isoforms changed in a gene-specific pattern during nitrogen starvation. Circular RNA may be an ancient, conserved feature of eukaryotic gene expression programs.

  11. Recombinant AAV Vectors for Enhanced Expression of Authentic IgG.

    PubMed

    Fuchs, Sebastian P; Martinez-Navio, José M; Gao, Guangping; Desrosiers, Ronald C

    2016-01-01

    Adeno-associated virus (AAV) has become a vector of choice for the treatment of a variety of genetic diseases that require safe and long-term delivery of a missing protein. Muscle-directed gene transfer for delivery of protective antibodies against AIDS viruses and other pathogens has been used experimentally in mice and monkeys. Here we examined a number of variations to AAV vector design for the ability to produce authentic immunoglobulin G (IgG) molecules. Expression of rhesus IgG from a single single-stranded AAV (ssAAV) vector (one vector approach) was compared to expression from two self-complementary AAV (scAAV) vectors, one for heavy chain and one for light chain (two vector approach). Both the one vector and the two vector approaches yielded considerable levels of expressed full-length IgG. A number of modifications to the ssAAV expression system were then examined for their ability to increase the efficiency of IgG expression. Inclusion of a furin cleavage sequence with a linker peptide just upstream of the 2A self-cleaving sequence from foot-and-mouth disease virus (F2A) increased IgG expression approximately 2 fold. Inclusion of these sequences also helped to ensure a proper sequence at the C-terminal end of the heavy chain. Inclusion of the post-transcriptional regulatory element from woodchuck hepatitis virus (WPRE) further increased IgG expression 1.5-2.0 fold. IgG1 versions of the two rhesus IgGs that were examined consistently expressed better than the IgG2 forms. In contrast to what has been reported for AAV2-mediated expression of other proteins, introduction of capsid mutations Y445F and Y731F did not increase ssAAV1-mediated expression of IgG as determined by transduction experiments in cell culture. Our findings provide a rational basis for AAV vector design for expression of authentic IgG. PMID:27332822

  12. Recombinant AAV Vectors for Enhanced Expression of Authentic IgG

    PubMed Central

    Fuchs, Sebastian P.; Martinez-Navio, José M.; Gao, Guangping; Desrosiers, Ronald C.

    2016-01-01

    Adeno-associated virus (AAV) has become a vector of choice for the treatment of a variety of genetic diseases that require safe and long-term delivery of a missing protein. Muscle-directed gene transfer for delivery of protective antibodies against AIDS viruses and other pathogens has been used experimentally in mice and monkeys. Here we examined a number of variations to AAV vector design for the ability to produce authentic immunoglobulin G (IgG) molecules. Expression of rhesus IgG from a single single-stranded AAV (ssAAV) vector (one vector approach) was compared to expression from two self-complementary AAV (scAAV) vectors, one for heavy chain and one for light chain (two vector approach). Both the one vector and the two vector approaches yielded considerable levels of expressed full-length IgG. A number of modifications to the ssAAV expression system were then examined for their ability to increase the efficiency of IgG expression. Inclusion of a furin cleavage sequence with a linker peptide just upstream of the 2A self-cleaving sequence from foot-and-mouth disease virus (F2A) increased IgG expression approximately 2 fold. Inclusion of these sequences also helped to ensure a proper sequence at the C-terminal end of the heavy chain. Inclusion of the post-transcriptional regulatory element from woodchuck hepatitis virus (WPRE) further increased IgG expression 1.5–2.0 fold. IgG1 versions of the two rhesus IgGs that were examined consistently expressed better than the IgG2 forms. In contrast to what has been reported for AAV2-mediated expression of other proteins, introduction of capsid mutations Y445F and Y731F did not increase ssAAV1-mediated expression of IgG as determined by transduction experiments in cell culture. Our findings provide a rational basis for AAV vector design for expression of authentic IgG. PMID:27332822

  13. Complex Effects of Deletions in the 5′ Untranslated Region of Primate Foamy Virus on Viral Gene Expression and RNA Packaging

    PubMed Central

    Heinkelein, Martin; Thurow, Jana; Dressler, Marco; Imrich, Horst; Neumann-Haefelin, Dieter; McClure, Myra O.; Rethwilm, Axel

    2000-01-01

    Due to various advantageous features there is current interest in retroviral vectors derived from primate foamy viruses (PFVs). Two PFV cis-acting sequences have been mapped in the 5′ region of the RNA (pre-)genome and in the 3′ pol genomic region. In order to genetically separate PFV packaging constructs from vector constructs, we investigated the effect of deletions in the 5′ untranslated region (UTR) of PFV packaging constructs and vectors on gene expression and RNA incorporation into viral particles. Our results indicate that the 5′ UTR serves different previously unknown functions. First, the R region of the long terminal repeat was found to be required for PFV gag gene expression. This regulation of gene expression appeared to be mainly posttranscriptional. Second, constructs with sequence deletions between the R region and the gag gene start codon packaged as much viral mRNA into particles as the undeleted construct, and RNA from such a 5′-UTR-deleted packaging construct was copackaged into vector-virus particles, together with vector RNA which was preferentialy packaged. Finally, in the U5 region a sequence was identified that was required to allow cleavage of the Gag precursor protein by the pol gene-encoded protease, suggesting a role of RNA in PFV particle formation. Taken together, the results indicate that complex interactions of the viral RNA, capsid, and polymerase proteins take place during PFV particle formation and that a clear separation of PFV vector and packaging construct sequences may be difficult to achieve. PMID:10708430

  14. miRNA expression atlas in male rat

    PubMed Central

    Minami, Keiichi; Uehara, Takeki; Morikawa, Yuji; Omura, Ko; Kanki, Masayuki; Horinouchi, Akira; Ono, Atsushi; Yamada, Hiroshi; Ohno, Yasuo; Urushidani, Tetsuro

    2014-01-01

    MicroRNAs (miRNAs) are small (~22 nucleotide) noncoding RNAs that play pivotal roles in regulation of gene expression. The value of miRNAs as circulating biomarkers is now broadly recognized; such tissue-specific biomarkers can be used to monitor tissue injury and several pathophysiological conditions in organs. In addition, miRNA profiles of normal organs and tissues are important for obtaining a better understanding of the source of modulated miRNAs in blood and how those modulations reflect various physiological and toxicological conditions. This work was aimed at creating an miRNA atlas in rats, as part of a collaborative effort with the Toxicogenomics Informatics Project in Japan (TGP2). We analyzed genome-wide miRNA profiles of 55 different organs and tissues obtained from normal male rats using miRNA arrays. The work presented herein represents a comprehensive dataset derived from normal samples profiled in a single study. Here we present the whole dataset with miRNA profiles of multiple organs, as well as precise information on experimental procedures and organ-specific miRNAs identified in this dataset. PMID:25977763

  15. Scaffolds for Artificial miRNA Expression in Animal Cells.

    PubMed

    Calloni, Raquel; Bonatto, Diego

    2015-10-01

    Artificial miRNAs (amiRNAs) are molecules that have been developed to promote gene silencing in a similar manner to naturally occurring miRNAs. amiRNAs are generally constructed by replacing the mature miRNA sequence in the pre-miRNA stem-loop with a sequence targeting a gene of interest. These molecules offer an interesting alternative to silencing approaches that are based on shRNAs and siRNAs because they present the same efficiency as these options and are less cytotoxic. amiRNAs have mostly been applied to gene knockdown in plants; they have been examined to a lesser extent in animal cells. Therefore, this article reviews the amiRNAs that have been developed for animal cells and focuses on the miRNA scaffolds that can already be applied to construct the artificial counterparts, as well as on the different approaches that have been described to promote amiRNA expression and silencing efficiency. Furthermore, the availability of amiRNA libraries and other tools that can be used to design and construct these molecules is briefly discussed, along with an overview of the therapeutic applications for which amiRNAs have already been evaluated.

  16. Tightly regulated vectors for the cloning and expression of toxic genes.

    PubMed

    Anthony, Larry C; Suzuki, Hideki; Filutowicz, Marcin

    2004-08-01

    A series of low-copy expression vectors that permits the stable maintenance and regulated expression of highly toxic gene products has been developed. These vectors utilize the lactose promoter/operator system, and protect against read-through transcription from other promoters on the plasmid by placement of the rrnB T1T2 terminators upstream of the lactose promoter. For additional regulatory control, the vectors utilize low-copy origins of replication. Either the pMPP6 origin (pSC101-derived) is used for cloning into Escherichia coli or related species, or the broad-host-range RK2 origin of replication is utilized for cloning into the majority of Gram-negative bacteria. The resulting plasmids have no detectable leaky expression. To test these vectors, the genes for the bacteriocidal colicins D, E3, and E7 were cloned and stably maintained in the absence of their immunity genes. Upon induction with isopropyl-beta-D-thiogalactopyranoside (IPTG), cell death was observed, indicating expression of each colicin. These low-copy expression vectors will be useful for the cloning and expression of toxic genes in bacterial systems. PMID:15234522

  17. Development of Virus-Induced Gene Expression and Silencing Vector Derived from Grapevine Algerian Latent Virus

    PubMed Central

    Park, Sang-Ho; Choi, Hoseong; Kim, Semin; Cho, Won Kyong; Kim, Kook-Hyung

    2016-01-01

    Grapevine Algerian latent virus (GALV) is a member of the genus Tombusvirus in the Tombusviridae and infects not only woody perennial grapevine plant but also herbaceous Nicotiana benthamiana plant. In this study, we developed GALV-based gene expression and virus-induced gene silencing (VIGS) vectors in N. benthamiana. The GALV coat protein deletion vector, pGMG, was applied to express the reporter gene, green fluorescence protein (GFP), but the expression of GFP was not detected due to the necrotic cell death on the infiltrated leaves. The p19 silencing suppressor of GALV was engineered to inactivate its expression and GFP was successfully expressed with unrelated silencing suppressor, HC-Pro, from soybean mosaic virus. The pGMG vector was used to knock down magnesium chelatase (ChlH) gene in N. benthamaina and the silencing phenotype was clearly observed on systemic leaves. Altogether, the GALV-derived vector is expected to be an attractive tool for useful gene expression and VIGS vectors in grapevine as well as N. benthamiana. PMID:27493613

  18. Development of Virus-Induced Gene Expression and Silencing Vector Derived from Grapevine Algerian Latent Virus.

    PubMed

    Park, Sang-Ho; Choi, Hoseong; Kim, Semin; Cho, Won Kyong; Kim, Kook-Hyung

    2016-08-01

    Grapevine Algerian latent virus (GALV) is a member of the genus Tombusvirus in the Tombusviridae and infects not only woody perennial grapevine plant but also herbaceous Nicotiana benthamiana plant. In this study, we developed GALV-based gene expression and virus-induced gene silencing (VIGS) vectors in N. benthamiana. The GALV coat protein deletion vector, pGMG, was applied to express the reporter gene, green fluorescence protein (GFP), but the expression of GFP was not detected due to the necrotic cell death on the infiltrated leaves. The p19 silencing suppressor of GALV was engineered to inactivate its expression and GFP was successfully expressed with unrelated silencing suppressor, HC-Pro, from soybean mosaic virus. The pGMG vector was used to knock down magnesium chelatase (ChlH) gene in N. benthamaina and the silencing phenotype was clearly observed on systemic leaves. Altogether, the GALV-derived vector is expected to be an attractive tool for useful gene expression and VIGS vectors in grapevine as well as N. benthamiana. PMID:27493613

  19. Novel vectors for the expression of antibody molecules using variable regions generated by polymerase chain reaction.

    PubMed

    Coloma, M J; Hastings, A; Wims, L A; Morrison, S L

    1992-07-31

    A new family of vectors has been produced which facilitates the cloning and expression of immunoglobulin variable regions cloned by polymerase chain reaction (PCR). The vectors are designed to express the cloned variable regions joined to human constant regions and take advantage of priming in the leader sequence so that no amino acid changes will be introduced into the mature antibody molecule. Both the heavy chain and light chain vectors utilize a murine VH promoter provided with an EcoRV restriction site so that the amplified variable regions can be directly cloned into a functional promoter. For the heavy chain an NheI restriction site has been generated at the first two amino acids of CH1 and the cloned leader and variable region are fused directly to the CH1 domain of the constant region. When the leader and variable regions of the light chain were fused directly to C kappa, no expression was observed. Therefore the light chain expression vector was designed with a SalI restriction site for cloning into a splice junction 3' of the variable region; VL then is joined to C kappa by splicing. Both vectors direct the expression of functional, fully assembled immunoglobulin molecules with the expected molecular weight. Use of redundant oligomers to prime the PCR permits the cloning and expression of recombinant antibodies without any prior information as to their sequence and makes it possible to rapidly generate recombinant antibodies from any monoclonal antibody producing cell line.

  20. A modified TMV-based vector facilitates the expression of longer foreign epitopes in tobacco.

    PubMed

    Jiang, Lubin; Li, Qiaoli; Li, Mangmang; Zhou, Zhiai; Wu, Ligang; Fan, Jihua; Zhang, Qingqi; Zhu, Huihui; Xu, Zhengkai

    2006-01-12

    Based upon a mutant isolated from tobacco infected with a recombinant tobacco mosaic virus (TMV), a new TMV-based vector was developed in which four to six C-terminal amino acid residues were deleted from the viral coat protein (CP) subunit. The new vector was quite similar to the original TMV-based vector, which all expressed a well characterized epitope peptide F11 (P(142)-A(152)) of VP1 from foot-and-mouth disease virus (FMDV) serotype O in tobacco, in the infectivity, yield of the virus particles and more importantly protective activity of F11 in guinea pigs and swine against the FMDV. Furthermore, the capacity of the length of foreign peptide encoded by this new vector was much improved to successfully express a peptide F25 containing two fused epitopes F14 (R(200)-L(213)) and F11 of FMDV VP1, which was failed using the original vector in tobacco. Although animal assays indicated that such expressed F25 was not as efficient as F11 in the immunity, possibly due to lack of a spacer arm between the two fused epitopes, the new TMV-based vector may meet the requirement of expressing longer foreign peptides for different vaccines and other medicines. PMID:16337317

  1. Induction of humoral responses to BHV-1 glycoprotein D expressed by HSV-1 amplicon vectors

    PubMed Central

    Blanc, Andrea Maria; Berois, Mabel Beatriz; Tomé, Lorena Magalí; Epstein, Alberto L.

    2012-01-01

    Herpes simplex virus type-1 (HSV-1) amplicon vectors are versatile and useful tools for transferring genes into cells that are capable of stimulating a specific immune response to their expressed antigens. In this work, two HSV-1-derived amplicon vectors were generated. One of these expressed the full-length glycoprotein D (gD) of bovine herpesvirus 1 while the second expressed the truncated form of gD (gDtr) which lacked the trans-membrane region. After evaluating gD expression in the infected cells, the ability of both vectors to induce a specific gD immune response was tested in BALB/c mice that were intramuscularly immunized. Specific serum antibody responses were detected in mice inoculated with both vectors, and the response against truncated gD was higher than the response against full-length gD. These results reinforce previous findings that HSV-1 amplicon vectors can potentially deliver antigens to animals and highlight the prospective use of these vectors for treating infectious bovine rhinotracheitis disease. PMID:22437537

  2. Quality assurance of RNA expression profiling in clinical laboratories.

    PubMed

    Tang, Weihua; Hu, Zhiyuan; Muallem, Hind; Gulley, Margaret L

    2012-01-01

    RNA expression profiles are increasingly used to diagnose and classify disease, based on expression patterns of as many as several thousand RNAs. To ensure quality of expression profiling services in clinical settings, a standard operating procedure incorporates multiple quality indicators and controls, beginning with preanalytic specimen preparation and proceeding thorough analysis, interpretation, and reporting. Before testing, histopathological examination of each cellular specimen, along with optional cell enrichment procedures, ensures adequacy of the input tissue. Other tactics include endogenous controls to evaluate adequacy of RNA and exogenous or spiked controls to evaluate run- and patient-specific performance of the test system, respectively. Unique aspects of quality assurance for array-based tests include controls for the pertinent outcome signatures that often supersede controls for each individual analyte, built-in redundancy for critical analytes or biochemical pathways, and software-supported scrutiny of abundant data by a laboratory physician who interprets the findings in a manner facilitating appropriate medical intervention. Access to high-quality reagents, instruments, and software from commercial sources promotes standardization and adoption in clinical settings, once an assay is vetted in validation studies as being analytically sound and clinically useful. Careful attention to the well-honed principles of laboratory medicine, along with guidance from government and professional groups on strategies to preserve RNA and manage large data sets, promotes clinical-grade assay performance.

  3. Dual role for argonautes in microRNA processing and posttranscriptional regulation of microRNA expression.

    PubMed

    Diederichs, Sven; Haber, Daniel A

    2007-12-14

    MicroRNAs are small endogenous noncoding RNAs involved in posttranscriptional gene regulation. During microRNA biogenesis, Drosha and Dicer process the primary transcript (pri-miRNA) through a precursor hairpin (pre-miRNA) to the mature miRNA. The miRNA is incorporated into the RNA-Induced Silencing Complex (RISC) with Argonaute proteins, the effector molecules in RNA interference (RNAi). Here, we show that all Argonautes elevate mature miRNA expression posttranscriptionally, independent of RNase activity. Also, we identify a role for the RISC slicer Argonaute2 (Ago2) in cleaving the pre-miRNA to an additional processing intermediate, termed Ago2-cleaved precursor miRNA or ac-pre-miRNA. This endogenous, on-pathway intermediate results from cleavage of the pre-miRNA hairpin 12 nucleotides from its 3'-end. By analogy to siRNA processing, Ago2 cleavage may facilitate removal of the nicked passenger strand from RISC after maturation. The multiple roles of Argonautes in the RNAi effector phase and miRNA biogenesis and maturation suggest coordinate regulation of microRNA expression and function.

  4. Silencing of hepatitis C virus replication by a non-viral vector based on solid lipid nanoparticles containing a shRNA targeted to the internal ribosome entry site (IRES).

    PubMed

    Torrecilla, Josune; Del Pozo-Rodríguez, Ana; Solinís, María Ángeles; Apaolaza, Paola S; Berzal-Herranz, Beatriz; Romero-López, Cristina; Berzal-Herranz, Alfredo; Rodríguez-Gascón, Alicia

    2016-10-01

    Gene silencing mediated by RNAi has gained increasing interest as an alternative for the treatment of infectious diseases such as refractory hepatitis C virus (HCV) infection. In this work we have designed and evaluated a non-viral vector based on solid lipid nanoparticles (SLN) bearing hyaluronic acid, protamine and a short hairpin RNA (shRNA74) targeted to the Internal Ribosome Entry Site (IRES) of the HCV. The vector was able to inhibit the expression of the HCV IRES in Huh-7 cells, with the inhibition level dependent on the shRNA74 to SLN ratio and on the shRNA74 dose added to the culture cells. The nanocarrier was also able to inhibit the replication in human hepatoma cells supporting a subgenomic HCV replicon (Huh-7 NS3-3'). The vector was quickly and efficiently internalized by the cells, and endocytosis was the most productive uptake mechanism for silencing. Clathrin-mediated endocytosis and to a lesser extent caveolae/lipid raft-mediated endocytosis were identified as endocytic mechanisms involved in the cell uptake. Internalization via the CD44 receptor was also involved, although this entry route seems to be less productive for silencing than endocytosis. The vector did not induce either hemolysis or agglutination of red cells in vitro, which was indicative of good biocompatibility. In summary, we have shown for the first time the ability of a non-viral SLN-based vector to silence a HCV replicon.

  5. Silencing of hepatitis C virus replication by a non-viral vector based on solid lipid nanoparticles containing a shRNA targeted to the internal ribosome entry site (IRES).

    PubMed

    Torrecilla, Josune; Del Pozo-Rodríguez, Ana; Solinís, María Ángeles; Apaolaza, Paola S; Berzal-Herranz, Beatriz; Romero-López, Cristina; Berzal-Herranz, Alfredo; Rodríguez-Gascón, Alicia

    2016-10-01

    Gene silencing mediated by RNAi has gained increasing interest as an alternative for the treatment of infectious diseases such as refractory hepatitis C virus (HCV) infection. In this work we have designed and evaluated a non-viral vector based on solid lipid nanoparticles (SLN) bearing hyaluronic acid, protamine and a short hairpin RNA (shRNA74) targeted to the Internal Ribosome Entry Site (IRES) of the HCV. The vector was able to inhibit the expression of the HCV IRES in Huh-7 cells, with the inhibition level dependent on the shRNA74 to SLN ratio and on the shRNA74 dose added to the culture cells. The nanocarrier was also able to inhibit the replication in human hepatoma cells supporting a subgenomic HCV replicon (Huh-7 NS3-3'). The vector was quickly and efficiently internalized by the cells, and endocytosis was the most productive uptake mechanism for silencing. Clathrin-mediated endocytosis and to a lesser extent caveolae/lipid raft-mediated endocytosis were identified as endocytic mechanisms involved in the cell uptake. Internalization via the CD44 receptor was also involved, although this entry route seems to be less productive for silencing than endocytosis. The vector did not induce either hemolysis or agglutination of red cells in vitro, which was indicative of good biocompatibility. In summary, we have shown for the first time the ability of a non-viral SLN-based vector to silence a HCV replicon. PMID:27451369

  6. Subtypes of asthma defined by epithelial cell expression of messenger RNA and microRNA.

    PubMed

    Woodruff, Prescott G

    2013-12-01

    Human asthma can be subcategorized in several ways, but one powerful approach is to subtype asthma on the basis of underlying cellular and molecular mechanisms. Groups of patients with a disease that share a common underlying biology are termed an "endotype." Endotypes of asthma have been studied at both the cellular level (by cytological examination of induced sputum) and, increasingly, at the molecular level. Genome-wide analyses of mRNA expression within the lung have been useful in the identification of molecular endotypes of asthma and point to protein biomarkers of those endotypes that can be measured in the blood. More recently, studies of microRNA expression in airway epithelial cells in asthma have identified additional candidate biomarkers of asthma endotypes. One potentially valuable property of microRNAs is that they can also be measured in extracellular fluids and therefore have the potential to serve directly as noninvasively measured biomarkers.

  7. An Integrated Analysis of MicroRNA and mRNA Expression Profiles to Identify RNA Expression Signatures in Lambskin Hair Follicles in Hu Sheep.

    PubMed

    Lv, Xiaoyang; Sun, Wei; Yin, Jinfeng; Ni, Rong; Su, Rui; Wang, Qingzeng; Gao, Wen; Bao, Jianjun; Yu, Jiarui; Wang, Lihong; Chen, Ling

    2016-01-01

    Wave patterns in lambskin hair follicles are an important factor determining the quality of sheep's wool. Hair follicles in lambskin from Hu sheep, a breed unique to China, have 3 types of waves, designated as large, medium, and small. The quality of wool from small wave follicles is excellent, while the quality of large waves is considered poor. Because no molecular and biological studies on hair follicles of these sheep have been conducted to date, the molecular mechanisms underlying the formation of different wave patterns is currently unknown. The aim of this article was to screen the candidate microRNAs (miRNA) and genes for the development of hair follicles in Hu sheep. Two-day-old Hu lambs were selected from full-sib individuals that showed large, medium, and small waves. Integrated analysis of microRNA and mRNA expression profiles employed high-throughout sequencing technology. Approximately 13, 24, and 18 differentially expressed miRNAs were found between small and large waves, small and medium waves, and medium and large waves, respectively. A total of 54, 190, and 81 differentially expressed genes were found between small and large waves, small and medium waves, and medium and large waves, respectively, by RNA sequencing (RNA-seq) analysis. Differentially expressed genes were classified using gene ontology and pathway analyses. They were found to be mainly involved in cell differentiation, proliferation, apoptosis, growth, immune response, and ion transport, and were associated with MAPK and the Notch signaling pathway. Reverse transcription-polymerase chain reaction (RT-PCR) analyses of differentially-expressed miRNA and genes were consistent with sequencing results. Integrated analysis of miRNA and mRNA expression indicated that, compared to small waves, large waves included 4 downregulated miRNAs that had regulatory effects on 8 upregulated genes and 3 upregulated miRNAs, which in turn influenced 13 downregulated genes. Compared to small waves, medium

  8. An Integrated Analysis of MicroRNA and mRNA Expression Profiles to Identify RNA Expression Signatures in Lambskin Hair Follicles in Hu Sheep

    PubMed Central

    Lv, Xiaoyang; Sun, Wei; Yin, Jinfeng; Ni, Rong; Su, Rui; Wang, Qingzeng; Gao, Wen; Bao, Jianjun; Yu, Jiarui; Wang, Lihong; Chen, Ling

    2016-01-01

    Wave patterns in lambskin hair follicles are an important factor determining the quality of sheep’s wool. Hair follicles in lambskin from Hu sheep, a breed unique to China, have 3 types of waves, designated as large, medium, and small. The quality of wool from small wave follicles is excellent, while the quality of large waves is considered poor. Because no molecular and biological studies on hair follicles of these sheep have been conducted to date, the molecular mechanisms underlying the formation of different wave patterns is currently unknown. The aim of this article was to screen the candidate microRNAs (miRNA) and genes for the development of hair follicles in Hu sheep. Two-day-old Hu lambs were selected from full-sib individuals that showed large, medium, and small waves. Integrated analysis of microRNA and mRNA expression profiles employed high-throughout sequencing technology. Approximately 13, 24, and 18 differentially expressed miRNAs were found between small and large waves, small and medium waves, and medium and large waves, respectively. A total of 54, 190, and 81 differentially expressed genes were found between small and large waves, small and medium waves, and medium and large waves, respectively, by RNA sequencing (RNA-seq) analysis. Differentially expressed genes were classified using gene ontology and pathway analyses. They were found to be mainly involved in cell differentiation, proliferation, apoptosis, growth, immune response, and ion transport, and were associated with MAPK and the Notch signaling pathway. Reverse transcription-polymerase chain reaction (RT-PCR) analyses of differentially-expressed miRNA and genes were consistent with sequencing results. Integrated analysis of miRNA and mRNA expression indicated that, compared to small waves, large waves included 4 downregulated miRNAs that had regulatory effects on 8 upregulated genes and 3 upregulated miRNAs, which in turn influenced 13 downregulated genes. Compared to small waves

  9. Gene Expression Profile Changes After Short-activating RNA-mediated Induction of Endogenous Pluripotency Factors in Human Mesenchymal Stem Cells

    PubMed Central

    Voutila, Jon; Sætrom, Pål; Mintz, Paul; Sun, Guihua; Alluin, Jessica; Rossi, John J; Habib, Nagy A; Kasahara, Noriyuki

    2012-01-01

    It is now recognized that small noncoding RNA sequences have the ability to mediate transcriptional activation of specific target genes in human cells. Using bioinformatics analysis and functional screening, we screened short-activating RNA (saRNA) oligonucleotides designed to target the promoter regions of the pluripotency reprogramming factors, Kruppel-like factor 4 (KLF4) and c-MYC. We identified KLF4 and c-MYC promoter-targeted saRNA sequences that consistently induced increases in their respective levels of nascent mRNA and protein expression in a time- and dose-dependent manner, as compared with scrambled sequence control oligonucleotides. The functional consequences of saRNA-induced activation of each targeted reprogramming factor were then characterized by comprehensively profiling changes in gene expression by microarray analysis, which revealed significant increases in mRNA levels of their respective downstream pathway genes. Notably, the microarray profile after saRNA-mediated induction of endogenous KLF4 and c-MYC showed similar gene expression patterns for stem cell- and cell cycle-related genes as compared with lentiviral vector-mediated overexpression of exogenous KLF4 and c-MYC transgenes, while divergent gene expression patterns common to viral vector-mediated transgene delivery were also noted. The use of promoter-targeted saRNAs for the activation of pluripotency reprogramming factors could have broad implications for stem cell research. PMID:23344177

  10. Modification of Globin Gene Expression by RNA Targeting Strategies

    PubMed Central

    Shen, Tong-Jian; Rogers, Heather; Yu, Xiaobing; Lin, Felix; Noguchi, Constance T.; Ho, Chien

    2007-01-01

    Objective Sickle cell anemia is a genetic blood disease resulting from production of mutant β-globin (βS) and has severe clinical consequences. It is known that a higher cellular γ-globin level, e.g., higher ratio of cellular γ-globin to βS-globin (γ/βS ratio), inhibits sickle hemoglobin (HbS) polymerization tendency. Hence, therapeutic treatment of sickle cell anemia has been focused on introducing γ-globin gene into red blood cells to increase the cellular γ/βS ratio. Here, we have introduced ribozymes and small interfering RNAs (siRNAs) against βS-globin mRNA into blood cells as a means to increase the γ/βS ratio. Methods Single and multi-ribozymes against βS-globin mRNA have been tested in vitro and in human erythroleukemia K562βS cells that stably express exogenous βS-globin gene. Primary human hematopoietic progenitor cells were also transfected with multi-ribozyme and the γ/(γ+β) ratio determined and compared with cells transfected with long hairpin β-globin cDNA and synthetic siRNA genes. Results We have found that the multi-ribozyme zb21A containing two ribozyme units effectively reduces βS-globin mRNA both in vitro and in K562βS cells. The γ-globin mRNA to βS-globin mRNA ratio in the multi-ribozyme transfected cells is about a factor of 2 more than that in the control cells. We have also found that the γ/(γ+β) ratio in the transfected hematopoietic progenitor cells is increased by more than 2-fold in cells treated with multi-ribozyme zb21A or siRNA ib5. Conclusion Our results suggest that introducing multi-ribozymes or siRNAs into red blood cells are comparable in their effectiveness to increase the ratio of cellular γ-globin mRNA to β- or βS-globin mRNA, providing possible strategies to increase the effectiveness of γ-globin gene transfer as gene therapy for treatment of patients with sickle cell anemia. PMID:17662889

  11. MicroRNA-Offset RNA Alters Gene Expression and Cell Proliferation

    PubMed Central

    Zhao, Jin; Schnitzler, Gavin R.; Iyer, Lakshmanan K.; Aronovitz, Mark J.; Baur, Wendy E.; Karas, Richard H.

    2016-01-01

    MicroRNA-offset RNAs (moRs) were first identified in simple chordates and subsequently in mouse and human cells by deep sequencing of short RNAs. MoRs are derived from sequences located immediately adjacent to microRNAs (miRs) in the primary miR (pri-miR). Currently moRs are considered to be simply a by-product of miR biosynthesis that lack biological activity. Here we show for the first time that a moR is biologically active. We demonstrate that endogenous or over-expressed moR-21 significantly alters gene expression and inhibits the proliferation of vascular smooth muscle cells (VSMC). In addition, we find that miR-21 and moR-21 may regulate different genes in a given pathway and can oppose each other in regulating certain genes. We report that there is a “seed region” of moR-21 as well as a “seed match region” in the target gene 3’UTR that are indispensable for moR-21-mediated gene down-regulation. We further demonstrate that moR-21-mediated gene repression is Argonaute 2 (Ago2) dependent. Taken together, these findings provide the first evidence that microRNA offset RNA alters gene expression and is biologically active. PMID:27276022

  12. IRES-mediated Tricistronic vectors for enhancing generation of high monoclonal antibody expressing CHO cell lines.

    PubMed

    Ho, Steven C L; Bardor, Muriel; Feng, Huatao; Mariati; Tong, Yen Wah; Song, Zhiwei; Yap, Miranda G S; Yang, Yuansheng

    2012-01-01

    A Tricistronic vector utilizing internal ribosome entry site (IRES) elements to express the light chain (LC), heavy chain (HC), and a neomycin phosphotransferase (NPT) selection marker from one transcript is designed for generation of mAb expressing CHO cell lines. As compared to the commonly used vectors, benefits of this design include: (1) minimized non-expressing clones, (2) enhanced stable mAb productivity without gene amplification, (3) control of LC and HC expression at defined ratios, and (4) consistent product quality. After optimization of the LC and HC arrangement and increasing selection stringency by weakening the NPT selection marker, this Tricistronic vector is able to generate stably transfected pools with specific productivity (qmAb) greater than 5pg/cell/day (pcd) and titers over 150mg/L. 5% of clones from these pools have qmAb greater than 20pcd and titers ranging from 300 to more than 500mg/L under non-optimized shake flask batch cultures using commercially available protein-free medium. The mAb produced by these clones have low aggregation and consistent glycosylation profiles. The entire process of transfection to high-expressing clones requires only 6 months. The IRES-mediated Tricistronic vector provides an attractive alternative to commonly used vectors for fast generation of mAb CHO cell lines with high productivity. PMID:22024589

  13. Baculovirus as versatile vectors for protein expression in insect and mammalian cells.

    PubMed

    Kost, Thomas A; Condreay, J Patrick; Jarvis, Donald L

    2005-05-01

    Today, many thousands of recombinant proteins, ranging from cytosolic enzymes to membrane-bound proteins, have been successfully produced in baculovirus-infected insect cells. Yet, in addition to its value in producing recombinant proteins in insect cells and larvae, this viral vector system continues to evolve in new and unexpected ways. This is exemplified by the development of engineered insect cell lines to mimic mammalian cell glycosylation of expressed proteins, baculovirus display strategies and the application of the virus as a mammalian-cell gene delivery vector. Novel vector design and cell engineering approaches will serve to further enhance the value of baculovirus technology.

  14. Production of Transgenic Calves Expressing an shRNA Targeting Myostatin

    PubMed Central

    Tessanne, K; Golding, MC; Long, CR; Peoples, MD; Hannon, G; Westhusin, ME

    2012-01-01

    Myostatin (MSTN) is a well-known negative regulator of muscle growth. Animals that possess mutations within this gene display an enhanced muscling phenotype, a desirable agricultural trait. Increased neonatal morbidity is common, however, resulting from complications arising from the birth of offspring with increased fetal muscle mass. The objective of the current research was to generate an attenuated MSTN-null phenotype in a large-animal model using RNA interference to enhance muscle development without the detrimental consequences of an inactivating mutation. To this end, we identified a series of short interfering RNAs that demonstrated effective suppression of MSTN mRNA and protein levels. To produce transgenic offspring capable of stable MSTN suppression in vivo, a recombinant lentiviral vector expressing a short hairpin RNA (shRNA) targeting MSTN for silencing was introduced into bovine fetal fibroblasts. These cells were used as nucleus donors for somatic cell nuclear transfer (SCNT). Twenty blastocysts were transferred into seven recipient cows resulting in five pregnancies. One transgenic calf developed to term, but died following delivery by Caesarean-section. As an alternative strategy, microinjection of recombinant lentiviral particles into the perivitelline space of in vitro-produced bovine zygotes was utilized to produce 40 transgenic blastocysts that were transferred into 14 recipient cows, resulting in 7 pregnancies. Five transgenic calves were produced, of which three expressed the transgene. This is the first report of transgenic livestock produced by direct injection of a recombinant lentivirus, and expressing transgenes encoding shRNAs targeting an endogenous gene (myostatin) for silencing. PMID:22139943

  15. MicroRNA Expression Signature in Degenerative Aortic Stenosis

    PubMed Central

    2016-01-01

    Degenerative aortic stenosis, characterized by narrowing of the exit of the left ventricle of the heart, has become the most common valvular heart disease in the elderly. The aim of this study was to investigate the microRNA (miRNA) signature in degenerative AS. Through microarray analysis, we identified the miRNA expression signature in the tissue samples from healthy individuals (n = 4) and patients with degenerative AS (n = 4). Six miRNAs (hsa-miR-193a-3p, hsa-miR-29b-1-5p, hsa-miR-505-5p, hsa-miR-194-5p, hsa-miR-99b-3p, and hsa-miR-200b-3p) were overexpressed and 14 (hsa-miR-3663-3p, hsa-miR-513a-5p, hsa-miR-146b-5p, hsa-miR-1972, hsa-miR-718, hsa-miR-3138, hsa-miR-21-5p, hsa-miR-630, hsa-miR-575, hsa-miR-301a-3p, hsa-miR-636, hsa-miR-34a-3p, hsa-miR-21-3p, and hsa-miR-516a-5p) were downregulated in aortic tissue from AS patients. GeneSpring 13.1 was used to identify potential human miRNA target genes by comparing a 3-way comparison of predictions from TargetScan, PITA, and microRNAorg databases. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis were performed to identify potential pathways and functional annotations associated with AS. Twenty miRNAs were significantly differentially expressed between patients with AS samples and normal controls and identified potential miRNA targets and molecular pathways associated with this morbidity. This study describes the miRNA expression signature in degenerative AS and provides an improved understanding of the molecular pathobiology of this disease. PMID:27579316

  16. MicroRNA Expression Signature in Degenerative Aortic Stenosis.

    PubMed

    Shi, Jing; Liu, Hui; Wang, Hui; Kong, Xiangqing

    2016-01-01

    Degenerative aortic stenosis, characterized by narrowing of the exit of the left ventricle of the heart, has become the most common valvular heart disease in the elderly. The aim of this study was to investigate the microRNA (miRNA) signature in degenerative AS. Through microarray analysis, we identified the miRNA expression signature in the tissue samples from healthy individuals (n = 4) and patients with degenerative AS (n = 4). Six miRNAs (hsa-miR-193a-3p, hsa-miR-29b-1-5p, hsa-miR-505-5p, hsa-miR-194-5p, hsa-miR-99b-3p, and hsa-miR-200b-3p) were overexpressed and 14 (hsa-miR-3663-3p, hsa-miR-513a-5p, hsa-miR-146b-5p, hsa-miR-1972, hsa-miR-718, hsa-miR-3138, hsa-miR-21-5p, hsa-miR-630, hsa-miR-575, hsa-miR-301a-3p, hsa-miR-636, hsa-miR-34a-3p, hsa-miR-21-3p, and hsa-miR-516a-5p) were downregulated in aortic tissue from AS patients. GeneSpring 13.1 was used to identify potential human miRNA target genes by comparing a 3-way comparison of predictions from TargetScan, PITA, and microRNAorg databases. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis were performed to identify potential pathways and functional annotations associated with AS. Twenty miRNAs were significantly differentially expressed between patients with AS samples and normal controls and identified potential miRNA targets and molecular pathways associated with this morbidity. This study describes the miRNA expression signature in degenerative AS and provides an improved understanding of the molecular pathobiology of this disease. PMID:27579316

  17. Use of a Sindbis virus DNA-based expression vector for induction of protective immunity against pseudorabies virus in pigs.

    PubMed

    Dufour, Vinciane; De Boisséson, Claire

    2003-06-20

    Injection of plasmid DNA encoding pseudorabies virus (PRV) glycoproteins into pig muscle has been shown to result in protective immunity against lethal infection. Nevertheless, such DNA vaccines are still less efficient than some attenuated or killed live vaccines. One way to increase DNA vaccine efficacy is to improve the vectorisation system at the molecular level, thereby enhancing the rate of in vivo-produced immunogen protein and consequently specific acquired immunity. The present study compared the effectiveness of the protein expression system depending on Sindbis virus (SIN) replicase [J. Virol. 70 (1996) 508] with that of more classical pcDNA3 plasmid. Pigs were vaccinated twice at 3-week interval with a mixture of three pcDNA3 plasmids expressing gB, gC and gD (designated as PRV-pcDNA3) or a mixture of three SIN plasmids expressing the same glycoproteins (PRV-pSINCP), and were challenged with a highly virulent PRV strain. The two DNA vaccines induced PRV-specific T cell-mediated immune response characterized by very low levels of IFN-gamma mRNA in PBMC after in vitro antigen-specific stimulation. Very low levels of neutralizing antibodies (NAb) were also obtained in sera following DNA injection(s). A second DNA injection did not boost immune responses. After a lethal challenge, high levels of IFN-gamma mRNA and high NAb response were induced in all DNA-vaccinated pigs, regardless of the vector used. Therefore, the two eukaryotic expression systems showed comparable efficacy in inducing antiviral immunity and clinical protection against PRV in pigs. This suggests that SIN DNA-based vector immunizing potential may differ according to antigen and/or host. PMID:12814698

  18. Integrated miRNA and mRNA Expression Profiling in Inflamed Colon of Patients with Ulcerative Colitis

    PubMed Central

    Van der Goten, Jan; Vanhove, Wiebe; Lemaire, Katleen; Van Lommel, Leentje; Machiels, Kathleen; Wollants, Willem-Jan; De Preter, Vicky; De Hertogh, Gert; Ferrante, Marc; Van Assche, Gert; Rutgeerts, Paul; Schuit, Frans; Vermeire, Séverine; Arijs, Ingrid

    2014-01-01

    Background Ulcerative colitis (UC) is associated with differential colonic expression of genes involved in immune response (e.g. IL8) and barrier integrity (e.g. cadherins). MicroRNAs (miRNAs) are regulators of gene expression and are involved in various immune-related diseases. In this study, we investigated (1) if miRNA expression in UC mucosa is altered and (2) if any of these changes correlate with mucosal mRNA expression. Integration of mRNA and miRNA expression profiling may allow the identification of functional links between dysregulated miRNAs and their target mRNA. Methodology Colonic mucosal biopsies were obtained from 17 UC (10 active and 7 inactive) patients and 10 normal controls. Total RNA was used to analyze miRNA and mRNA expression via Affymetrix miRNA 2.0 and Affymetrix Human Gene 1.0ST arrays, respectively. Both miRNA and gene expression profiles were integrated by correlation analysis to identify dysregulated miRNAs with their corresponding predicted target mRNA. Microarray data were validated with qRT-PCR. Regulation of IL8 and CDH11 expression by hsa-miR-200c-3p was determined by luciferase reporter assays. Results When comparing active UC patients vs. controls, 51 miRNAs and 1543 gene probe sets gave significantly different signals. In contrast, in inactive UC vs. controls, no significant miRNA expression differences were found while 155 gene probe sets had significantly different signals. We then identified potential target genes of the significantly dysregulated miRNAs and genes in active UC vs. controls and found a highly significant inverse correlation between hsa-miR-200c-3p and IL8, an inflammatory marker, and between hsa-miR-200c-3p and CDH11, a gene related to intestinal epithelial barrier function. We could demonstrate that hsa-miR-200c-3p directly regulates IL8 and CDH11 expression. Conclusion Differential expression of immune- and barrier-related genes in inflamed UC mucosa may be influenced by altered expression of mi

  19. PmiRExAt: plant miRNA expression atlas database and web applications

    PubMed Central

    Gurjar, Anoop Kishor Singh; Panwar, Abhijeet Singh; Gupta, Rajinder; Mantri, Shrikant S.

    2016-01-01

    High-throughput small RNA (sRNA) sequencing technology enables an entirely new perspective for plant microRNA (miRNA) research and has immense potential to unravel regulatory networks. Novel insights gained through data mining in publically available rich resource of sRNA data will help in designing biotechnology-based approaches for crop improvement to enhance plant yield and nutritional value. Bioinformatics resources enabling meta-analysis of miRNA expression across multiple plant species are still evolving. Here, we report PmiRExAt, a new online database resource that caters plant miRNA expression atlas. The web-based repository comprises of miRNA expression profile and query tool for 1859 wheat, 2330 rice and 283 maize miRNA. The database interface offers open and easy access to miRNA expression profile and helps in identifying tissue preferential, differential and constitutively expressing miRNAs. A feature enabling expression study of conserved miRNA across multiple species is also implemented. Custom expression analysis feature enables expression analysis of novel miRNA in total 117 datasets. New sRNA dataset can also be uploaded for analysing miRNA expression profiles for 73 plant species. PmiRExAt application program interface, a simple object access protocol web service allows other programmers to remotely invoke the methods written for doing programmatic search operations on PmiRExAt database. Database URL: http://pmirexat.nabi.res.in. PMID:27081157

  20. PmiRExAt: plant miRNA expression atlas database and web applications.

    PubMed

    Gurjar, Anoop Kishor Singh; Panwar, Abhijeet Singh; Gupta, Rajinder; Mantri, Shrikant S

    2016-01-01

    High-throughput small RNA (sRNA) sequencing technology enables an entirely new perspective for plant microRNA (miRNA) research and has immense potential to unravel regulatory networks. Novel insights gained through data mining in publically available rich resource of sRNA data will help in designing biotechnology-based approaches for crop improvement to enhance plant yield and nutritional value. Bioinformatics resources enabling meta-analysis of miRNA expression across multiple plant species are still evolving. Here, we report PmiRExAt, a new online database resource that caters plant miRNA expression atlas. The web-based repository comprises of miRNA expression profile and query tool for 1859 wheat, 2330 rice and 283 maize miRNA. The database interface offers open and easy access to miRNA expression profile and helps in identifying tissue preferential, differential and constitutively expressing miRNAs. A feature enabling expression study of conserved miRNA across multiple species is also implemented. Custom expression analysis feature enables expression analysis of novel miRNA in total 117 datasets. New sRNA dataset can also be uploaded for analysing miRNA expression profiles for 73 plant species. PmiRExAt application program interface, a simple object access protocol web service allows other programmers to remotely invoke the methods written for doing programmatic search operations on PmiRExAt database.Database URL:http://pmirexat.nabi.res.in. PMID:27081157

  1. Regulation of adeno-associated virus gene expression in 293 cells: control of mRNA abundance and translation

    SciTech Connect

    Trempe, J.P.; Carter, B.J.

    1988-01-01

    The authors studied the effects of the adeno-associated virus (AAV) rep gene on the control of gene expression from the AAV p/sub 40/ promoter in 293 cells in the absence of an adenovirus coinfection. AAV vectors containing the chloramphenicol acetyltransferase (cat) gene were used to measure the levels of cat expression and steady-state mRNA from p/sub 40/. When the rep gene was present in cis or in trans, cat expression from p/sub 40/ was decreased 3- to 10-fold, but there was a 2- to 10-fold increase in the level of p/sub 40/ mRNA. Conversely, cat expression increased and the p/sub 40/ mRNA level decreased in the absence of the rep gene. Both wild-type and carboxyl-terminal truncated Rep proteins were capable of eliciting both effects. These data suggest two roles for the pleiotropic AAV rep gene: as a translational inhibitor and as a positive regulator of p/sub 40/ mRNA levels. They also provide additional evidence for a cis-acting negative regulatory region which decreases RNA from the AAV p/sub 5/ promoter in a fashion independent of rep.

  2. Production of human beta interferon in insect cells infected with a Baculovirus expression vector

    SciTech Connect

    Smith, G.E.; Summers, M.D.; Fraser, M.J.

    1983-12-01

    Autographa californica nuclear polyhedrosis virus (AcNPV) was used as an expression vector for human beta interferon. By using specially constructed plasmids, the protein-coding sequences for interferon were linked to the AcNPV promoter for the gene encoding for polyhedrin, the major occlusion protein. The interferon gene was inserted at various locations relative to the AcNPV polyhedrin transcriptional and translational signals, and the interferon-polyhedrin hybrid genes were transferred to infectious AcNPV expression vectors. Biologically active interferon was produced, and greater than 95% was secreted from infected insect cells. A maximum of ca. 5 x 10/sup 6/ U of interferon activity was produced by 10/sup 6/ infected cells. These results demonstrate that AcNPV should be suitable for use as a eucaryotic expression vector for the production of products from cloned genes.

  3. Construction of a directional T vector for cloning PCR products and expression in Escherichia coli.

    PubMed

    Liang, Xiu-Yi; Liang, Zhi-Cheng; Zhang, Zhi; Zhou, Jiao-Jiao; Liu, Shi-Yu; Tian, Sheng-Li

    2015-05-01

    In order to clone PCR products and express them effectively in Escherichia coli, a directional cloning system was constructed by generating a T vector based on pQE-30Xa. The vector was prepared by inserting an XcmI cassette containing an endonuclease XcmI site, a kanamycin selective marker, a multiple-cloning-site (MCS) region and an opposite endonuclease XcmI site into the vector pQE-30Xa. The T vector pQE-T with single overhanging dT residues at both 3' ends was obtained by digesting with the restriction enzyme XcmI. For directional cloning, a BamHI site was introduced to the ends of the PCR products. A BamHI site was also located on the multiple cloning site of pQE-T. The PCR products were ligated with pQE-T. The directionally inserted recombinants were distinguished by using BamHI to digest the recombinants because there are two BamHI sites located on the both sides of PCR fragment. In order to identify the T-vector functions, the 14-3-3-ZsGreen and hRBP genes were amplified and a BamHI site was added to the ends of the genes to confirm this vector by ligation with pQE-T. Results showed that the 14-3-3-ZsGreen and hRBP were cloned to the vector pQE-T directly and corresponding proteins were successfully produced. It was here demonstrated that this directional vector is capable of gene cloning and is used to manipulate gene expression very easily. The methodology proposed here involves easy incorporation of the construct into other vectors in various hosts.

  4. Tight regulation, modulation, and high-level expression by vectors containing the arabinose PBAD promoter.

    PubMed

    Guzman, L M; Belin, D; Carson, M J; Beckwith, J

    1995-07-01

    We have constructed a series of plasmid vectors (pBAD vectors) containing the PBAD promoter of the araBAD (arabinose) operon and the gene encoding the positive and negative regulator of this promoter, araC. Using the phoA gene and phoA fusions to monitor expression in these vectors, we show that the ratio of induction/repression can be 1,200-fold, compared with 50-fold for PTAC-based vectors. phoA expression can be modulated over a wide range of inducer (arabinose) concentrations and reduced to extremely low levels by the presence of glucose, which represses expression. Also, the kinetics of induction and repression are very rapid and significantly affected by the ara allele in the host strain. Thus, the use of this system which can be efficiently and rapidly turned on and off allows the study of important aspects of bacterial physiology in a very simple manner and without changes of temperature. We have exploited the tight regulation of the PBAD promoter to study the phenotypes of null mutations of essential genes and explored the use of pBAD vectors as an expression system.

  5. RNA1-Independent Replication and GFP Expression from Tomato marchitez virus Isolate M Cloned cDNA.

    PubMed

    Ferriol, I; Turina, M; Zamora-Macorra, E J; Falk, B W

    2016-05-01

    Tomato marchitez virus (ToMarV; synonymous with Tomato apex necrosis virus) is a positive-strand RNA virus in the genus Torradovirus within the family Secoviridae. ToMarV is an emergent whitefly-transmitted virus that causes important diseases in tomato (Solanum lycopersicum) in Mexico. Here, the genome sequence of the ToMarV isolate M (ToMarV-M) was determined. We engineered full-length cDNA clones of the ToMarV-M genomic RNA (RNA1 and RNA2), separately, into a binary vector. Coinfiltration of both triggered systemic infections in Nicotiana benthamiana, tomato, and tomatillo (Physalis philadelphica) plants and recapitulated the biological activity of the wild-type virus. The viral progeny generated from tomato and tomatillo plants were transmissible by the whitefly Bemisia tabaci biotype B. Also, we assessed whether these infectious clones could be used for screening tomato cultivars for resistance to ToMarV and our results allowed us to differentiate resistant and susceptible tomato lines. We demonstrated that RNA1 of ToMarV-M is required for the replication of RNA2, and it can replicate independently of RNA2. From this, ToMarV-M RNA2 was used to express the green fluorescent protein in N. benthamiana plants, which allowed us to track cell-to-cell movement. The construction of full-length infectious cDNA clones of ToMarV-M provides an excellent tool to investigate virus-host-vector interactions and elucidate the functions of torradovirus-encoded proteins or the mechanisms of replication of torradovirus genomic RNA. PMID:26756828

  6. RNA1-Independent Replication and GFP Expression from Tomato marchitez virus Isolate M Cloned cDNA.

    PubMed

    Ferriol, I; Turina, M; Zamora-Macorra, E J; Falk, B W

    2016-05-01

    Tomato marchitez virus (ToMarV; synonymous with Tomato apex necrosis virus) is a positive-strand RNA virus in the genus Torradovirus within the family Secoviridae. ToMarV is an emergent whitefly-transmitted virus that causes important diseases in tomato (Solanum lycopersicum) in Mexico. Here, the genome sequence of the ToMarV isolate M (ToMarV-M) was determined. We engineered full-length cDNA clones of the ToMarV-M genomic RNA (RNA1 and RNA2), separately, into a binary vector. Coinfiltration of both triggered systemic infections in Nicotiana benthamiana, tomato, and tomatillo (Physalis philadelphica) plants and recapitulated the biological activity of the wild-type virus. The viral progeny generated from tomato and tomatillo plants were transmissible by the whitefly Bemisia tabaci biotype B. Also, we assessed whether these infectious clones could be used for screening tomato cultivars for resistance to ToMarV and our results allowed us to differentiate resistant and susceptible tomato lines. We demonstrated that RNA1 of ToMarV-M is required for the replication of RNA2, and it can replicate independently of RNA2. From this, ToMarV-M RNA2 was used to express the green fluorescent protein in N. benthamiana plants, which allowed us to track cell-to-cell movement. The construction of full-length infectious cDNA clones of ToMarV-M provides an excellent tool to investigate virus-host-vector interactions and elucidate the functions of torradovirus-encoded proteins or the mechanisms of replication of torradovirus genomic RNA.

  7. Development of Lentiviral Vectors Simultaneously Expressing Multiple siRNAs Against CCR5, vif and tat/rev Genes for an HIV-1 Gene Therapy Approach

    PubMed Central

    Spanevello, Francesca; Calistri, Arianna; Del Vecchio, Claudia; Mantelli, Barbara; Frasson, Chiara; Basso, Giuseppe; Palù, Giorgio; Cavazzana, Marina; Parolin, Cristina

    2016-01-01

    Gene therapy holds considerable promise for the functional cure of HIV-1 infection and, in this context, RNA interference (RNAi)-based approaches represent powerful strategies. Stable expression of small interfering RNAs (siRNAs) targeting HIV genes or cellular cofactors has the potential to render HIV-1 susceptible cells resistant to infection. To inhibit different steps of virus life cycle, self-inactivating lentiviral vectors expressing multiple siRNAs targeting the CCR5 cellular gene as well as vif and tat/rev viral transcripts, under the control of different RNA polymerase III promoters (U6, 7SK, H1) were developed. The use of a single RNA polymerase III promoter driving the expression of a sequence giving rise to three siRNAs directed against the selected targets (e-shRNA) was also investigated. Luciferase assay and inhibition of HIV-1 replication in human Jurkat T-cell line were adopted to select the best combination of promoter/siRNA. The efficacy of selected developed combinatorial vectors in interfering with viral replication was evaluated in human primary CD4+ T lymphocytes. We identified two effective anti-HIV combinatorial vectors that conferred protection against R5- and X4- tropic viruses. Overall, our results showed that the antiviral effect is influenced by different factors, including the promoter used to express the RNAi molecules and the selected cassette combination. These findings contribute to gain further insights in the design of RNAi-based gene therapy approaches against HIV-1 for clinical application. PMID:27093170

  8. Identifying Cancer Subtypes from miRNA-TF-mRNA Regulatory Networks and Expression Data

    PubMed Central

    Liu, Lin; Wang, Rujing; Sun, Bingyu; Li, Jiuyong

    2016-01-01

    Background Identifying cancer subtypes is an important component of the personalised medicine framework. An increasing number of computational methods have been developed to identify cancer subtypes. However, existing methods rarely use information from gene regulatory networks to facilitate the subtype identification. It is widely accepted that gene regulatory networks play crucial roles in understanding the mechanisms of diseases. Different cancer subtypes are likely caused by different regulatory mechanisms. Therefore, there are great opportunities for developing methods that can utilise network information in identifying cancer subtypes. Results In this paper, we propose a method, weighted similarity network fusion (WSNF), to utilise the information in the complex miRNA-TF-mRNA regulatory network in identifying cancer subtypes. We firstly build the regulatory network where the nodes represent the features, i.e. the microRNAs (miRNAs), transcription factors (TFs) and messenger RNAs (mRNAs) and the edges indicate the interactions between the features. The interactions are retrieved from various interatomic databases. We then use the network information and the expression data of the miRNAs, TFs and mRNAs to calculate the weight of the features, representing the level of importance of the features. The feature weight is then integrated into a network fusion approach to cluster the samples (patients) and thus to identify cancer subtypes. We applied our method to the TCGA breast invasive carcinoma (BRCA) and glioblastoma multiforme (GBM) datasets. The experimental results show that WSNF performs better than the other commonly used computational methods, and the information from miRNA-TF-mRNA regulatory network contributes to the performance improvement. The WSNF method successfully identified five breast cancer subtypes and three GBM subtypes which show significantly different survival patterns. We observed that the expression patterns of the features in some miRNA-TF-mRNA

  9. Tolerance and responsive gene expression of Sogatella furcifera under extreme temperature stresses are altered by its vectored plant virus.

    PubMed

    Xu, Donglin; Zhong, Ting; Feng, Wendi; Zhou, Guohui

    2016-01-01

    Southern rice black-streaked dwarf virus (SRBSDV), a newly emerged fijivirus causing great loss to rice production in eastern and southeastern Asian countries in recent years, is efficiently transmitted by a rice pest, white-backed planthopper (WBPH, Sogatella furcifera) in a persistent, circulative propagative manner and can be considered as an insect virus. In this study, SRBSDV infection in WBPH was found to increase the vector's death rate under extreme cold stress but improve its survival rate under extreme heat stress. Digital gene expression profiling based on RNA-Seq revealed different gene regulation patterns in WBPH under viral and/or temperature stress. Under cold stress, the virus infection upregulated 1540 genes and downregulated 131 genes in the insect, most of which were related to membrane properties and biological processes of actin and cytoskeleton; whereas under heat stress, it upregulated 363 genes and downregulated 548 genes, most of which were associated to metabolism and intracellular organelles. Several types of stress-responsive genes involving intestinal mucin, cuticle protein, ubiquitin protease, immune response, RNA interference and heat shock response, were largely upregulated under cold stress, but largely downregulated under heat stress, by SRBSDV infection. Our results suggest two distinct mechanisms of virus-altered vector insect tolerance to temperature stress. PMID:27531640

  10. Rabies Virus Vector Transgene Expression Level and Cytotoxicity Improvement Induced by Deletion of Glycoprotein Gene

    PubMed Central

    Ohara, Shinya; Sato, Sho; Oyama, Kei; Tsutsui, Ken-Ichiro; Iijima, Toshio

    2013-01-01

    The glycoprotein (G) of rabies virus (RV) is required for binding to neuronal receptors and for viral entry. G-deleted RV vector is a powerful tool for investigating the organization and function of the neural circuits. It gives the investigator the ability to genetically target initial infection to particular neurons and to control trans-synaptic propagation. In this study we have quantitatively evaluated the effect of G gene deletion on the cytotoxicity and transgene expression level of the RV vector. We compared the characteristics of the propagation-competent RV vector (rHEP5.0-CVSG-mRFP) and the G-deleted RV vector (rHEP5.0-ΔG-mRFP), both of which are based on the attenuated HEP-Flury strain and express monomeric red fluorescent protein (mRFP) as a transgene. rHEP5.0-ΔG-mRFP showed lower cytotoxicity than rHEP5.0-CVSG-mRFP, and within 16 days of infection we found no change in the basic electrophysiological properties of neurons infected with the rHEP5.0-ΔG-mRFP. The mRFP expression level of rHEP5.0-ΔG-mRFP was much higher than that of rHEP5.0-CVSG-mRFP, and 3 days after infection the retrogradely infected neurons were clearly visualized by the expressed fluorescent protein without any staining. This may be due to the low cytotoxicity and/or the presumed change in the polymerase gene (L) expression level of the G-deleted RV vector. Although the mechanisms remains to be clarified, the results of this study indicate that deletion of the G gene greatly improves the usability of the RV vector for studying the organization and function of the neural circuits by decreasing the cytotoxicity and increasing the transgene expression level. PMID:24244660

  11. Biochemical properties of full-length hepatitis C virus RNA-dependent RNA polymerase expressed in insect cells.

    PubMed

    Choi, Han-Byul; Kim, Yeon-Gu; Oh, Jong-Won

    2003-12-31

    The hepatitis C virus (HCV) RNA-dependent RNA polymerase, NS5B protein, is the key viral enzyme responsible for replication of the HCV viral RNA genome. Although several full-length and truncated forms of the HCV NS5B proteins have been expressed previously in insect cells, contamination of host terminal transferase (TNTase) has hampered analysis of the RNA synthesis initiation mechanism using natural HCV RNA templates. We have expressed the HCV NS5B protein in insect cells using a recombinant baculovirus and purified it to near homogeneity without contaminated TNTase. The highly purified recombinant HCV NS5B was capable of copying 9.6-kb full-length HCV RNA template, and mini-HCV RNA carrying both 5'- and 3'-untranslated regions (UTRs) of the HCV genome. In the absence of a primer, and other cellular and viral factors, the NS5B could elongate over HCV RNA templates, but the synthesized products were primarily in the double stranded form, indicating that no cyclic replication occurred with NS5B alone. RNA synthesis using RNA templates representing the 3'-end region of HCV minus-strand RNA and the X-RNA at the 3'-end of HCV RNA genome was also initiated de novo. No formation of dimer-size self-primed RNA products resulting from extension of the 3'-end hydroxyl group was observed. Despite the internal de novo initiation from the X-RNA, the NS5B could not initiate RNA synthesis from the internal region of oligouridylic acid (U)(20), suggesting that HCV RNA polymerase initiates RNA synthesis from the selected region in the 3'-UTR of HCV genome.

  12. LncRNA ontology: inferring lncRNA functions based on chromatin states and expression patterns

    PubMed Central

    Li, Yongsheng; Chen, Hong; Pan, Tao; Jiang, Chunjie; Zhao, Zheng; Wang, Zishan; Zhang, Jinwen; Xu, Juan; Li, Xia

    2015-01-01

    Accumulating evidences suggest that long non-coding RNAs (lncRNAs) perform important functions. Genome-wide chromatin-states area rich source of information about cellular state, yielding insights beyond what is typically obtained by transcriptome profiling. We propose an integrative method for genome-wide functional predictions of lncRNAs by combining chromatin states data with gene expression patterns. We first validated the method using protein-coding genes with known function annotations. Our validation results indicated that our integrative method performs better than co-expression analysis, and is accurate across different conditions. Next, by applying the integrative model genome-wide, we predicted the probable functions for more than 97% of human lncRNAs. The putative functions inferred by our method match with previously annotated by the targets of lncRNAs. Moreover, the linkage from the cellular processes influenced by cancer-associated lncRNAs to the cancer hallmarks provided a “lncRNA point-of-view” on tumor biology. Our approach provides a functional annotation of the lncRNAs, which we developed into a web-based application, LncRNA Ontology, to provide visualization, analysis, and downloading of lncRNA putative functions. PMID:26485761

  13. Engineered Expression Vectors Significantly Enhanced the Production of 2-Keto-D-gluconic Acid by Gluconobacter oxidans.

    PubMed

    Shi, Yuan-yuan; Li, Ke-fei; Lin, Jin-ping; Yang, Sheng-li; Wei, Dong-zhi

    2015-06-10

    2-Keto-D-gluconic acid (2KGA), a precursor of the important food antioxidant erythorbic acid, can be produced by Gluconobacter oxidans. To genetically engineer G. oxidans for improved 2KGA production, six new expression vectors with increased copy numbers based on pBBR1MCS-5 were constructed via rational mutagenesis. The utility of the mutant vectors was demonstrated by the increased ga2dh mRNA abundance, enzyme activity, and 2KGA production when the ga2dh gene was overexpressed using these vectors. Among the obtained constructs, G. oxidans/pBBR-3510-ga2dh displayed the highest oxidative activity toward gluconic acid (GA). In a batch biotransformation process, the G. oxidans/pBBR-3510-ga2dh strain exhibited 2KGA productivity (0.63 g/g CWW/h) higher than that obtained using strain G. oxidans/pBBR-ga2dh (0.40 g/g CWW/h). When sufficient oxygen was supplied during the biotransformation, up to 480 g/L GA was exhausted in 45 h by the G. oxidans/pBBR-3510-ga2dh strain and approximately 486 g/L 2KGA was produced, generating the productivity of 0.54 g/g CWW/h.

  14. Graph based fusion of miRNA and mRNA expression data improves clinical outcome prediction in prostate cancer

    PubMed Central

    2011-01-01

    Background One of the main goals in cancer studies including high-throughput microRNA (miRNA) and mRNA data is to find and assess prognostic signatures capable of predicting clinical outcome. Both mRNA and miRNA expression changes in cancer diseases are described to reflect clinical characteristics like staging and prognosis. Furthermore, miRNA abundance can directly affect target transcripts and translation in tumor cells. Prediction models are trained to identify either mRNA or miRNA signatures for patient stratification. With the increasing number of microarray studies collecting mRNA and miRNA from the same patient cohort there is a need for statistical methods to integrate or fuse both kinds of data into one prediction model in order to find a combined signature that improves the prediction. Results Here, we propose a new method to fuse miRNA and mRNA data into one prediction model. Since miRNAs are known regulators of mRNAs we used the correlations between them as well as the target prediction information to build a bipartite graph representing the relations between miRNAs and mRNAs. This graph was used to guide the feature selection in order to improve the prediction. The method is illustrated on a prostate cancer data set comprising 98 patient samples with miRNA and mRNA expression data. The biochemical relapse was used as clinical endpoint. It could be shown that the bipartite graph in combination with both data sets could improve prediction performance as well as the stability of the feature selection. Conclusions Fusion of mRNA and miRNA expression data into one prediction model improves clinical outcome prediction in terms of prediction error and stable feature selection. The R source code of the proposed method is available in the supplement. PMID:22188670

  15. Construction of baculovirus expression vector of miRNAs and its expression in insect cells.

    PubMed

    Huang, Yong; Zou, Quan; Shen, Xing Jia; Yu, Xue Li; Wang, Zhan Bin; Cheng, Xiang Chao

    2012-01-01

    MicroRNAs (miRNAs) are endogenous small non-protein coding RNAs that play important regulatory roles in animals and plants by binding to target transcripts for cleavage or translational repression. The miR-9a is very conservative in animals from flies to humans. Studies indicated that miR-9a is involved in the regulation of neurogenesis in animals. In our study, the baculovirus expression system was used to transcribe a recombinant vector containing miR-9a for further analysis the function ofmiR-9a. The sequence ofpre-miR-9a from silkworm DNA was first cloned into the donor pFastBac. The enhanced green fluorescent protein (EGFP) was used as reporter gene. The recombinant donor plasmid pFastBac-miR-9a was transformed into E.coli DH10Bac/AcNPV forming Bacmid-9a which was transfected into insect cells with cational lipofectin. The transcription of mature miR-9a was detected by Real-time PCR. The results show the recombinant Bacmid-9a was successfully constructed and effectively transcribed miR-9a in infected Sf21 insect cells. PMID:22937569

  16. Deregulated messenger RNA expression during T cell apoptosis.

    PubMed Central

    Kerkhoff, E; Ziff, E B

    1995-01-01

    The IL-2 dependent murine cytotoxic T cell line CTLL-2 undergoes programmed cell death when deprived of its specific cytokine. We analyzed the expression of cell cycle related genes after IL-2 deprivation. Here we show that a generalized decrease and re elevation of the levels of mRNA takes place as part of the apoptotic program. The levels of several mRNAs encoding cell cycle functions, including cyclin D2, cyclin D3, cyclin B1, c-myc and max all declined at 1.5-3 h following IL-2 deprivation. Notably, the maxmRNA, which was shown to be expressed in proliferating, growth arrested and differentiated cells, is down regulated with the same kinetics as the other mRNAs. Surprisingly, the mRNAs whose levels declined at 1.5-3 h rose again at 10-14 h, a time which closely followed the time of the first detection of apoptotic DNA degradation, at 8 h, but which precedes actual loss of viability, at 14 h, as measured by trypan blue exclusion. Of all analyzed genes only the expression of the S-phase specific histone H4 gene resists the initial decrease and declines gradually over the course of cell death. Measurement of c-Myc protein synthesis at a late stage of the apoptotic program revealed that the accumulated reinduced mRNA is not translated into protein. Because transcriptional regulation has been shown to be dependent on the chromatin structure, the reinduction may be triggered by relaxation of the chromatin caused by alterations in the chromatin structure of apoptotic cells. Images PMID:8532529

  17. BayMiR: inferring evidence for endogenous miRNA-induced gene repression from mRNA expression profiles

    PubMed Central

    2013-01-01

    Background Popular miRNA target prediction techniques use sequence features to determine the functional miRNA target sites. These techniques commonly ignore the cellular conditions in which miRNAs interact with their targets in vivo. Gene expression data are rich resources that can complement sequence features to take into account the context dependency of miRNAs. Results We introduce BayMiR, a new computational method, that predicts the functionality of potential miRNA target sites using the activity level of the miRNAs inferred from genome-wide mRNA expression profiles. We also found that mRNA expression variation can be used as another predictor of functional miRNA targets. We benchmarked BayMiR, the expression variation, Cometa, and the TargetScan “context scores” on two tasks: predicting independently validated miRNA targets and predicting the decrease in mRNA abundance in miRNA overexpression assays. BayMiR performed better than all other methods in both benchmarks and, surprisingly, the variation index performed better than Cometa and some individual determinants of the TargetScan context scores. Furthermore, BayMiR predicted miRNA target sets are more consistently annotated with GO and KEGG terms than similar sized random subsets of genes with conserved miRNA seed regions. BayMiR gives higher scores to target sites residing near the poly(A) tail which strongly favors mRNA degradation using poly(A) shortening. Our work also suggests that modeling multiplicative interactions among miRNAs is important to predict endogenous mRNA targets. Conclusions We develop a new computational method for predicting the target mRNAs of miRNAs. BayMiR applies a large number of mRNA expression profiles and successfully identifies the mRNA targets and miRNA activities without using miRNA expression data. The BayMiR package is publicly available and can be readily applied to any mRNA expression data sets. PMID:24001276

  18. Successful transgene expression with serial doses of aerosolized rAAV2 vectors in rhesus macaques.

    PubMed

    Fischer, Anne C; Beck, Suzanne E; Smith, Carolina I; Laube, Beth L; Askin, Frederic B; Guggino, Sandra E; Adams, Robert J; Flotte, Terence R; Guggino, William B

    2003-12-01

    Bronchoscopic microspraying of recombinant adeno-associated viral (rAAV) vectors targets high doses of vector directly to pulmonary epithelium. Single-dose endobronchial gene therapy trials have been accomplished in cystic fibrosis patients; however, repeated dosing strategies are likely essential for lifetime correction. These studies address whether serial redosing with rAAV2 vectors results in an antiserotypic response and, furthermore, whether it triggers an inflammatory response prohibitive to transgene expression. Serial redosing of 9 x 10(11) infectious units of aerosolized rAAV2 vectors to rhesus macaques resulted in successful gene transfer by quantitative PCR (1.43 x 10(9) copies/g tissue) and transgene expression. Additionally, confocal microscopy and immunohistochemical analysis demonstrated in situ expression localized to the pulmonary epithelium. Although serial redosing did induce a heightened anti-neutralizing antibody response in sera, gene transfer prevailed with resultant expression. This study is the first to demonstrate successful gene transfer subsequent to repeated aerosolized doses of rAAV2 in immunocompetent nonhuman primates without associated inflammatory responses prohibitive to transgene expression. PMID:14664794

  19. Improved Production Efficiency of Virus-Like Particles by the Baculovirus Expression Vector System.

    PubMed

    López-Vidal, Javier; Gómez-Sebastián, Silvia; Bárcena, Juan; Nuñez, Maria del Carmen; Martínez-Alonso, Diego; Dudognon, Benoit; Guijarro, Eva; Escribano, José M

    2015-01-01

    Vaccines based on virus-like particles (VLPs) have proven effective in humans and animals. In this regard, the baculovirus expression vector system (BEVS) is one of the technologies of choice to generate such highly immunogenic vaccines. The extended use of these vaccines for human and animal populations is constrained because of high production costs, therefore a significant improvement in productivity is crucial to ensure their commercial viability. Here we describe the use of the previously described baculovirus expression cassette, called TB, to model the production of two VLP-forming vaccine antigens in insect cells. Capsid proteins from porcine circovirus type 2 (PCV2 Cap) and from the calicivirus that causes rabbit hemorrhagic disease (RHDV VP60) were expressed in insect cells using baculoviruses genetically engineered with the TB expression cassette. Productivity was compared to that obtained using standard counterpart vectors expressing the same proteins under the control of the polyhedrin promoter. Our results demonstrate that the use of the TB expression cassette increased the production yields of these vaccine antigens by around 300% with respect to the standard vectors. The recombinant proteins produced by TB-modified vectors were fully functional, forming VLPs identical in size and shape to those generated by the standard baculoviruses, as determined by electron microscopy analysis. The use of the TB expression cassette implies a simple modification of the baculovirus vectors that significantly improves the cost efficiency of VLP-based vaccine production, thereby facilitating the commercial viability and broad application of these vaccines for human and animal health. PMID:26458221

  20. Improved Production Efficiency of Virus-Like Particles by the Baculovirus Expression Vector System

    PubMed Central

    Bárcena, Juan; Nuñez, Maria del Carmen; Martínez-Alonso, Diego; Dudognon, Benoit; Guijarro, Eva; Escribano, José M.

    2015-01-01

    Vaccines based on virus-like particles (VLPs) have proven effective in humans and animals. In this regard, the baculovirus expression vector system (BEVS) is one of the technologies of choice to generate such highly immunogenic vaccines. The extended use of these vaccines for human and animal populations is constrained because of high production costs, therefore a significant improvement in productivity is crucial to ensure their commercial viability. Here we describe the use of the previously described baculovirus expression cassette, called TB, to model the production of two VLP-forming vaccine antigens in insect cells. Capsid proteins from porcine circovirus type 2 (PCV2 Cap) and from the calicivirus that causes rabbit hemorrhagic disease (RHDV VP60) were expressed in insect cells using baculoviruses genetically engineered with the TB expression cassette. Productivity was compared to that obtained using standard counterpart vectors expressing the same proteins under the control of the polyhedrin promoter. Our results demonstrate that the use of the TB expression cassette increased the production yields of these vaccine antigens by around 300% with respect to the standard vectors. The recombinant proteins produced by TB-modified vectors were fully functional, forming VLPs identical in size and shape to those generated by the standard baculoviruses, as determined by electron microscopy analysis. The use of the TB expression cassette implies a simple modification of the baculovirus vectors that significantly improves the cost efficiency of VLP-based vaccine production, thereby facilitating the commercial viability and broad application of these vaccines for human and animal health. PMID:26458221

  1. Lentiviral Vectors Mediate Long-Term and High Efficiency Transgene Expression in HEK 293T cells

    PubMed Central

    Mao, Yingying; Yan, Renhe; Li, Andrew; Zhang, Yanling; Li, Jinlong; Du, Hongyan; Chen, Baihong; Wei, Wenjin; Zhang, Yi; Sumners, Colin; Zheng, Haifa; Li, Hongwei

    2015-01-01

    Objectives:Lentiviral vectors have been used successfully to rapidly produce decigram quantities of active recombinant proteins in mammalian cell lines. To optimize the protein production platform, the roles of Ubiquitous Chromatin Opening Element (UCOE), an insulator, and selected promoters were evaluated based on efficiency and stability of foreign gene expression mediated by lentiviral vectors. Methods: Five lentiviral vectors, pFIN-EF1α-GFP-2A-mCherH-WPRE containing EF1α promoter and HS4 insulator, p'HR.cppt.3'1.2kb-UCOE-SFFV-eGFP containing SFFV promoter and UCOE, pTYF-CMV(β-globin intron)-eGFP containing CMV promoter and β-globin intron, pTYF-CMV-eGFP containing CMV promoter, and pTYF-EF1α-eGFP with EF1α promoter were packaged, titered, and then transduced into 293T cells (1000 viral genomes per cell). The transduced cells were passaged once every three days at a ratio of 1:10. Expression level and stability of the foreign gene, green fluorescence protein (GFP), was evaluated using fluorescent microscopy and flow cytometry. Furthermore, we constructed a hepatitis C virus (HCV) E1 recombinant lentiviral vector, pLV-CMV-E1, driven by the CMV promoter. This vector was packaged and transduced into 293T cells, and the recombinant cell lines with stable expression of E1 protein were established by limiting dilution. Results:GFP expression in 293T cells transduced with the five lentiviral vectors peaked between passages 3 and 5 and persisted for more than 5 weeks. The expression was prolonged in the cells transduced with TYF-CMV (β-globin intron)-eGFP or TYF-CMV-eGFP, demonstrating less than a 50% decrease even at 9 weeks post transduction (p>0.05). The TYF-CMV-eGFP-transduced cells began with a higher level of GFP expression than other vectors did. The percentage of GFP positive cells for any of the five lentiviral vectors sustained over time. Moreover, the survival rates of all transfected cells exceeded 80% at both 5 and 9 weeks post transduction

  2. AAV Vectors Expressing LDLR Gain-of-Function Variants Demonstrate Increased Efficacy in Mouse Models of Familial Hypercholesterolemia

    PubMed Central

    Somanathan, Suryanarayan; Jacobs, Frank; Wang, Qiang; Hanlon, Alexandra L; Wilson, James M; Rader, Daniel J

    2014-01-01

    Rationale Familial hypercholesterolemia (FH) is a genetic disorder that arises due to loss-of-function mutations in the low-density lipoprotein receptor (LDLR) and homozygous FH (hoFH) is a candidate for gene therapy using adeno-associated viral (AAV) vectors. Proprotein convertase subtilisin/kexin type 9 (PCSK9) and inducible degrader of LDLR (IDOL) negatively regulate LDLR protein and could dampen AAV encoded LDLR expression. Objective We sought to create vectors expressing gain-of-function human LDLR variants that are resistant to degradation by human PCSK9 and IDOL and thereby enhance hepatic LDLR protein abundance and plasma LDL cholesterol reduction. Methods and Results Amino acid substitutions were introduced into the coding sequence of human LDLR cDNA to reduce interaction with hPCSK9 and hIDOL. A panel of mutant hLDLRs was initially screened in vitro for escape from PCSK9. The variant hLDLR-L318D was further evaluated using a mouse model of hoFH lacking endogenous LDLR and apolipoprotein B mRNA editing enzyme, APOBEC-1 (DKO). Administration of wild type hLDLR to DKO mice, expressing hPCSK9, led to diminished LDLR activity. However, LDLR-L318D was resistant to hPCSK9 mediated degradation and effectively reduced cholesterol levels. Similarly, the LDLR-K809R\\C818A construct avoided hIDOL regulation and achieved stable reductions in serum cholesterol. An AAV8.LDLR-L318D\\K809R\\C818A vector that carried all three amino acid substitutions conferred partial resistance to both hPCSK9 and hIDOL mediated degradation. Conclusion Amino acid substitutions in the human LDLR confer partial resistance to PCSK9 and IDOL regulatory pathways with improved reduction in cholesterol levels and improve upon a potential gene therapeutic approach to treat homozygous FH subjects. PMID:25023731

  3. Comprehensive analysis of human small RNA sequencing data provides insights into expression profiles and miRNA editing

    PubMed Central

    Gong, Jing; Wu, Yuliang; Zhang, Xiantong; Liao, Yifang; Sibanda, Vusumuzi Leroy; Liu, Wei; Guo, An-Yuan

    2014-01-01

    MicroRNAs (miRNAs) play key regulatory roles in various biological processes and diseases. A comprehensive analysis of large scale small RNA sequencing data (smRNA-seq) will be very helpful to explore tissue or disease specific miRNA markers and uncover miRNA variants. Here, we systematically analyzed 410 human smRNA-seq datasets, which samples are from 24 tissue/disease/cell lines. We tested the mapping strategies and found that it was necessary to make multiple-round mappings with different mismatch parameters. miRNA expression profiles revealed that on average ∼70% of known miRNAs were expressed at low level or not expressed (RPM < 1) in a sample and only ∼9% of known miRNAs were relatively highly expressed (RPM > 100). About 30% known miRNAs were not expressed in all of our used samples. The miRNA expression profiles were compiled into an online database (HMED, http://bioinfo.life.hust.edu.cn/smallRNA/). Dozens of tissue/disease specific miRNAs, disease/control dysregulated miRNAs and miRNAs with arm switching events were discovered. Further, we identified some highly confident editing sites including 24 A-to-I sites and 23 C-to-U sites. About half of them were widespread miRNA editing sites in different tissues. We characterized that the 2 types of editing sites have different features with regard to location, editing level and frequency. Our analyses for expression profiles, specific miRNA markers, arm switching, and editing sites, may provide valuable information for further studies of miRNA function and biomarker finding. PMID:25692236

  4. Sequence and expression of ferredoxin mRNA in barley

    SciTech Connect

    Zielinski, R.; Funder, P.M.; Ling, V. )

    1990-05-01

    We have isolated and structurally characterized a full-length cDNA clone encoding ferredoxin from a {lambda}gt10 cDNA library prepared from barley leaf mRNA. The ferredoxin clone (pBFD-1) was fused head-to-head with a partial-length cDNA clone encoding calmodulin, and was fortuitously isolated by screening the library with a calmodulin-specific oligonucleotide probe. The mRNA sequence from which pBFD-1 was derived is expressed exclusively in the leaf tissues of 7-d old barley seedlings. Barley pre-ferredoxin has a predicted size of 15.3 kDal, of which 4.6 kDal are accounted for by the transit peptide. The polypeptide encoded by pBFD-1 is identical to wheat ferredoxin, and shares slightly more amino acid sequence similarity with spinach ferredoxin I than with ferredoxin II. Ferredoxin mRNA levels are rapidly increased 10-fold by white light in etiolated barley leaves.

  5. Enhancing chemosensitivity in oral squamous cell carcinoma by lentivirus vector-mediated RNA interference targeting EGFR and MRP2

    PubMed Central

    Chen, Ying-Ju; Chen, Shiuan-Yin; Lovel, Ronald; Ku, Yi-Chu; Lai, Yi-Hui; Hung, Chiao-Ling; Li, Yu-Fen; Lu, Yin-Che; Tai, Chien-Kuo

    2016-01-01

    Oral cancer is the eighth most common type of cancer among men worldwide, with an age-standardized rate of 6.3 per 100,000, and is the fourth leading cause of cancer-associated mortality among men in Taiwan. Cisplatin and 5-fluorouracil (5-FU) are two of the most frequently utilized chemotherapy drugs for the treatment of oral cancer. Although oral cancer patients initially benefit from chemotherapy with these drugs, they may develop resistance to them, which worsens their prognosis and reduces survival rates. It has been reported that increased levels of epidermal growth factor receptor (EGFR) and multidrug resistance-associated protein 2 (MRP2) induce drug resistance in numerous types of human cancer. Therefore, the present study employed lentivirus vector-mediated RNA interference (RNAi) in order to target the genes encoding EGFR and MRP2 in the oral squamous cell carcinoma cell line OC2. It was observed that RNAi-mediated downregulation of EGFR or MRP2 increased the sensitivity to 5-FU and cisplatin in OC2 cells. Downregulation of EGFR resulted in significant suppression of OC2 tumor growth following 5-FU administration. However, simultaneous downregulation of the two genes did not further suppress the tumor growth, indicating that MRP2 does not have a significant role in the chemosensitivity of EGFR-downregulated cells to 5-FU. In contrast, downregulation of MRP2 was demonstrated to significantly enhance the therapeutic effects of cisplatin in EGFR-downregulated OC2 tumors. The observation that the expression of MRP2 was positively correlated with the level of cisplatin resistance in cells suggests that RNAi-mediated downregulation of MRP2 may be applicable as a therapeutic approach toward reversing MRP2-dependent cisplatin resistance in oral cancer. PMID:27602148

  6. Changes in apoptotic microRNA and mRNA expression profiling in Caenorhabditis elegans during the Shenzhou-8 mission

    PubMed Central

    Gao, Ying; Li, Shuai; Xu, Dan; Wang, Junjun; Sun, Yeqing

    2015-01-01

    Radiation and microgravity exposure have been proven to induce abnormal apoptosis in microRNA (miRNA) and mRNA expression, but whether space conditions, including radiation and microgravity, activate miRNAs to regulate the apoptosis is undetermined. For that purpose, we investigated miRNome and mRNA expression in the ced-1 Caenorhabditis elegans mutant vs the wild-type, both of which underwent spaceflight, spaceflight 1g-centrifuge control and ground control conditions during the Shenzhou-8 mission. Results showed that no morphological changes in the worms were detected, but differential miRNA expression increased from 43 (ground control condition) to 57 and 91 in spaceflight and spaceflight control conditions, respectively. Microgravity altered miRNA expression profiling by decreasing the number and significance of differentially expressed miRNA compared with 1 g incubation during spaceflight. Alterations in the miRNAs were involved in alterations in apoptosis, neurogenesis larval development, ATP metabolism and GTPase-mediated signal transduction. Among these, 17 altered miRNAs potentially involved in apoptosis were screened and showed obviously different expression signatures between space conditions. By integrated analysis of miRNA and mRNA, miR-797 and miR-81 may be involved in apoptosis by targeting the genes ced-10 and both drp-1 and hsp-1, respectively. Compared with ground condition, space conditions regulated apoptosis though a different manner on transcription, by altering expression of seven core apoptotic genes in spaceflight condition, and eight in spaceflight control condition. Results indicate that, miRNA of Caenorhabditis elegans probably regulates apoptotic gene expression in response to space environmental stress, and shows different behavior under microgravity condition compared with 1 g condition in the presence of space radiation. PMID:26286471

  7. Hepatic miRNA expression reprogrammed by Plasmodium chabaudi malaria.

    PubMed

    Delić, Denis; Dkhil, Mohamed; Al-Quraishy, Saleh; Wunderlich, Frank

    2011-05-01

    Evidence is accumulating that miRNAs are critically implicated in the outcome of diseases, but little information is available for infectious diseases. This study investigates the hepatic miRNA signature in female C57BL/6 mice infected with self-healing Plasmodium chabaudi malaria. Primary infections result in approximately 50% peak parasitemia on day 8 p.i., approximately 80% survival, and development of protective immunity. The latter is evidenced as 100% survival and 1.5% peak parasitemia upon homolog re-infections of those mice which are still alive on day 56 after primary infection. Such immune mice exhibit increased levels of IgG2a and IgG2b isotypes and still contain P. chabaudi-infected erythrocytes in their livers as revealed by light microscopy and PCR analysis. Primary infections, but not secondary infections, induce an upregulation of hepatic mRNAs encoding IL-1β, TNFα, IFNγ, NF-κB, and iNOS, and a downregulation of mRNAs for CYP7A1 and SULT2A2, respectively. Using miRXplore microarrays containing 634 mouse miRNAs in combination with quantitative RT-PCR, the liver is found to respond to primary infections with an upregulation of the three miRNA species miR-26b, MCMV-miR-M23-1-5p, and miR-1274a, and a downregulation of the 16 miRNA species miR-101b, let-7a, let-7g, miR-193a-3p, miR-192, miR-142-5p, miR-465d, miR-677, miR-98, miR-694, miR-374(*), miR-450b-5p, miR-464, miR-377, miR-20a(*), and miR-466d-3p, respectively. Surprisingly, about the same pattern of miRNA expression is revealed in immune mice, and this pattern is even sustained upon homolog re-infections of immune mice. These data suggest that development of protective immunity against malarial blood stages of P. chabaudi is associated with a reprogramming of the expression of distinct miRNA species in the female mouse liver.

  8. Screening of mRNA Chemical Modification to Maximize Protein Expression with Reduced Immunogenicity

    PubMed Central

    Uchida, Satoshi; Kataoka, Kazunori; Itaka, Keiji

    2015-01-01

    Chemical modification of nucleosides in mRNA is an important technology to regulate the immunogenicity of mRNA. In this study, various previously reported mRNA formulations were evaluated by analyzing in vitro protein expression and immunogenicity in multiple cell lines. For the macrophage-derived cell line, RAW 264.7, modified mRNA tended to have reduced immunogenicity and increased protein expression compared to the unmodified mRNA. In contrast, in some cell types, such as hepatocellular carcinoma cells (HuH-7) and mouse embryonic fibroblasts (MEFs), protein expression was decreased by mRNA modification. Further analyses revealed that mRNA modifications decreased translation efficiency but increased nuclease stability. Thus, mRNA modification is likely to exert both positive and negative effects on the efficiency of protein expression in transfected cells and optimal mRNA formulation should be determined based on target cell types and transfection purposes. PMID:26213960

  9. Screening of mRNA Chemical Modification to Maximize Protein Expression with Reduced Immunogenicity.

    PubMed

    Uchida, Satoshi; Kataoka, Kazunori; Itaka, Keiji

    2015-01-01

    Chemical modification of nucleosides in mRNA is an important technology to regulate the immunogenicity of mRNA. In this study, various previously reported mRNA formulations were evaluated by analyzing in vitro protein expression and immunogenicity in multiple cell lines. For the macrophage-derived cell line, RAW 264.7, modified mRNA tended to have reduced immunogenicity and increased protein expression compared to the unmodified mRNA. In contrast, in some cell types, such as hepatocellular carcinoma cells (HuH-7) and mouse embryonic fibroblasts (MEFs), protein expression was decreased by mRNA modification. Further analyses revealed that mRNA modifications decreased translation efficiency but increased nuclease stability. Thus, mRNA modification is likely to exert both positive and negative effects on the efficiency of protein expression in transfected cells and optimal mRNA formulation should be determined based on target cell types and transfection purposes. PMID:26213960

  10. Distribution of miRNA expression across human tissues.

    PubMed

    Ludwig, Nicole; Leidinger, Petra; Becker, Kurt; Backes, Christina; Fehlmann, Tobias; Pallasch, Christian; Rheinheimer, Steffi; Meder, Benjamin; Stähler, Cord; Meese, Eckart; Keller, Andreas

    2016-05-01

    We present a human miRNA tissue atlas by determining the abundance of 1997 miRNAs in 61 tissue biopsies of different organs from two individuals collected post-mortem. One thousand three hundred sixty-four miRNAs were discovered in at least one tissue, 143 were present in each tissue. To define the distribution of miRNAs, we utilized a tissue specificity index (TSI). The majority of miRNAs (82.9%) fell in a middle TSI range i.e. were neither specific for single tissues (TSI > 0.85) nor housekeeping miRNAs (TSI < 0.5). Nonetheless, we observed many different miRNAs and miRNA families that were predominantly expressed in certain tissues. Clustering of miRNA abundances revealed that tissues like several areas of the brain clustered together. Considering -3p and -5p mature forms we observed miR-150 with different tissue specificity. Analysis of additional lung and prostate biopsies indicated that inter-organism variability was significantly lower than inter-organ variability. Tissue-specific differences between the miRNA patterns appeared not to be significantly altered by storage as shown for heart and lung tissue. MiRNAs TSI values of human tissues were significantly (P = 10(-8)) correlated with those of rats; miRNAs that were highly abundant in certain human tissues were likewise abundant in according rat tissues. We implemented a web-based repository enabling scientists to access and browse the data (https://ccb-web.cs.uni-saarland.de/tissueatlas).

  11. Preproglucagon mRNA expression in adult rat submandibular glands.

    PubMed

    Egéa, J C; Hirtz, C; Deville de Périère, D

    2003-04-01

    Salivary glands of various animal species have been reported to contain and suggested to produce glucagon or glucagon-like material, but the origin and the nature of this salivary peptide are still doubtful. The present study was undertaken to ascertain whether the glucagon gene is expressed in rat submandibular glands and in an immortalized murine cell line derived from salivary glands (SCA-9 cell line). For this purpose, total RNA was isolated from submandibular glands or cultured cells and submitted to reverse transcription. The cDNAs obtained were amplified by a nested polymerase chain reaction using preproglucagon primers. The results showed that the preproglucagon mRNA was expressed in adult rat submandibular glands but not in the SCA-9 cell line. Determination of cyclic DNA (cDNA) sequence established identity with the coding regions of rat pancreatic pre-proglucagon gene. In conclusion, these results strongly support the idea that rat submandibular glands could represent a source of extrapancreatic glucagon or of its precursor's peptide.

  12. Targeting alpha-synuclein with a microRNA-embedded silencing vector in the rat substantia nigra: positive and negative effects

    PubMed Central

    Khodr, Christina E.; Becerra, Amanda; Han, Ye; Bohn, Martha C.

    2014-01-01

    Background Alpha-synuclein (SNCA) downregulation shows therapeutic potential for synucleinopathies, including Parkinson’s disease (PD). Previously we showed that human (h)SNCA gene silencing using a short hairpin (sh)RNA in rat substantia nigra (SN) protects against a hSNCA-induced forelimb deficit, but not dopamine (DA) neuron loss. Further, the mir-embedded hSNCA gene silencing shRNA increases cell death in vitro, but the same target sequence embedded in a microRNA30 transcript (mir30-hSNCA) does not. Objective Examine hSNCA gene silencing using mir30-hSNCA in vivo. Methods Rats were stereotaxically injected into one SN with adeno-associated virus serotype 2/8 (AAV)-hSNCA, AAV-hSNCA plus AAV-mir30-SNCA or AAV-hSNCA plus a control non-silencing mir30-embedded siRNA and DA neuron markers and associated behavior were examined. Results AAV2/8-mediated SN hSNCA expression induces a forelimb deficit and tyrosine hydroxylase-immunoreactive (TH-IR) neuron loss. hSNCA gene silencing using mir30-hSNCA protects against this forelimb deficit at 2m and ameliorates TH-IR neuron loss. Striatal (ST) TH-IR fiber density and DA markers, assessed by western blot, are unaffected by AAV-hSNCA alone. Co-expression of either silencing vector reduces ST TH-IR fibers, panTH in SN and Ser40 phosphorylated TH in SN and ST, but does not affect vesicular monoamine transporter-2. However, hSNCA gene silencing promotes partial TH-IR fiber recovery by 2m. Co-expression of either silencing vector also induces SN inflammation, although some recovery was observed by 2m in hSNCA-silenced SN. Conclusion hSNCA gene silencing with AAV-mir30-hSNCA has positive effects on forelimb behavior and SN DA neurons, which are compromised by inflammation and reduced TH expression, suggesting that AAV2/8-mir30-hSNCA-mediated gene silencing, although promising in vitro, is not a candidate for therapeutic translation for PD. PMID:24463035

  13. Development of an adenoviral vector with robust expression driven by p53

    SciTech Connect

    Bajgelman, Marcio C.; Strauss, Bryan E.

    2008-02-05

    Here we introduce a new adenoviral vector where transgene expression is driven by p53. We first developed a synthetic promoter, referred to as PGTx{beta}, containing a p53-responsive element, a minimal promoter and the first intron of the rabbit {beta}-globin gene. Initial assays using plasmid-based vectors indicated that expression was tightly controlled by p53 and was 5-fold stronger than the constitutive CMV immediate early promoter/enhancer. The adenoviral vector, AdPG, was also shown to offer p53-responsive expression in prostate carcinoma cells LNCaP (wt p53), DU-145 (temperature sensitive mutant of p53) and PC3 (p53-null, but engineered to express temperature-sensitive p53 mutants). AdPG served as a sensor of p53 activity in LNCaP cells treated with chemotherapeutic agents. Since p53 can be induced by radiotherapy and chemotherapy, this new vector could be further developed for use in combination with conventional therapies to bring about cooperation between the genetic and pharmacologic treatment modalities.

  14. Gene expression in midgut tissues of Diaphorina citri: Application to biology and vector control

    Technology Transfer Automated Retrieval System (TEKTRAN)

    We produced a gene expression dataset from the midgut tissues of the Asian citrus psyllid (AsCP), Diaphorina citri (Hemiptera: Psyllidae). The AsCP is the primary vector associated with the spread of a devastating citrus trees disease, huanglongbing (HLB). The occurrence and spread of the AsCP and H...

  15. A high-throughput protein expression system in Pichia pastoris using a newly developed episomal vector

    Technology Transfer Automated Retrieval System (TEKTRAN)

    We describe here the construction of a Gateway-compatible vector, pBGP1-DEST, for rapid and convenient preparation of expression plasmids for production of secretory proteins in Pichia pastoris. pBGP1-DEST directs the synthesis of a fusion protein consisting of the N-terminal signal and pro-sequence...

  16. Subcellular distribution of small interfering RNA: directed delivery through RNA polymerase III expression cassettes and localization by in situ hybridization.

    PubMed

    Paul, Cynthia P

    2005-01-01

    Reduction in the expression of specific genes through small interfering RNAs (siRNAs) is dependent on the colocalization of siRNAs with other components of the RNA interference (RNAi) pathways within the cell. The expression of siRNAs within cells from cassettes that are derived from genes transcribed by RNA polymerase III (pol III) and provide for selective subcellular distribution of their products can be used to direct siRNAs to the cellular pathways. Expression from the human U6 promoter, resulting in siRNA accumulation in the nucleus, is effective in reducing gene expression, whereas cytoplasmic and nucleolar localization of the siRNA when expressed from the 5S or 7 SL promoters is not effective. The distribution of siRNA within the cell is determined by fluorescence in situ hybridization. Although the long uninterrupted duplex of siRNA makes it difficult to detect with DNA oligonucleotide probes, labeled oligonucleotide probes with 2'-O-methyl RNA backbones provide the stability needed for a strong signal. These methods contribute to studies of the interconnected cellular RNAi pathways and are useful in adapting RNAi as a tool to determine gene function and develop RNA-based therapeutics. PMID:15644179

  17. Lentiviral vectors with CMV or MHCII promoters administered in vivo: immune reactivity versus persistence of expression.

    PubMed

    Kimura, Takahiro; Koya, Richard C; Anselmi, Laura; Sternini, Catia; Wang, He-Jing; Comin-Anduix, Begonya; Prins, Robert M; Faure-Kumar, Emmanuelle; Rozengurt, Nora; Cui, Yan; Kasahara, Noriyuki; Stripecke, Renata

    2007-07-01

    Lentiviral vectors (LVs) are potential tools for genetic vaccination. To improve the safety of LV vaccines, we evaluated the selectivity, bio-distribution, persistence of expression, and immune potency of vesicular stomatitis virus G (VSV-G)-pseudotyped vectors transcriptionally targeted to antigen presenting cells (APCs) through a major histocompatibility complex class II (MHCII) promoter. Control vectors contained the ubiquitous cytomegalovirus (CMV) promoter. Bio-distribution studies after intravenous injections of LVs expressing green fluorescent protein (GFP) or luciferase were conducted by a combination of flow cytometry, immunofluorescence, real-time quantitative polymerase chain reaction (RT-Q-PCR) and whole-body bioluminescence analyses. GFP-expressing vectors showed selective expression in MHCII(+) cells of spleen and LV-CMV-GFP administration produced noticeable spleen inflammation, whereas LV-MHCII-GFP did not. Long-term optical imaging analyses of C57BL/6 mice injected with LV-CMV-LUC showed diminishing luciferase expression in the liver and spleen over time. Vaccination/boost with LV-CMV expressing the melanoma antigen tyrosinase-related protein 2 (TRP2) yielded dose-dependent antigen-specific CD8(+) T-cell reactivity and high protection against B16 melanoma challenge. Unexpectedly, administration of LVs containing the MHCII promoter resulted in persistence of luciferase expression and viral integration in MHCII(+) splenocytes and virtually no CD8(+) T-cell responses against TRP2. These studies reveal that APC transduction by LVs could lead to immune reactivity (LV-CMV) or persistence of transgene expression (LV-MHCII), providing a relevant paradigm for vaccination and gene replacement approaches. PMID:17505480

  18. Expression of the glycoprotein gene from a fish rhabdovirus by using baculovirus vectors

    SciTech Connect

    Koener, J.F.; Leong, J.A.C. )

    1990-01-01

    A cDNA fragment containing the gene encoding the glycoprotein of infectious hematopoietic necrosis virus was inserted into Autographa californica baculovirus vectors under the control of the polyhedrin promoter. A 66-kilodalton protein, identical in size to the glycosylated glycoprotein of infectious hematopoietic necrosis virus, was expressed at high levels in Spodoptera frugiperda cells infected with the recombinant viruses. The expressed protein reacted with antiserum to the glycoprotein on Western blots.

  19. Novel methods for expression of foreign antigens in live vector vaccines

    PubMed Central

    Wang, Jin Yuan; Harley, Regina H.; Galen, James E.

    2013-01-01

    Bacterial live vector vaccines represent a vaccine development strategy that offers exceptional flexibility. In this approach, genes encoding protective antigens of unrelated bacterial, viral or parasitic pathogens are expressed in an attenuated bacterial vaccine strain that delivers these foreign antigens to the immune system, thereby eliciting relevant immune responses. Rather than expressing these antigens using low copy expression plasmids, here we pursue expression of foreign proteins from the live vector chromosome. Our strategy is designed to compensate for the inherent disadvantage of loss of gene dosage (vs. plasmid-based expression) by integrating antigen-encoding gene cassettes into multiple chromosomal sites already inactivated in an attenuated Salmonella enterica serovar Typhi vaccine candidate. We tested expression of a cassette encoding the green fluorescent protein (GFPuv) integrated separately into native guaBA, htrA or clyA chromosomal loci. Using single integrations, we show that expression levels of GFPuv are significantly affected by the site of integration, regardless of the inclusion of additional strong promoters within the incoming cassette. Using cassettes integrated into both guaBA and htrA, we observe cumulative synthesis levels from two integration sites superior to single integrations. Most importantly, we observe that GFPuv expression increases in a growth phase-dependent manner, suggesting that foreign antigen synthesis may be “tuned” to the physiology of the live vaccine. We expect this novel platform expression technology to prove invaluable in the development of a wide variety of multivalent live vector vaccines, capable of expressing multiple antigens from both chromosomal and plasmid-based expression systems within a single strain. PMID:23406777

  20. Gene structure and expression of nanos (nos) and oskar (osk) orthologues of the vector mosquito, Culex quinquefasciatus.

    PubMed

    Juhn, J; Marinotti, O; Calvo, E; James, A A

    2008-09-01

    The products of the maternal-effect genes, nanos (nos) and oskar (osk), are important for the development of germ cells in insects. Furthermore, these genes have been proposed as candidates for donating functional DNA regulatory sequences for use in gene drive systems to control transmission of mosquito-borne pathogens. The nos and osk genes of the cosmopolitan vector mosquito, Culex quinquefasciatus, encode proteins with domains common to orthologues found in other mosquitoes. Expression analyses support the conclusion that the role of these genes is conserved generally among members of the nematocera. Hybridization in situ analyses reveal differences in mRNA distribution in early embryos in comparison with the cyclorraphan, Drosophila melanogaster, highlighting a possible feature in the divergence of the clades each insect represents.

  1. MicroRNA Expression Profile in Penile Cancer Revealed by Next-Generation Small RNA Sequencing

    PubMed Central

    Zhang, Yuanwei; Xu, Bo; Zhou, Jun; Fan, Song; Hao, Zongyao; Shi, Haoqiang; Zhang, Xiansheng; Kong, Rui; Xu, Lingfan; Gao, Jingjing; Zou, Duohong; Liang, Chaozhao

    2015-01-01

    Penile cancer (PeCa) is a relatively rare tumor entity but possesses higher morbidity and mortality rates especially in developing countries. To date, the concrete pathogenic signaling pathways and core machineries involved in tumorigenesis and progression of PeCa remain to be elucidated. Several studies suggested miRNAs, which modulate gene expression at posttranscriptional level, were frequently mis-regulated and aberrantly expressed in human cancers. However, the miRNA profile in human PeCa has not been reported before. In this present study, the miRNA profile was obtained from 10 fresh penile cancerous tissues and matched adjacent non-cancerous tissues via next-generation sequencing. As a result, a total of 751 and 806 annotated miRNAs were identified in normal and cancerous penile tissues, respectively. Among which, 56 miRNAs with significantly different expression levels between paired tissues were identified. Subsequently, several annotated miRNAs were selected randomly and validated using quantitative real-time PCR. Compared with the previous publications regarding to the altered miRNAs expression in various cancers and especially genitourinary (prostate, bladder, kidney, testis) cancers, the most majority of deregulated miRNAs showed the similar expression pattern in penile cancer. Moreover, the bioinformatics analyses suggested that the putative target genes of differentially expressed miRNAs between cancerous and matched normal penile tissues were tightly associated with cell junction, proliferation, growth as well as genomic instability and so on, by modulating Wnt, MAPK, p53, PI3K-Akt, Notch and TGF-β signaling pathways, which were all well-established to participate in cancer initiation and progression. Our work presents a global view of the differentially expressed miRNAs and potentially regulatory networks of their target genes for clarifying the pathogenic transformation of normal penis to PeCa, which research resource also provides new insights

  2. Vector algebra in the analysis of genome-wide expression data

    PubMed Central

    Kuruvilla, Finny G; Park, Peter J; Schreiber, Stuart L

    2002-01-01

    Background Data from thousands of transcription-profiling experiments in organisms ranging from yeast to humans are now publicly available. How best to analyze these data remains an important challenge. A variety of tools have been used for this purpose, including hierarchical clustering, self-organizing maps and principal components analysis. In particular, concepts from vector algebra have proven useful in the study of genome-wide expression data. Results Here we present a framework based on vector algebra for the analysis of transcription profiles that is geometrically intuitive and computationally efficient. Concepts in vector algebra such as angles, magnitudes, subspaces, singular value decomposition, bases and projections have natural and powerful interpretations in the analysis of microarray data. Angles in particular offer a rigorous method of defining 'similarity' and are useful in evaluating the claims of a microarray-based study. We present a sample analysis of cells treated with rapamycin, an immunosuppressant whose effects have been extensively studied with microarrays. In addition, the algebraic concept of a basis for a space affords the opportunity to simplify data analysis and uncover a limited number of expression vectors to span the transcriptional range of cell behavior. Conclusions This framework represents a compact, powerful and scalable construction for analysis and computation. As the amount of microarray data in the public domain grows, these vector-based methods are relevant in determining statistical significance. These approaches are also well suited to extract biologically meaningful information in the analysis of signaling networks. PMID:11897023

  3. Brain Gene Expression Signatures From Cerebrospinal Fluid Exosome RNA Profiling

    NASA Technical Reports Server (NTRS)

    Zanello, S. B.; Stevens, B.; Calvillo, E.; Tang, R.; Gutierrez Flores, B.; Hu, L.; Skog, J.; Bershad, E.

    2016-01-01

    While the Visual Impairment and Intracranial Pressure (VIIP) syndrome observations have focused on ocular symptoms, spaceflight has been also associated with a number of other performance and neurologic signs, such as headaches, cognitive changes, vertigo, nausea, sleep/circadian disruption and mood alterations, which, albeit likely multifactorial, can also result from elevation of intracranial pressure (ICP). We therefore hypothesize that these various symptoms are caused by disturbances in the neurophysiology of the brain structures and are correlated with molecular markers in the cerebrospinal fluid (CSF) as indicators of neurophysiological changes. Exosomes are 30-200 nm microvesicles shed into all biofluids, including blood, urine, and CSF, carrying a highly rich source of intact protein and RNA cargo. Exosomes have been identified in human CSF, and their proteome and RNA pool is a potential new reservoir for biomarker discovery in neurological disorders. The purpose of this study is to investigate changes in brain gene expression via exosome analysis in patients suffering from ICP elevation of varied severity (idiopathic intracranial hypertension -IIH), a condition which shares some of the neuroophthalmological features of VIIP, as a first step toward obtaining evidence suggesting that cognitive function and ICP levels can be correlated with biomarkers in the CSF. Our preliminary work, reported last year, validated the exosomal technology applicable to CSF analysis and demonstrated that it was possible to obtain gene expression evidence of inflammation processes in traumatic brain injury patients. We are now recruiting patients with suspected IIH requiring lumbar puncture at Baylor College of Medicine. Both CSF (5 ml) and human plasma (10 ml) are being collected in order to compare the pattern of differentially expressed genes observed in CSF and in blood. Since blood is much more accessible than CSF, we would like to determine whether plasma biomarkers for

  4. [Construction of the oocyte-specific expressing phiC31 integrase vector pZP3-INT and its expression in mouse oocytes].

    PubMed

    Xu, Huan-Yu; Gong, Xiu-Li; Guo, Xin-Bing; Ma, Qing-Wen; Zeng, Yi-Tao

    2009-06-01

    Streptomyces phage phiC31 integrase is a site-specific recombinase, which can catalyze site-specific, unidirectional recombination between the attP site and attB site. To explore whether it can be used to mediate the recombination of specific gene in oocytes, GV-stage oocytes were collected from 3-week-old Kunming White mice by puncturing antral follocles with a sharp needle, and micro-injected with oocyte-specific expressing phiC31 integrase vector pZP3-INT and site -specific recombination detection vector pBCPB+. phiC31 integrase mRNA were detected by RT-PCR and the recombination of pBCPB+ was evaluated by PCR in mouse oocytes at 48 h after injection. Both can get corresponding bands. These results indicated that the expression of phiC31 integrase can be driven by ZP3 promoter efficiently and phiC31 integrase can mediate the site-specific recombination between attP site and attB site in mouse GV-stage oocytes. It could be a powerful tool for the study of recombination of specific gene in mouse oocytes and would provide an alternative way for the mouse oocyte genome manipulation.

  5. Regulation of Gene Expression in Plants through miRNA Inactivation

    PubMed Central

    Zhang, Yuanji; Ziegler, Todd E.; Roberts, James K.; Heck, Gregory R.

    2011-01-01

    Eukaryotic organisms possess a complex RNA-directed gene expression regulatory network allowing the production of unique gene expression patterns. A recent addition to the repertoire of RNA-based gene regulation is miRNA target decoys, endogenous RNA that can negatively regulate miRNA activity. miRNA decoys have been shown to be a valuable tool for understanding the function of several miRNA families in plants and invertebrates. Engineering and precise manipulation of an endogenous RNA regulatory network through modification of miRNA activity also affords a significant opportunity to achieve a desired outcome of enhanced plant development or response to environmental stresses. Here we report that expression of miRNA decoys as single or heteromeric non-cleavable microRNA (miRNA) sites embedded in either non-protein-coding or within the 3′ untranslated region of protein-coding transcripts can regulate the expression of one or more miRNA targets. By altering the sequence of the miRNA decoy sites, we were able to attenuate miRNA inactivation, which allowed for fine regulation of native miRNA targets and the production of a desirable range of plant phenotypes. Thus, our results demonstrate miRNA decoys are a flexible and robust tool, not only for studying miRNA function, but also for targeted engineering of gene expression in plants. Computational analysis of the Arabidopsis transcriptome revealed a number of potential miRNA decoys, suggesting that endogenous decoys may have an important role in natural modulation of expression in plants. PMID:21731706

  6. Use of Adeno-Associated and Herpes Simplex Viral Vectors for In Vivo Neuronal Expression in Mice

    PubMed Central

    Penrod, Rachel D.; Wells, Audrey M.; Carlezon, William A.; Cowan, Christopher W.

    2015-01-01

    Adeno-associated viruses and the herpes simplex virus are the two most widely used vectors for the in vivo expression of exogenous genes. Advances in the development of these vectors have enabled remarkable temporal and spatial control of gene expression. This unit provides methods for storing, delivering, and verifying expression of adeno-associated and herpes simplex viruses in the adult mouse brain. It also describes important considerations for experiments using in vivo expression of these viral vectors, including serotype and promoter selection, as well as timing of expression. Additional protocols are provided that describe methods for preliminary experiments to determine the appropriate conditions for in vivo delivery. PMID:26426386

  7. Use of Adeno-Associated and Herpes Simplex Viral Vectors for In Vivo Neuronal Expression in Mice.

    PubMed

    Penrod, Rachel D; Wells, Audrey M; Carlezon, William A; Cowan, Christopher W

    2015-01-01

    Adeno-associated viruses and the herpes simplex virus are the two most widely used vectors for the in vivo expression of exogenous genes. Advances in the development of these vectors have enabled remarkable temporal and spatial control of gene expression. This unit provides methods for storing, delivering, and verifying expression of adeno-associated and herpes simplex viruses in the adult mouse brain. It also describes important considerations for experiments using in vivo expression of these viral vectors, including serotype and promoter selection, as well as timing of expression. Additional protocols are provided that describe methods for preliminary experiments to determine the appropriate conditions for in vivo delivery.

  8. Integration of Expressed Sequence Tag Data Flanking Predicted RNA Secondary Structures Facilitates Novel Non-Coding RNA Discovery

    PubMed Central

    Krzyzanowski, Paul M.; Price, Feodor D.; Muro, Enrique M.; Rudnicki, Michael A.; Andrade-Navarro, Miguel A.

    2011-01-01

    Many computational methods have been used to predict novel non-coding RNAs (ncRNAs), but none, to our knowledge, have explicitly investigated the impact of integrating existing cDNA-based Expressed Sequence Tag (EST) data that flank structural RNA predictions. To determine whether flanking EST data can assist in microRNA (miRNA) prediction, we identified genomic sites encoding putative miRNAs by combining functional RNA predictions with flanking ESTs data in a model consistent with miRNAs undergoing cleavage during maturation. In both human and mouse genomes, we observed that the inclusion of flanking ESTs adjacent to and not overlapping predicted miRNAs significantly improved the performance of various methods of miRNA prediction, including direct high-throughput sequencing of small RNA libraries. We analyzed the expression of hundreds of miRNAs predicted to be expressed during myogenic differentiation using a customized microarray and identified several known and predicted myogenic miRNA hairpins. Our results indicate that integrating ESTs flanking structural RNA predictions improves the quality of cleaved miRNA predictions and suggest that this strategy can be used to predict other non-coding RNAs undergoing cleavage during maturation. PMID:21698286

  9. Cassette vectors directing expression of T cell receptor genes in transgenic mice.

    PubMed

    Kouskoff, V; Signorelli, K; Benoist, C; Mathis, D

    1995-03-27

    We describe a pair of cassette vectors that can be used to express rearranged T cell receptor genes in transgenic mice. Short DNA fragments containing rearranged V alpha and V beta segments are readily amplified from T cells and introduced between artificial cloning sites. Transgene-derived mRNAs are transcribed under the control of the natural TCR alpha and -beta promoter/enhancer elements. Using this vector, we have obtained transgenic mouse lines which display transgene-encoded TCR alpha and beta chains on a majority of T cells. PMID:7714342

  10. Synergistic effect of a tissue kallikrein 1 and tissue inhibitor of matrix metalloproteinase 1 co‑expression vector on the proliferation of rat vascular smooth muscle cells.

    PubMed

    Zhu, Pengli; Yu, Huizhen; Huang, Shujie; Xiang, Hong; Li, Feng; Zheng, Weiping

    2015-10-01

    Tissue kallikrein 1 (TK1) and tissue inhibitor of matrix metalloproteinase 1 (TIMP1) are important in inhibiting vascular smooth muscle cell (VSMC) proliferation and improving vascular remodeling, respectively. It was hypothesized that a combination of TK1 and TIMP1 genes, mediated by an adenovirus vector could augment or act in synergy to enhance the inhibitory effects. The promoter, mCMV carrying hTIMP1 cDNA was subcloned into pDC316‑hTK1 to construct a recombinant plasmid carrying hTK1 and hTIMP1 genes. Subsequently, the double gene plasmid and adenovirus backbone plasmid were packaged into HEK293A cells. Gene transcription and protein expression were examined, respectively using reverse transcription‑quantitative polymerase chain reaction (PCR) and western blotting assays. VSMC proliferation was assessed using cell counting and methyl‑thiazolyl‑tetrazoliuin methods. The constructed plasmid containing hTK1 and hTIMP1 genes was correctly identified by means of PCR, double digestion and sequencing analysis. The co‑expression vector, Ad‑hTK1‑hTIMP1 was successfully constructed and packaged into HEK293A cells. When VSMCs were transfected with the co‑expression vector, the mRNA transcription and protein expression of hTK1 and hTIMP1 exhibited abundant expression in a concentration‑dependent and time‑dependent manner, independently. In conclusion, the co‑expression vector synergistically inhibited the cell growth and proliferation induced by platelet‑derived growth factor‑BB compared with the single gene vector.

  11. Long Noncoding RNA Expression during Human B-Cell Development.

    PubMed

    Petri, Andreas; Dybkær, Karen; Bøgsted, Martin; Thrue, Charlotte Albæk; Hagedorn, Peter H; Schmitz, Alexander; Bødker, Julie Støve; Johnsen, Hans Erik; Kauppinen, Sakari

    2015-01-01

    Long noncoding RNAs (lncRNAs) have emerged as important regulators of diverse cellular processes, but their roles in the developing immune system are poorly understood. In this study, we analysed lncRNA expression during human B-cell development by array-based expression profiling of eleven distinct flow-sorted B-cell subsets, comprising pre-B1, pre-B2, immature, naive, memory, and plasma cells from bone marrow biopsies (n = 7), and naive, centroblast, centrocyte, memory, and plasmablast cells from tonsil tissue samples (n = 6), respectively. A remapping strategy was used to assign the array probes to 37630 gene-level probe sets, reflecting recent updates in genomic and transcriptomic databases, which enabled expression profiling of 19579 long noncoding RNAs, comprising 3947 antisense RNAs, 5277 lincRNAs, 7625 pseudogenes, and 2730 additional lncRNAs. As a first step towards inferring the functions of the identified lncRNAs in developing B-cells, we analysed their co-expression with well-characterized protein-coding genes, a method known as "guilt by association". By using weighted gene co-expression network analysis, we identified 272 lincRNAs, 471 antisense RNAs, 376 pseudogene RNAs, and 64 lncRNAs within seven sub-networks associated with distinct stages of B-cell development, such as early B-cell development, B-cell proliferation, affinity maturation of antibody, and terminal differentiation. These data provide an important resource for future studies on the functions of lncRNAs in development of the adaptive immune response, and the pathogenesis of B-cell malignancies that originate from distinct B-cell subpopulations. PMID:26394393

  12. Vector for regulated expression of cloned genes in a wide range of gram-negative bacteria.

    PubMed Central

    Mermod, N; Ramos, J L; Lehrbach, P R; Timmis, K N

    1986-01-01

    A pKT231-based broad-host-range plasmid vector was constructed which enabled regulation of expression of cloned genes in a wide range of gram-negative bacteria. This vector, pNM185, contained upstream of its EcoRI, SstI, and SstII cloning sites the positively activated pm twin promoters of the TOL plasmid and xylS, the gene of the positive regulator of these promoters. Expression of cloned genes was induced with micromolar quantities of benzoate or m-toluate, the inexpensive coinducers of the pm promoters. Expression of a test gene, xylE, which specifies catechol 2,3-dioxygenase, cloned in this vector was tested in representative strains of a variety of gram-negative bacteria. Regulated expression of xylE was observed in most strains examined, and induced levels of enzyme representing up to 5% of total cellular protein and ratios of induced:noninduced levels of enzyme up to a factor of 600 were observed. The level of xylE gene expression in different bacteria tended to be correlated with their phylogenetic distance from Pseudomonas putida. Images PMID:3525513

  13. High-level overproduction of Thermobifida enzyme in Streptomyces lividans using a novel expression vector.

    PubMed

    Li, Jun-Xia; Zhao, Long-Mei; Wu, Ru-Juan; Zheng, Zhao-Jun; Zhang, Ri-Jun

    2013-09-10

    In this study, we constructed a novel Streptomyces-E.coli shuttle vector pZRJ362 combining the xylose isomerase promoter and amylase terminator. A gene encoding the endoglucanase Cel6A in Thermobifida fusca was amplified by PCR, cloned into Streptomyces lividans host strain using the novel expression vector and Pichia pastoris GS115 host strain using the vector pPICZα-C, respectively. Afterwards, the expression pattern and the maximum expression level were comparatively studied in both expression systems. The maximum enzyme activity of Cel6A-(His)₆ secreted in S. lividans supernatant after 84-h of cultivation amounted to 5.56 U/mL, which was dramatically higher than that secreted in P. pastoris about 1.4 U/mL after 96-h of cultivation. The maximum expression level of Cel6A-(His)₆ in S. lividans supernatant reached up to 173 mg/L after 84-h of cultivation. The endoglucanase activity staining SDS-PAGE showed that there were some minor proteins in S. lividans supernatant which may be the Cel6A derivant by proteolytic degradation, while there was no proteolytic product detected in supernatant of P. pastoris.

  14. New alkane-responsive expression vectors for Escherichia coli and pseudomonas.

    PubMed

    Smits, T H; Seeger, M A; Witholt, B; van Beilen, J B

    2001-07-01

    We have developed Escherichia coli and Pseudomonas expression vectors based on the alkane-responsive Pseudomonas putida (oleovorans) GPo1 promoter PalkB. The expression vectors were tested in several E. coli strains, P. putida GPo12 and P. fluorescens KOB2Delta1 with catechol-2,3-dioxygenase (XylE). Induction factors ranged between 100 and 2700 for pKKPalk in E. coli and pCom8 in Pseudomonas strains, but were clearly lower for pCom8, pCom9, and pCom10 in E. coli. XylE expression levels of more than 10% of total cell protein were obtained for E. coli as well as for Pseudomonas strains. PMID:11535032

  15. Bacterial control of host gene expression through RNA polymerase II

    PubMed Central

    Lutay, Nataliya; Ambite, Ines; Hernandez, Jenny Grönberg; Rydström, Gustav; Ragnarsdóttir, Bryndís; Puthia, Manoj; Nadeem, Aftab; Zhang, Jingyao; Storm, Petter; Dobrindt, Ulrich; Wullt, Björn; Svanborg, Catharina

    2013-01-01

    The normal flora furnishes the host with ecological barriers that prevent pathogen attack while maintaining tissue homeostasis. Urinary tract infections (UTIs) constitute a highly relevant model of microbial adaptation in which some patients infected with Escherichia coli develop acute pyelonephritis, while other patients with bacteriuria exhibit an asymptomatic carrier state similar to bacterial commensalism. It remains unclear if the lack of destructive inflammation merely reflects low virulence or if carrier strains actively inhibit disease-associated responses in the host. Here, we identify a new mechanism of bacterial adaptation through broad suppression of RNA polymerase II–dependent (Pol II–dependent) host gene expression. Over 60% of all genes were suppressed 24 hours after human inoculation with the prototype asymptomatic bacteriuria (ABU) strain E. coli 83972, and inhibition was verified by infection of human cells. Specific repressors and activators of Pol II–dependent transcription were modified, Pol II phosphorylation was inhibited, and pathogen-specific signaling was suppressed in cell lines and inoculated patients. An increased frequency of strains inhibiting Pol II was epidemiologically verified in ABU and fecal strains compared with acute pyelonephritis, and a Pol II antagonist suppressed the disease-associated host response. These results suggest that by manipulating host gene expression, ABU strains promote tissue integrity while inhibiting pathology. Such bacterial modulation of host gene expression may be essential to sustain asymptomatic bacterial carriage by ensuring that potentially destructive immune activation will not occur. PMID:23728172

  16. Expression and functional studies on the noncoding RNA, PRINS.

    PubMed

    Szegedi, Krisztina; Göblös, Anikó; Bacsa, Sarolta; Antal, Mária; Németh, István Balázs; Bata-Csörgő, Zsuzsanna; Kemény, Lajos; Dobozy, Attila; Széll, Márta

    2012-12-21

    PRINS, a noncoding RNA identified earlier by our research group, contributes to psoriasis susceptibility and cellular stress response. We have now studied the cellular and histological distribution of PRINS by using in situ hybridization and demonstrated variable expressions in different human tissues and a consistent staining pattern in epidermal keratinocytes and in vitro cultured keratinocytes. To identify the cellular function(s) of PRINS, we searched for a direct interacting partner(s) of this stress-induced molecule. In HaCaT and NHEK cell lysates, the protein proved to be nucleophosmin (NPM) protein as a potential physical interactor with PRINS. Immunohistochemical experiments revealed an elevated expression of NPM in the dividing cells of the basal layers of psoriatic involved skin samples as compared with healthy and psoriatic uninvolved samples. Others have previously shown that NPM is a ubiquitously expressed nucleolar phosphoprotein which shuttles to the nucleoplasm after UV-B irradiation in fibroblasts and cancer cells. We detected a similar translocation of NPM in UV-B-irradiated cultured keratinocytes. The gene-specific silencing of PRINS resulted in the retention of NPM in the nucleolus of UV-B-irradiated keratinocytes; suggesting that PRINS may play a role in the NPM-mediated cellular stress response in the skin.

  17. Large-Scale microRNA Expression Profiling Identifies Putative Retinal miRNA-mRNA Signaling Pathways Underlying Form-Deprivation Myopia in Mice.

    PubMed

    Tkatchenko, Andrei V; Luo, Xiaoyan; Tkatchenko, Tatiana V; Vaz, Candida; Tanavde, Vivek M; Maurer-Stroh, Sebastian; Zauscher, Stefan; Gonzalez, Pedro; Young, Terri L

    2016-01-01

    Development of myopia is associated with large-scale changes in ocular tissue gene expression. Although differential expression of coding genes underlying development of myopia has been a subject of intense investigation, the role of non-coding genes such as microRNAs in the development of myopia is largely unknown. In this study, we explored myopia-associated miRNA expression profiles in the retina and sclera of C57Bl/6J mice with experimentally induced myopia using microarray technology. We found a total of 53 differentially expressed miRNAs in the retina and no differences in miRNA expression in the sclera of C57BL/6J mice after 10 days of visual form deprivation, which induced -6.93 ± 2.44 D (p < 0.000001, n = 12) of myopia. We also identified their putative mRNA targets among mRNAs found to be differentially expressed in myopic retina and potential signaling pathways involved in the development of form-deprivation myopia using miRNA-mRNA interaction network analysis. Analysis of myopia-associated signaling pathways revealed that myopic response to visual form deprivation in the retina is regulated by a small number of highly integrated signaling pathways. Our findings highlighted that changes in microRNA expression are involved in the regulation of refractive eye development and predicted how they may be involved in the development of myopia by regulating retinal gene expression. PMID:27622715

  18. Large-Scale microRNA Expression Profiling Identifies Putative Retinal miRNA-mRNA Signaling Pathways Underlying Form-Deprivation Myopia in Mice

    PubMed Central

    Vaz, Candida; Tanavde, Vivek M.; Maurer-Stroh, Sebastian; Zauscher, Stefan; Gonzalez, Pedro; Young, Terri L.

    2016-01-01

    Development of myopia is associated with large-scale changes in ocular tissue gene expression. Although differential expression of coding genes underlying development of myopia has been a subject of intense investigation, the role of non-coding genes such as microRNAs in the development of myopia is largely unknown. In this study, we explored myopia-associated miRNA expression profiles in the retina and sclera of C57Bl/6J mice with experimentally induced myopia using microarray technology. We found a total of 53 differentially expressed miRNAs in the retina and no differences in miRNA expression in the sclera of C57BL/6J mice after 10 days of visual form deprivation, which induced -6.93 ± 2.44 D (p < 0.000001, n = 12) of myopia. We also identified their putative mRNA targets among mRNAs found to be differentially expressed in myopic retina and potential signaling pathways involved in the development of form-deprivation myopia using miRNA-mRNA interaction network analysis. Analysis of myopia-associated signaling pathways revealed that myopic response to visual form deprivation in the retina is regulated by a small number of highly integrated signaling pathways. Our findings highlighted that changes in microRNA expression are involved in the regulation of refractive eye development and predicted how they may be involved in the development of myopia by regulating retinal gene expression. PMID:27622715

  19. Altered Gene Expression Associated with microRNA Binding Site Polymorphisms

    PubMed Central

    Võsa, Urmo; Esko, Tõnu; Kasela, Silva; Annilo, Tarmo

    2015-01-01

    Allele-specific gene expression associated with genetic variation in regulatory regions can play an important role in the development of complex traits. We hypothesized that polymorphisms in microRNA (miRNA) response elements (MRE-SNPs) that either disrupt a miRNA binding site or create a new miRNA binding site can affect the allele-specific expression of target genes. By integrating public expression quantitative trait locus (eQTL) data, miRNA binding site predictions, small RNA sequencing, and Argonaute crosslinking immunoprecipitation (AGO-CLIP) datasets, we identified genetic variants that can affect gene expression by modulating miRNA binding efficiency. We also identified MRE-SNPs located in regions associated with complex traits, indicating possible causative mechanisms associated with these loci. The results of this study expand the current understanding of gene expression regulation and help to interpret the mechanisms underlying eQTL effects. PMID:26496489

  20. The prognostic value of epidermal growth factor receptor mRNA expression in primary ovarian cancer.

    PubMed Central

    Bartlett, J. M.; Langdon, S. P.; Simpson, B. J.; Stewart, M.; Katsaros, D.; Sismondi, P.; Love, S.; Scott, W. N.; Williams, A. R.; Lessells, A. M.; Macleod, K. G.; Smyth, J. F.; Miller, W. R.

    1996-01-01

    The expression of mRNA for the epidermal growth factor (EGF) receptor, EGF and transforming growth factor alpha (TGF-alpha) was determined in 76 malignant, six borderline and 15 benign primary ovarian tumours using the reverse transcriptase-polymerase chain reaction and related to clinical and pathological parameters. Of the malignant tumours, 70% (53/76) expressed EGF receptor mRNA, 31% (23/75) expressed EGF mRNA and 35% (26/75) expressed TGF-alpha mRNA. For the borderline tumours, four of six (67%) expressed EGF receptor mRNA, 1/6 (17%) expressed TGF-alpha mRNA and none expressed EGF mRNA. Finally, 33% (5/15) of the benign tumours expressed EGF receptor mRNA, whereas 40% (6/15) expressed EGF mRNA and 7% (1/15) expressed TGF-alpha mRNA. The presence of the EGF receptor in malignant tumours was associated with that of TGF-alpha (P = 0.0015) but not with EGF (P = 1.00), whereas there was no relationship between the presence of EGF and TGF-alpha (P = 1.00). EGF receptor mRNA expression was significantly and positively associated with serous histology (P = 0.006) but not with stage or grade. Neither EGF nor TGF-alpha showed any link with histological subtype or stage. The survival of patients with malignant tumours possessing EGF receptor mRNA was significantly reduced compared with that of patients whose tumours were negative (P = 0.030 for all malignant tumours; P = 0.007 for malignant epithelial tumours only). In contrast, neither the expression of TGF-alpha nor EGF was related to survival. These data suggest that the presence of EGF receptor mRNA is associated with poor prognosis in primary ovarian cancer. Images Figure 1 PMID:8562334

  1. Endothelin-1 mRNA expression in the rat kidney.

    PubMed Central

    Nunez, D J; Taylor, E A; Oh, V M; Schofield, J P; Brown, M J

    1991-01-01

    Cultured pig and bovine endothelial cells are capable of synthesizing endothelin-1 (ET-1). Thus the observation that the kidney contains a large number of binding sites for ET distributed in close proximity to endothelial cells suggests that ET-1 may be released from the endothelium to act locally on these receptors. In support of this hypothesis, using the technique of reverse transcription with specific amplification of cDNA, we report here that ET-1 mRNA is expressed in the rat kidney. The partial sequence of the amplified rat ET-1 cDNA confirms that the mature rat peptide is identical to that of the mouse, man and pig, but with some differences in codon usage. PMID:2039460

  2. Hierarchical Generative Biclustering for MicroRNA Expression Analysis

    NASA Astrophysics Data System (ADS)

    Caldas, José; Kaski, Samuel

    Clustering methods are a useful and common first step in gene expression studies, but the results may be hard to interpret. We bring in explicitly an indicator of which genes tie each cluster, changing the setup to biclustering. Furthermore, we make the indicators hierarchical, resulting in a hierarchy of progressively more specific biclusters. A non-parametric Bayesian formulation makes the model rigorous and yet flexible, and computations feasible. The formulation additionally offers a natural information retrieval relevance measure that allows relating samples in a principled manner. We show that the model outperforms other four biclustering procedures in a large miRNA data set. We also demonstrate the model's added interpretability and information retrieval capability in a case study that highlights the potential and novel role of miR-224 in the association between melanoma and non-Hodgkin lymphoma. Software is publicly available.

  3. ProteoMirExpress: Inferring MicroRNA and Protein-centered Regulatory Networks from High-throughput Proteomic and mRNA Expression Data*

    PubMed Central

    Qin, Jing; Li, Mulin Jun; Wang, Panwen; Wong, Nai Sum; Wong, Maria P.; Xia, Zhengyuan; Tsao, George S. W.; Zhang, Michael Q.; Wang, Junwen

    2013-01-01

    MicroRNAs (miRNAs) regulate gene expression through translational repression and RNA degradation. Recently developed high-throughput proteomic methods measure gene expression changes at protein level and therefore can reveal the direct effects of miRNAs' translational repression. Here, we present a web server, ProteoMirExpress, that integrates proteomic and mRNA expression data together to infer miRNA-centered regulatory networks. With both types of high-throughput data from the users, ProteoMirExpress is able to discover not only miRNA targets that have decreased mRNA, but also subgroups of targets with suppressed proteins whose mRNAs are not significantly changed or with decreased mRNA whose proteins are not significantly changed, which are usually ignored by most current methods. Furthermore, both direct and indirect targets of miRNAs can be detected. Therefore, ProteoMirExpress provides more comprehensive miRNA-centered regulatory networks. We used several published data to assess the quality of our inferred networks and prove the value of our server. ProteoMirExpress is available online, with free access to academic users. PMID:23924514

  4. Development of a versatile shuttle vector for gene expression in Geobacillus spp.

    PubMed

    Taylor, Mark P; Esteban, Carlos D; Leak, David J

    2008-07-01

    An improved, versatile shuttle vector has been created for the metabolic engineering of Geobacillus spp. As kanamycin is the most thermo-tolerant of commonly used antibiotics, the gene encoding a thermostable kanamycin nucleotidyltransferase, together with the origin of replication from the G. stearothermophilus plasmid pBST1 were cloned into the Escherichia coli cloning vector pUC18. The resulting vector, named pUCG18, replicated in both organisms and could be transformed with an efficiency of 1 x 10(4) transformants per microg of DNA in G. thermoglucosidasius and was stable up to 68 degrees C with antibiotic selection. It was used to demonstrate expression of the pyruvate decarboxylase (pdc) gene from Zymomonas palmae in G. thermoglucosidasius at 45 degrees C. Sequence analysis of the pBST1 derived origin of replication revealed homology with a family of theta replicons that have previously only been found in strains of Bacillus megaterium. PMID:18501964

  5. Conserved expression of lincRNA during human and macaque prefrontal cortex development and maturation.

    PubMed

    He, Zhisong; Bammann, Hindrike; Han, Dingding; Xie, Gangcai; Khaitovich, Philipp

    2014-07-01

    The current annotation of the human genome includes more than 12,000 long intergenic noncoding RNAs (lincRNA). While a handful of lincRNA have been shown to play important regulatory roles, the functionality of most remains unclear. Here, we examined the expression conservation and putative functionality of lincRNA in human and macaque prefrontal cortex (PFC) development and maturation. We analyzed transcriptome sequence (RNA-seq) data from 38 human and 40 macaque individuals covering the entire postnatal development interval. Using the human data set, we detected the expression of 5835 lincRNA annotated in GENCODE and further identified 1888 novel lincRNA. Most of these lincRNA show low DNA sequence conservation, as well as low expression levels. Remarkably, developmental expression patterns of these lincRNA were as conserved between humans and macaques as those of protein-coding genes. Transfection of development-associated lincRNA into human SH-SY5Y cells affected gene expression, indicating their regulatory potential. In brain, expression of these putative target genes correlated with the expression of the corresponding lincRNA during human and macaque PFC development. These results support the potential functionality of lincRNA in primate PFC development.

  6. Successful disabling of the 5' UTR of HCV using adeno-associated viral vectors to deliver modular multimeric primary microRNA mimics.

    PubMed

    Bourhill, Tarryn; Arbuthnot, Patrick; Ely, Abdullah

    2016-09-01

    Chronic hepatitis C virus (HCV) infection is a major health concern and is strongly associated with cirrhosis, hepatocellular carcinoma and liver-related mortality. The HCV genome is the template for both protein translation and viral replication and, being RNA, is amenable to direct genetic silencing by RNA interference (RNAi). HCV is a highly mutable virus and is capable of escaping RNAi-mediated silencing. This has highlighted the importance of developing RNAi-based therapy that simultaneously targets multiple regions of the HCV genome. To develop a multi-targeting RNAi activator, a novel approach for the generation of anti-HCV gene therapy was investigated. Five artificial primary miRNA (pri-miR) were each designed to mimic the naturally occurring monomeric pri-miR-31. Potent knockdown of an HCV reporter was seen with four of the five constructs and were processed according to the intended design. The design of the individual pri-miR mimics enabled the modular assembly into multimeric mimics of any possible conformation. Consequently the four potent pri-miR mimics were used to generate polycistronic cassettes, which showed impressive silencing of an HCV target. To further their application as a gene therapy, recombinant adeno-associated viral (rAAV) vectors that express the polycistronic pri-miR mimics were generated. All AAV-delivered anti-HCV pri-miR mimics significantly knocked down the expression of an HCV target and showed inhibition of HCV replicon replication. Here we describe a protocol for the generation of therapeutic rAAVs that express modular polycistronic pri-miR cassettes allowing for rapid alteration and generation of tailored therapeutic constructs against HCV. PMID:27181212

  7. Successful disabling of the 5' UTR of HCV using adeno-associated viral vectors to deliver modular multimeric primary microRNA mimics.

    PubMed

    Bourhill, Tarryn; Arbuthnot, Patrick; Ely, Abdullah

    2016-09-01

    Chronic hepatitis C virus (HCV) infection is a major health concern and is strongly associated with cirrhosis, hepatocellular carcinoma and liver-related mortality. The HCV genome is the template for both protein translation and viral replication and, being RNA, is amenable to direct genetic silencing by RNA interference (RNAi). HCV is a highly mutable virus and is capable of escaping RNAi-mediated silencing. This has highlighted the importance of developing RNAi-based therapy that simultaneously targets multiple regions of the HCV genome. To develop a multi-targeting RNAi activator, a novel approach for the generation of anti-HCV gene therapy was investigated. Five artificial primary miRNA (pri-miR) were each designed to mimic the naturally occurring monomeric pri-miR-31. Potent knockdown of an HCV reporter was seen with four of the five constructs and were processed according to the intended design. The design of the individual pri-miR mimics enabled the modular assembly into multimeric mimics of any possible conformation. Consequently the four potent pri-miR mimics were used to generate polycistronic cassettes, which showed impressive silencing of an HCV target. To further their application as a gene therapy, recombinant adeno-associated viral (rAAV) vectors that express the polycistronic pri-miR mimics were generated. All AAV-delivered anti-HCV pri-miR mimics significantly knocked down the expression of an HCV target and showed inhibition of HCV replicon replication. Here we describe a protocol for the generation of therapeutic rAAVs that express modular polycistronic pri-miR cassettes allowing for rapid alteration and generation of tailored therapeutic constructs against HCV.

  8. DsRNA-mediated targeting of ribosomal transcripts RPS6 and RPL26 induces long-lasting and significant reductions in fecundity of the vector Aedes aegypti.

    PubMed

    Estep, A S; Sanscrainte, N D; Becnel, J J

    2016-07-01

    Ribosomal transcripts produce critical proteins that are involved in most cellular production processes. Targeting ribosomal transcripts has produced mortality in mites and ticks but the effect of ribosomal transcript knockdown has not been thoroughly examined in mosquitoes. We examine the effects of triggers targeting four ribosomal proteins (RP) transcripts. Although no significant mortality was observed after dsRNA microinjection and subsequent blood feeding, significant contrasts were observed on fecundity. Triggers targeting RPS6 and RPL26 effectively reduced gene expression but more importantly, reduced reproductive output by more than 96% and 91% at the first oviposition while triggers targeting RPL1 and RPS2 did not cause a reduction although gene expression was reduced. Significantly reduced fecundity continued through a second oviposition cycle in dsRPS6 and dsRPL26 cohorts, although the effect was not as strong. Relative gene expression levels confirmed specific transcript knockdown up to 20days post-injection in mosquitoes that did not oviposit or produced reduced clutch sizes. Dissections at 36h post-blood meal indicated defects in oocyte provisioning. The strong phenotype produced by dsRPS6 allowed us to examine the effects in various tissues as well as the dose response, trigger format, delivery method and trigger specificity in Aedes aegypti. Strong knockdown was observed in the abdomen and the ovaries. Greater than 50ng of dsRPS6 significantly reduced fecundity but not when delivered in a sugar meal or as an siRNA. Similar bioassays with mutated dsRPS6 triggers indicates that up to three mismatches per possible siRNA are still effective in reducing fecundity. These studies indicate that while active and effective triggers can be developed for vector species, the lack of an efficient delivery method is the biggest barrier to use as a potential control method. PMID:27180677

  9. Antagonism Pattern Detection between MicroRNA and Target Expression in Ewing’s Sarcoma

    PubMed Central

    Martignetti, Loredana; Laud-Duval, Karine; Tirode, Franck; Pierron, Gaelle; Reynaud, Stéphanie; Barillot, Emmanuel; Delattre, Olivier; Zinovyev, Andrei

    2012-01-01

    MicroRNAs (miRNAs) have emerged as fundamental regulators that silence gene expression at the post-transcriptional and translational levels. The identification of their targets is a major challenge to elucidate the regulated biological processes. The overall effect of miRNA is reflected on target mRNA expression, suggesting the design of new investigative methods based on high-throughput experimental data such as miRNA and transcriptome profiles. We propose a novel statistical measure of non-linear dependence between miRNA and mRNA expression, in order to infer miRNA-target interactions. This approach, which we name antagonism pattern detection, is based on the statistical recognition of a triangular-shaped pattern in miRNA-target expression profiles. This pattern is observed in miRNA-target expression measurements since their simultaneously elevated expression is statistically under-represented in the case of miRNA silencing effect. The proposed method enables miRNA target prediction to strongly rely on cellular context and physiological conditions reflected by expression data. The procedure has been assessed on synthetic datasets and tested on a set of real positive controls. Then it has been applied to analyze expression data from Ewing’s sarcoma patients. The antagonism relationship is evaluated as a good indicator of real miRNA-target biological interaction. The predicted targets are consistently enriched for miRNA binding site motifs in their 3′UTR. Moreover, we reveal sets of predicted targets for each miRNA sharing important biological function. The procedure allows us to infer crucial miRNA regulators and their potential targets in Ewing’s sarcoma disease. It can be considered as a valid statistical approach to discover new insights in the miRNA regulatory mechanisms. PMID:22848594

  10. Transgene expression in Penaeus monodon cells: evaluation of recombinant baculoviral vectors with shrimp specific hybrid promoters.

    PubMed

    Puthumana, Jayesh; Philip, Rosamma; Bright Singh, I S

    2016-08-01

    It has been realized that shrimp cell immortalization may not be accomplished without in vitro transformation by expressing immortalizing gene in cells. In this process, efficiency of transgene expression is confined to the ability of vectors to transmit gene of interests to the genome. Over the years, unavailability of such vectors has been hampering application of such a strategy in shrimp cells. We report the use of recombinant baculovirus mediated transduction using hybrid promoter system for transgene expression in lymphoid cells of Penaeus monodon. Two recombinant baculovirus vectors with shrimp viral promoters (WSSV-Ie1 and IHHNV-P2) were constructed (BacIe1-GFP and BacP2-GFP) and green fluorescent protein (GFP) used as the transgene. The GFP expression in cells under the control of hybrid promoters, PH-Ie1 or PH-P2, were analyzed and confirmed in shrimp cells. The results indicate that the recombinant baculovirus with shrimp specific viral promoters (hybrid) can be employed for delivery of foreign genes to shrimp cells for in vitro transformation.

  11. A Functionally Superior Second-Generation Vector Expressing an Aurora Kinase-A-Specific T-Cell Receptor for Anti-Leukaemia Adoptive Immunotherapy.

    PubMed

    Casey, Nicholas Paul; Fujiwara, Hiroshi; Tanimoto, Kazushi; Okamoto, Sachiko; Mineno, Junichi; Kuzushima, Kiyotaka; Shiku, Hiroshi; Yasukawa, Masaki

    2016-01-01

    Aurora Kinase A is a cancer-associated protein normally involved in the regulation of mitosis. Being over-expressed in a range of cancers, it is a suitable target for cell-based immunotherapy. Gene transfer of T-cell receptor sequences cognisant of HLA-A*0201-restricted Aurora Kinase A antigen has previously been shown to transfer specific immunoreactivity against the target peptide in a Human Lymphocyte Antigen-restricted manner. While T cell receptor gene-transfer has great potential in overcoming the difficulties of isolating and expanding tumour-reactive lymphocytes from a patient's own cells, one hurdle is potential mispairing and competition between exogenous and endogenous T cell receptor chains. We have used a retroviral vector design bearing a short-interfering RNA that downregulates endogenous T cell receptor chains, without affecting expression of the transgenic T cell receptor sequences. The T cell receptor expression cassette also includes a 2A self-cleaving peptide, resulting in equimolar expression of the T cell receptor alpha and beta chains, further enhancing formation of the desired T cell receptor. Via a simple, modular cloning method, we have cloned the alpha and beta chains of the anti-Aurora Kinase A-reactive T cell receptor into this 'siTCR' vector. We then compared the activity of this vector against the original, 'conventional' vector across a panel of assays. T cell receptors expressed from the siTCR-vector retained the cytotoxic functionality of the original vector, with evidence of reduced off-target reactivity. The rate of expression of correctly-formed T cell receptors was superior using the siTCR design, and this was achieved at lower vector copy numbers. Maintaining T cell receptor efficacy with a reduced vector copy number reduces the risk of genotoxicity. The siTCR design also reduces the risk of mispairing and cross-reactivity, while increasing the functional titre. Such improvements in the safety of T cell receptor gene

  12. A Functionally Superior Second-Generation Vector Expressing an Aurora Kinase-A-Specific T-Cell Receptor for Anti-Leukaemia Adoptive Immunotherapy

    PubMed Central

    Casey, Nicholas Paul; Fujiwara, Hiroshi; Tanimoto, Kazushi; Okamoto, Sachiko; Mineno, Junichi; Kuzushima, Kiyotaka; Shiku, Hiroshi; Yasukawa, Masaki

    2016-01-01

    Aurora Kinase A is a cancer-associated protein normally involved in the regulation of mitosis. Being over-expressed in a range of cancers, it is a suitable target for cell-based immunotherapy. Gene transfer of T-cell receptor sequences cognisant of HLA-A*0201-restricted Aurora Kinase A antigen has previously been shown to transfer specific immunoreactivity against the target peptide in a Human Lymphocyte Antigen-restricted manner. While T cell receptor gene-transfer has great potential in overcoming the difficulties of isolating and expanding tumour-reactive lymphocytes from a patient’s own cells, one hurdle is potential mispairing and competition between exogenous and endogenous T cell receptor chains. We have used a retroviral vector design bearing a short-interfering RNA that downregulates endogenous T cell receptor chains, without affecting expression of the transgenic T cell receptor sequences. The T cell receptor expression cassette also includes a 2A self-cleaving peptide, resulting in equimolar expression of the T cell receptor alpha and beta chains, further enhancing formation of the desired T cell receptor. Via a simple, modular cloning method, we have cloned the alpha and beta chains of the anti-Aurora Kinase A-reactive T cell receptor into this ‘siTCR’ vector. We then compared the activity of this vector against the original, ‘conventional’ vector across a panel of assays. T cell receptors expressed from the siTCR-vector retained the cytotoxic functionality of the original vector, with evidence of reduced off-target reactivity. The rate of expression of correctly-formed T cell receptors was superior using the siTCR design, and this was achieved at lower vector copy numbers. Maintaining T cell receptor efficacy with a reduced vector copy number reduces the risk of genotoxicity. The siTCR design also reduces the risk of mispairing and cross-reactivity, while increasing the functional titre. Such improvements in the safety of T cell receptor gene

  13. Modular Broad-Host-Range Expression Vectors for Single-Protein and Protein Complex Purification

    PubMed Central

    Fodor, Barna D.; Kovács, Ákos T.; Csáki, Róbert; Hunyadi-Gulyás, Éva; Klement, Éva; Maróti, Gergely; Mészáros, Lívia S.; Medzihradszky, Katalin F.; Rákhely, Gábor; Kovács, Kornél L.

    2004-01-01

    A set of modular broad-host-range expression vectors with various affinity tags (six-His-tag, FLAG-tag, Strep-tag II, T7-tag) was created. The complete nucleotide sequences of the vectors are known, and these small vectors can be mobilized by conjugation. They are useful in the purification of proteins and protein complexes from gram-negative bacterial species. The plasmids were easily customized for Thiocapsa roseopersicina, Rhodobacter capsulatus, and Methylococcus capsulatus by inserting an appropriate promoter. These examples demonstrate the versatility and flexibility of the vectors. The constructs harbor the T7 promoter for easy overproduction of the desired protein in an appropriate Escherichia coli host. The vectors were useful in purifying different proteins from T. roseopersicina. The FLAG-tag-Strep-tag II combination was utilized for isolation of the HynL-HypC2 protein complex involved in hydrogenase maturation. These tools should be useful for protein purification and for studying protein-protein interactions in a range of bacterial species. PMID:14766546

  14. MicroRNA-383 expression regulates proliferation, migration, invasion, and apoptosis in human glioma cells.

    PubMed

    Xu, Dawei; Ma, Pengju; Gao, Guojun; Gui, Yongkun; Niu, Xiaolu; Jin, Baozhe

    2015-09-01

    This study aims to evaluate microRNA-383 (miR-383) expression level in glioma cells and its influences on proliferation, migration, invasion, apoptosis, and cell cycle in glioma cells. miR-383 expression levels were determined by real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR). Thirty BALB/c-nu mice were randomly assigned into three groups: U87-miR-383 group, vector-control group, and blank group. Tumorigenicity experiment was conducted to confirm the function of miR-383. U251 and U87 glioma cells were divided into three groups: non-transfected control cells (NT group), glioma cells transfected with miR-383 (miR-383 group), and glioma cells transfected with negative sequence (NC group). Transfection efficiency was measured by qRT-PCR. Cell counting kit-8 (CCK-8) assay was used to detect cell proliferation. Cell migration and invasion were examined by utilizing a Transwell chamber. Cell cycle and apoptosis were analyzed by flow cytometry. The qRT-PCR results revealed that miR-383 expression was down-regulated in human glioma cells, and was negatively related to the pathological grading of glioma. The rates of tumor growth in vector-control group and blank group were significantly faster than that in U87-miR-383 group, and the average tumor volume and weight in vector-control group and blank group were increased as compared with U87-miR-383 group. Additionally, miR-383 levels in miR-383 group were higher than those in NT group and NC group. CCK-8 assay indicated lower cell viability in miR-383 group as compared with NT group and NC group. Flow cytometry implied that the percentages of cells in miR-383 group reduced, while the cell apoptosis rate enhanced compared with NT group and NC group. In conclusion, our findings suggest that miR-383 expression is down-regulated in glioma cells, inhibiting cell proliferation, migration, and invasion, affecting the cell cycle, and inducing cell apoptosis.

  15. MicroRNA-383 expression regulates proliferation, migration, invasion, and apoptosis in human glioma cells.

    PubMed

    Xu, Dawei; Ma, Pengju; Gao, Guojun; Gui, Yongkun; Niu, Xiaolu; Jin, Baozhe

    2015-09-01

    This study aims to evaluate microRNA-383 (miR-383) expression level in glioma cells and its influences on proliferation, migration, invasion, apoptosis, and cell cycle in glioma cells. miR-383 expression levels were determined by real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR). Thirty BALB/c-nu mice were randomly assigned into three groups: U87-miR-383 group, vector-control group, and blank group. Tumorigenicity experiment was conducted to confirm the function of miR-383. U251 and U87 glioma cells were divided into three groups: non-transfected control cells (NT group), glioma cells transfected with miR-383 (miR-383 group), and glioma cells transfected with negative sequence (NC group). Transfection efficiency was measured by qRT-PCR. Cell counting kit-8 (CCK-8) assay was used to detect cell proliferation. Cell migration and invasion were examined by utilizing a Transwell chamber. Cell cycle and apoptosis were analyzed by flow cytometry. The qRT-PCR results revealed that miR-383 expression was down-regulated in human glioma cells, and was negatively related to the pathological grading of glioma. The rates of tumor growth in vector-control group and blank group were significantly faster than that in U87-miR-383 group, and the average tumor volume and weight in vector-control group and blank group were increased as compared with U87-miR-383 group. Additionally, miR-383 levels in miR-383 group were higher than those in NT group and NC group. CCK-8 assay indicated lower cell viability in miR-383 group as compared with NT group and NC group. Flow cytometry implied that the percentages of cells in miR-383 group reduced, while the cell apoptosis rate enhanced compared with NT group and NC group. In conclusion, our findings suggest that miR-383 expression is down-regulated in glioma cells, inhibiting cell proliferation, migration, and invasion, affecting the cell cycle, and inducing cell apoptosis. PMID:25936342

  16. Expression of chicken parvovirus VP2 in chicken embryo fibroblasts requires codon optimization for production of naked DNA and vectored meleagrid herpesvirus type 1 vaccines.

    PubMed

    Spatz, Stephen J; Volkening, Jeremy D; Mullis, Robert; Li, Fenglan; Mercado, John; Zsak, Laszlo

    2013-10-01

    Meleagrid herpesvirus type 1 (MeHV-1) is an ideal vector for the expression of antigens from pathogenic avian organisms in order to generate vaccines. Chicken parvovirus (ChPV) is a widespread infectious virus that causes serious disease in chickens. It is one of the etiological agents largely suspected in causing Runting Stunting Syndrome (RSS) in chickens. Initial attempts to express the wild-type gene encoding the capsid protein VP2 of ChPV by insertion into the thymidine kinase gene of MeHV-1 were unsuccessful. However, transient expression of a codon-optimized synthetic VP2 gene cloned into the bicistronic vector pIRES2-Ds-Red2, could be demonstrated by immunocytochemical staining of transfected chicken embryo fibroblasts (CEFs). Red fluorescence could also be detected in these transfected cells since the red fluorescent protein gene is downstream from the internal ribosome entry site (IRES). Strikingly, fluorescence could not be demonstrated in cells transiently transfected with the bicistronic vector containing the wild-type or non-codon-optimized VP2 gene. Immunocytochemical staining of these cells also failed to demonstrate expression of wild-type VP2, indicating that the lack of expression was at the RNA level and the VP2 protein was not toxic to CEFs. Chickens vaccinated with a DNA vaccine consisting of the bicistronic vector containing the codon-optimized VP2 elicited a humoral immune response as measured by a VP2-specific ELISA. This VP2 codon-optimized bicistronic cassette was rescued into the MeHV-1 genome generating a vectored vaccine against ChPV disease.

  17. Potential for cellular stress response to hepatic factor VIII expression from AAV vector

    PubMed Central

    Zolotukhin, Irene; Markusic, David M; Palaschak, Brett; Hoffman, Brad E; Srikanthan, Meera A; Herzog, Roland W

    2016-01-01

    Hemophilia A and B are coagulation disorders resulting from the loss of functional coagulation factor VIII (FVIII) or factor IX proteins, respectively. Gene therapy for hemophilia with adeno-associated virus vectors has shown efficacy in hemophilia B patients. Although hemophilia A patients are more prevalent, the development of therapeutic adeno-associated virus vectors has been impeded by the size of the F8 cDNA and impaired secretion of FVIII protein. Further, it has been reported that over-expression of the FVIII protein induces endoplasmic reticulum stress and activates the unfolded protein response pathway both in vitro and in hepatocytes in vivo, presumably due to retention of misfolded FVIII protein within the endoplasmic reticulum. Engineering of the F8 transgene, including removal of the B domain (BDD-FVIII) and codon optimization, now allows for the generation of adeno-associated virus vectors capable of expressing therapeutic levels of FVIII. Here we sought to determine if the risks of inducing the unfolded protein response in murine hepatocytes extend to adeno-associated virus gene transfer. Although our data show a mild activation of unfolded protein response markers following F8 gene delivery at a certain vector dose in C57BL/6 mice, it was not augmented upon further elevated dosing, did not induce liver pathology or apoptosis, and did not impact FVIII immunogenicity. PMID:27738644

  18. Inhibition of Sox2 Expression in the Adult Neural Stem Cell Niche In Vivo by Monocationic-based siRNA Delivery

    PubMed Central

    Remaud, Sylvie; López-Juárez, Silvia Alejandra; Bolcato-Bellemin, Anne-Laure; Neuberg, Patrick; Stock, Fabrice; Bonnet, Marie-Elise; Ghaddab, Rym; Clerget-Froidevaux, Marie Stéphanie; Pierre-Simons, Jacqueline; Erbacher, Patrick; Demeneix, Barbara A; Morvan-Dubois, Ghislaine

    2013-01-01

    RNA interference (RNAi) is a major tool for basic and applied investigations. However, obtaining RNAi data that have physiological significance requires investigation of regulations and therapeutic strategies in appropriate in vivo settings. To examine in vivo gene regulation and protein function in the adult neural stem cell (NSC) niche, we optimized a new non-viral vector for delivery of siRNA into the subventricular zone (SVZ). This brain region contains the neural stem and progenitor cells populations that express the stem cell marker, SOX2. Temporally and spatially controlled Sox2 knockdown was achieved using the monocationic lipid vector, IC10. siRNA/IC10 complexes were stable over time and smaller (<40 nm) than jetSi complexes (≈400 nm). Immunocytochemistry showed that siRNA/IC10 complexes efficiently target both the progenitor and stem cell populations in the adult SVZ. Injection of the complexes into the lateral brain ventricle resulted in specific knockdown of Sox2 in the SVZ. Furthermore, IC10-mediated transient in vivo knockdown of Sox2-modulated expression of several genes implicated in NSC maintenance. Taken together, these data show that IC10 cationic lipid formulation can efficiently vectorize siRNA in a specific area of the adult mouse brain, achieving spatially and temporally defined loss of function. PMID:23612115

  19. Inhibition of Sox2 Expression in the Adult Neural Stem Cell Niche In Vivo by Monocationic-based siRNA Delivery.

    PubMed

    Remaud, Sylvie; López-Juárez, Silvia Alejandra; Bolcato-Bellemin, Anne-Laure; Neuberg, Patrick; Stock, Fabrice; Bonnet, Marie-Elise; Ghaddab, Rym; Clerget-Froidevaux, Marie Stéphanie; Pierre-Simons, Jacqueline; Erbacher, Patrick; Demeneix, Barbara A; Morvan-Dubois, Ghislaine

    2013-04-23

    RNA interference (RNAi) is a major tool for basic and applied investigations. However, obtaining RNAi data that have physiological significance requires investigation of regulations and therapeutic strategies in appropriate in vivo settings. To examine in vivo gene regulation and protein function in the adult neural stem cell (NSC) niche, we optimized a new non-viral vector for delivery of siRNA into the subventricular zone (SVZ). This brain region contains the neural stem and progenitor cells populations that express the stem cell marker, SOX2. Temporally and spatially controlled Sox2 knockdown was achieved using the monocationic lipid vector, IC10. siRNA/IC10 complexes were stable over time and smaller (<40 nm) than jetSi complexes (≈400 nm). Immunocytochemistry showed that siRNA/IC10 complexes efficiently target both the progenitor and stem cell populations in the adult SVZ. Injection of the complexes into the lateral brain ventricle resulted in specific knockdown of Sox2 in the SVZ. Furthermore, IC10-mediated transient in vivo knockdown of Sox2-modulated expression of several genes implicated in NSC maintenance. Taken together, these data show that IC10 cationic lipid formulation can efficiently vectorize siRNA in a specific area of the adult mouse brain, achieving spatially and temporally defined loss of function.Molecular Therapy-Nucleic Acids (2013) 2, e89; doi:10.1038/mtna.2013.8; published online 23 April 2013.

  20. [Construction and Expression of RNase-Resisting His-Tagged Virus-Like Particles Containing FluA/B mRNA].

    PubMed

    Zhang, Jin; Xue, Xiaoning; Xu, Hefei; Zhu, Ke; Chen, Xiaoguang; Zhang, Juan; Zhang, Qi; Lin, Yuan

    2015-11-01

    To prepare virus-like particles containing FluA/B mRNA as RNA standard and control in Influenza RNA detection, the genes coding the coat protein and maturase of E. coli bacteriophage MS2 were amplified and cloned into D-pET32a vector. Then we inserted 6 histidines to MS2 coat protein by QuikChange Site-Directed Mutagenesis Kit to construct the universal expressing vector D-pET32a-CP-His. In addition, the partial gene fragments of FluA and FluB were cloned to the down-stream of expressing vector. The recombinant plasmid D-pET32a-CP-His-FluA/B was transformed to BL21 with induction by IPTG. The virus-like particles were purified by Ni+ chromatography. The virus-like particles can be detected by RT-PCR, but not PCR. They can be conserved stably for at least 3 months at both 4 degrees C and -20 degrees C. His-tagged virus-like particles are more stable and easier to purification. It can be used as RNA standard and control in Influenza virus RNA detection. PMID:26951007

  1. MicroRNA-33 suppresses CCL2 expression in chondrocytes.

    PubMed

    Wei, Meng; Xie, Qingyun; Zhu, Jun; Wang, Tao; Zhang, Fan; Cheng, Yue; Guo, Dongyang; Wang, Ying; Mo, Liweng; Wang, Shuai

    2016-06-01

    CCL2-mediated macrophage infiltration in articular tissues plays a pivotal role in the development of the osteoarthritis (OA). miRNAs regulate the onset and progression of diseases via controlling the expression of a series of genes. How the CCL2 gene was regulated by miRNAs was still not fully elucidated. In the present study, we demonstrated that the binding sites of miR-33 in the 3'UTR of CCL2 gene were conserved in human, mouse and rat species. By performing gain- or loss-of-function studies, we verified that miR-33 suppressed CCL2 expression in the mRNA and protein levels. We also found that miR-33 suppressed the CCL2 levels in the supernatant of cultured primary mouse chondrocytes. With reporter gene assay, we demonstrated that miR-33 targeted at AAUGCA in the 3'UTR of CCL2 gene. In transwell migration assays, we demonstrated that the conditional medium (CM) from miR-33 deficient chondrocytes potentiated the monocyte chemotaxis in a CCL2 dependent manner. Finally, we demonstrated that the level of miR-33 was decreased, whereas the CCL2 level was increased in the articular cartilage from the OA patients compared with the control group. In summary, we identified miR-33 as a novel suppressor of CCL2 in chondrocytes. The miR-33/CCL2 axis in chondrocytes regulates monocyte chemotaxis, providing a potential mechanism of macrophage infiltration in OA.

  2. Versatile and stable vectors for efficient gene expression in Ralstonia eutropha H16.

    PubMed

    Gruber, Steffen; Hagen, Jeremias; Schwab, Helmut; Koefinger, Petra

    2014-09-30

    The Gram-negative β-proteobacterium Ralstonia eutropha H16 is primarily known for polyhydroxybutyrate (PHB) production and its ability to grow chemolithoautotrophically by using CO2 and H2 as sole carbon and energy sources. The majority of metabolic engineering and heterologous expression studies conducted so far rely on a small number of suitable expression systems. Particularly the plasmid based expression systems already developed for the use in R. eutropha H16 suffer from high segregational instability and plasmid loss after a short time of fermentation. In order to develop efficient and highly stable plasmid expression vectors for the use in R. eutropha H16, a new plasmid design was created including the RP4 partitioning system, as well as various promoters and origins of replication. The application of minireplicons derived from broad-host-range plasmids RSF1010, pBBR1, RP4 and pSa for the construction of expression vectors and the use of numerous, versatile promoters extend the range of feasible expression levels considerably. In particular, the use of promoters derived from the bacteriophage T5 was described for the first time in this work, characterizing the j5 promoter as the strongest promoter yet to be applied in R. eutropha H16. Moreover, the implementation of the RP4 partition sequence in plasmid design increased plasmid stability significantly and enables fermentations with marginal plasmid loss of recombinant R. eutropha H16 for at least 96 h. The utility of the new vector family in R. eutropha H16 is demonstrated by providing expression data with different model proteins and consequently further raises the value of this organism as cell factory for biotechnological applications including protein and metabolite production.

  3. Reprint of "versatile and stable vectors for efficient gene expression in Ralstonia eutropha H16".

    PubMed

    Gruber, Steffen; Hagen, Jeremias; Schwab, Helmut; Koefinger, Petra

    2014-12-20

    The Gram-negative β-proteobacterium Ralstonia eutropha H16 is primarily known for polyhydroxybutyrate (PHB) production and its ability to grow chemolithoautotrophically by using CO2 and H2 as sole carbon and energy sources. The majority of metabolic engineering and heterologous expression studies conducted so far rely on a small number of suitable expression systems. Particularly the plasmid based expression systems already developed for the use in R. eutropha H16 suffer from high segregational instability and plasmid loss after a short time of fermentation. In order to develop efficient and highly stable plasmid expression vectors for the use in R. eutropha H16, a new plasmid design was created including the RP4 partitioning system, as well as various promoters and origins of replication. The application of minireplicons derived from broad-host-range plasmids RSF1010, pBBR1, RP4 and pSa for the construction of expression vectors and the use of numerous, versatile promoters extend the range of feasible expression levels considerably. In particular, the use of promoters derived from the bacteriophage T5 was described for the first time in this work, characterizing the j5 promoter as the strongest promoter yet to be applied in R. eutropha H16. Moreover, the implementation of the RP4 partition sequence in plasmid design increased plasmid stability significantly and enables fermentations with marginal plasmid loss of recombinant R. eutropha H16 for at least 96h. The utility of the new vector family in R. eutropha H16 is demonstrated by providing expression data with different model proteins and consequently further raises the value of this organism as cell factory for biotechnological applications including protein and metabolite production.

  4. [MicroRNA Target Prediction Based on Support Vector Machine Ensemble Classification Algorithm of Under-sampling Technique].

    PubMed

    Chen, Zhiru; Hong, Wenxue

    2016-02-01

    Considering the low accuracy of prediction in the positive samples and poor overall classification effects caused by unbalanced sample data of MicroRNA (miRNA) target, we proposes a support vector machine (SVM)-integration of under-sampling and weight (IUSM) algorithm in this paper, an under-sampling based on the ensemble learning algorithm. The algorithm adopts SVM as learning algorithm and AdaBoost as integration framework, and embeds clustering-based under-sampling into the iterative process, aiming at reducing the degree of unbalanced distribution of positive and negative samples. Meanwhile, in the process of adaptive weight adjustment of the samples, the SVM-IUSM algorithm eliminates the abnormal ones in negative samples with robust sample weights smoothing mechanism so as to avoid over-learning. Finally, the prediction of miRNA target integrated classifier is achieved with the combination of multiple weak classifiers through the voting mechanism. The experiment revealed that the SVM-IUSW, compared with other algorithms on unbalanced dataset collection, could not only improve the accuracy of positive targets and the overall effect of classification, but also enhance the generalization ability of miRNA target classifier.

  5. [MicroRNA Target Prediction Based on Support Vector Machine Ensemble Classification Algorithm of Under-sampling Technique].

    PubMed

    Chen, Zhiru; Hong, Wenxue

    2016-02-01

    Considering the low accuracy of prediction in the positive samples and poor overall classification effects caused by unbalanced sample data of MicroRNA (miRNA) target, we proposes a support vector machine (SVM)-integration of under-sampling and weight (IUSM) algorithm in this paper, an under-sampling based on the ensemble learning algorithm. The algorithm adopts SVM as learning algorithm and AdaBoost as integration framework, and embeds clustering-based under-sampling into the iterative process, aiming at reducing the degree of unbalanced distribution of positive and negative samples. Meanwhile, in the process of adaptive weight adjustment of the samples, the SVM-IUSM algorithm eliminates the abnormal ones in negative samples with robust sample weights smoothing mechanism so as to avoid over-learning. Finally, the prediction of miRNA target integrated classifier is achieved with the combination of multiple weak classifiers through the voting mechanism. The experiment revealed that the SVM-IUSW, compared with other algorithms on unbalanced dataset collection, could not only improve the accuracy of positive targets and the overall effect of classification, but also enhance the generalization ability of miRNA target classifier. PMID:27382743

  6. Real time facial expression recognition from image sequences using support vector machines

    NASA Astrophysics Data System (ADS)

    Kotsia, I.; Pitas, I.

    2005-07-01

    In this paper, a real-time method is proposed as a solution to the problem of facial expression classi cation in video sequences. The user manually places some of the Candide grid nodes to the face depicted at the rst frame. The grid adaptation system, based on deformable models, tracks the entire Candide grid as the facial expression evolves through time, thus producing a grid that corresponds to the greatest intensity of the facial expression, as shown at the last frame. Certain points that are involved into creating the Facial Action Units movements are selected. Their geometrical displacement information, de ned as the coordinates' dierence between the last and the rst frame, is extracted to be the input to a six class Support Vector Machine system. The output of the system is the facial expression recognized. The proposed real-time system, recognizes the 6 basic facial expressions with an approximately 98% accuracy.

  7. Engineering Hepadnaviruses as Reporter-Expressing Vectors: Recent Progress and Future Perspectives.

    PubMed

    Bai, Weiya; Cui, Xiaoxian; Xie, Youhua; Liu, Jing

    2016-01-01

    The Hepadnaviridae family of small, enveloped DNA viruses are characterized by a strict host range and hepatocyte tropism. The prototype hepatitis B virus (HBV) is a major human pathogen and constitutes a public health problem, especially in high-incidence areas. Reporter-expressing recombinant viruses are powerful tools in both studies of basic virology and development of antiviral therapeutics. In addition, the highly restricted tropism of HBV for human hepatocytes makes it an ideal tool for hepatocyte-targeting in vivo applications such as liver-specific gene delivery. However, compact genome organization and complex replication mechanisms of hepadnaviruses have made it difficult to engineer replication-competent recombinant viruses that express biologically-relevant cargo genes. This review analyzes difficulties associated with recombinant hepadnavirus vector development, summarizes and compares the progress made in this field both historically and recently, and discusses future perspectives regarding both vector design and application. PMID:27171106

  8. Engineering Hepadnaviruses as Reporter-Expressing Vectors: Recent Progress and Future Perspectives

    PubMed Central

    Bai, Weiya; Cui, Xiaoxian; Xie, Youhua; Liu, Jing

    2016-01-01

    The Hepadnaviridae family of small, enveloped DNA viruses are characterized by a strict host range and hepatocyte tropism. The prototype hepatitis B virus (HBV) is a major human pathogen and constitutes a public health problem, especially in high-incidence areas. Reporter-expressing recombinant viruses are powerful tools in both studies of basic virology and development of antiviral therapeutics. In addition, the highly restricted tropism of HBV for human hepatocytes makes it an ideal tool for hepatocyte-targeting in vivo applications such as liver-specific gene delivery. However, compact genome organization and complex replication mechanisms of hepadnaviruses have made it difficult to engineer replication-competent recombinant viruses that express biologically-relevant cargo genes. This review analyzes difficulties associated with recombinant hepadnavirus vector development, summarizes and compares the progress made in this field both historically and recently, and discusses future perspectives regarding both vector design and application. PMID:27171106

  9. The infectious BAC genomic DNA expression library: a high capacity vector system for functional genomics

    PubMed Central

    Lufino, Michele M. P.; Edser, Pauline A. H.; Quail, Michael A.; Rice, Stephen; Adams, David J.; Wade-Martins, Richard

    2016-01-01

    Gene dosage plays a critical role in a range of cellular phenotypes, yet most cellular expression systems use heterologous cDNA-based vectors which express proteins well above physiological levels. In contrast, genomic DNA expression vectors generate physiologically-relevant levels of gene expression by carrying the whole genomic DNA locus of a gene including its regulatory elements. Here we describe the first genomic DNA expression library generated using the high-capacity herpes simplex virus-1 amplicon technology to deliver bacterial artificial chromosomes (BACs) into cells by viral transduction. The infectious BAC (iBAC) library contains 184,320 clones with an average insert size of 134.5 kb. We show in a Chinese hamster ovary (CHO) disease model cell line and mouse embryonic stem (ES) cells that this library can be used for genetic rescue studies in a range of contexts including the physiological restoration of Ldlr deficiency, and viral receptor expression. The iBAC library represents an important new genetic analysis tool openly available to the research community. PMID:27353647

  10. New Recombinant Mycobacterium bovis BCG Expression Vectors: Improving Genetic Control over Mycobacterial Promoters

    PubMed Central

    Kanno, Alex I.; Goulart, Cibelly; Rofatto, Henrique K.; Oliveira, Sergio C.; Leite, Luciana C. C.

    2016-01-01

    The expression of many antigens, stimulatory molecules, or even metabolic pathways in mycobacteria such as Mycobacterium bovis BCG or M. smegmatis was made possible through the development of shuttle vectors, and several recombinant vaccines have been constructed. However, gene expression in any of these systems relied mostly on the selection of natural promoters expected to provide the required level of expression by trial and error. To establish a systematic selection of promoters with a range of strengths, we generated a library of mutagenized promoters through error-prone PCR of the strong PL5 promoter, originally from mycobacteriophage L5. These promoters were cloned upstream of the enhanced green fluorescent protein reporter gene, and recombinant M. smegmatis bacteria exhibiting a wide range of fluorescence levels were identified. A set of promoters was selected and identified as having high (pJK-F8), intermediate (pJK-B7, pJK-E6, pJK-D6), or low (pJK-C1) promoter strengths in both M. smegmatis and M. bovis BCG. The sequencing of the promoter region demonstrated that it was extensively modified (6 to 11%) in all of the plasmids selected. To test the functionality of the system, two different expression vectors were demonstrated to allow corresponding expression levels of the Schistosoma mansoni antigen Sm29 in BCG. The approach used here can be used to adjust expression levels for synthetic and/or systems biology studies or for vaccine development to maximize the immune response. PMID:26850295

  11. Construction and characterization of replication-competent simian immunodeficiency virus vectors that express gamma interferon.

    PubMed Central

    Giavedoni, L D; Yilma, T

    1996-01-01

    We report the construction and characterization of several replication-competent simian immunodeficiency virus (SIV) vectors with a deletion in the viral nef gene (SIV(delta nef)) that express gamma interferon (IFN-gamma). The expression of the cytokine gene was controlled either by the simian virus 40 early promoter or by the SIV 5' long terminal repeat regulatory sequences, utilizing the nef gene splice signals. To enhance the expression of IFN-gamma, the two in-frame nef start codons were mutated without altering the Env amino acid sequence (SIV(HyIFN)). Plasmids containing full-length proviral genomes were used to obtain high-titer stocks of each recombinant virus in cell cultures. Expression of IFN-gamma by SIV(HyIFN) reached levels as high as 10(6) U/ml after 11 days in culture. The IFN-gamma gene was unstable and sustained deletions after serial passage of SIV(delta nef) vectors in CEM-X-174 cells. The degree of instability appears to depend on size and orientation of the insert and the expression of IFN-gamma. Only one virus, SIV(HyIFN), expressed detectable levels of IFN-gamma up to the sixth passage. Prospects for the use of IFN-gamma and other lymphokines to enhance the safety and efficacy of live attenuated vaccines are discussed. PMID:8642649

  12. [An expression T-vector and its application at low temperatures].

    PubMed

    He, Yanbin; Qi, Yakun; Huang, Linting; Zhou, Rong; Shao, Weilan

    2015-12-01

    In modern biology and biotechnology research, recombinant gene expression has been the most popular method to obtain the target protein. In recent years, many foreign genes have been efficiently expressed in Escherichia coli. However, proteins encoded by animal, plant or mesophilic microbial genes often lose activities or become denatured within a few hours at regular growth temperatures for E. coli; some other target proteins are toxic to host cells and therefore difficult to be over-expressed. The new T-vector, pEXC-T, was constructed by combining TA cloning and cold-shock induction to obtain high expression levels with low costs. This paper reports the construction of pEXC-T and optimization of induction techniques for gene expression. Two instable proteins were tested and successfully expressed in soluble form by using pEXC vector. The development of pEXC-T offers a convenient technique for the preparations of recombinant proteins to be used in structure/function studies, or as diagnostic markers and medicinal proteins. PMID:27093840

  13. New Recombinant Mycobacterium bovis BCG Expression Vectors: Improving Genetic Control over Mycobacterial Promoters.

    PubMed

    Kanno, Alex I; Goulart, Cibelly; Rofatto, Henrique K; Oliveira, Sergio C; Leite, Luciana C C; McFadden, Johnjoe

    2016-04-01

    The expression of many antigens, stimulatory molecules, or even metabolic pathways in mycobacteria such as Mycobacterium bovis BCG or M. smegmatis was made possible through the development of shuttle vectors, and several recombinant vaccines have been constructed. However, gene expression in any of these systems relied mostly on the selection of natural promoters expected to provide the required level of expression by trial and error. To establish a systematic selection of promoters with a range of strengths, we generated a library of mutagenized promoters through error-prone PCR of the strong PL5 promoter, originally from mycobacteriophage L5. These promoters were cloned upstream of the enhanced green fluorescent protein reporter gene, and recombinant M. smegmatis bacteria exhibiting a wide range of fluorescence levels were identified. A set of promoters was selected and identified as having high (pJK-F8), intermediate (pJK-B7, pJK-E6, pJK-D6), or low (pJK-C1) promoter strengths in both M. smegmatis and M. bovisBCG. The sequencing of the promoter region demonstrated that it was extensively modified (6 to 11%) in all of the plasmids selected. To test the functionality of the system, two different expression vectors were demonstrated to allow corresponding expression levels of the Schistosoma mansoni antigen Sm29 in BCG. The approach used here can be used to adjust expression levels for synthetic and/or systems biology studies or for vaccine development to maximize the immune response. PMID:26850295

  14. A new Ebola virus nonstructural glycoprotein expressed through RNA editing.

    PubMed

    Mehedi, Masfique; Falzarano, Darryl; Seebach, Jochen; Hu, Xiaojie; Carpenter, Michael S; Schnittler, Hans-Joachim; Feldmann, Heinz

    2011-06-01

    Ebola virus (EBOV), an enveloped, single-stranded, negative-sense RNA virus, causes severe hemorrhagic fever in humans and nonhuman primates. The EBOV glycoprotein (GP) gene encodes the nonstructural soluble glycoprotein (sGP) but also produces the transmembrane glycoprotein (GP₁,₂) through transcriptional editing. A third GP gene product, a small soluble glycoprotein (ssGP), has long been postulated to be produced also as a result of transcriptional editing. To identify and characterize the expression of this new EBOV protein, we first analyzed the relative ratio of GP gene-derived transcripts produced during infection in vitro (in Vero E6 cells or Huh7 cells) and in vivo (in mice). The average percentages of transcripts encoding sGP, GP₁,₂, and ssGP were approximately 70, 25, and 5%, respectively, indicating that ssGP transcripts are indeed produced via transcriptional editing. N-terminal sequence similarity with sGP, the absence of distinguishing antibodies, and the abundance of sGP made it difficult to identify ssGP through conventional methodology. Optimized 2-dimensional (2D) gel electrophoresis analyses finally verified the expression and secretion of ssGP in tissue culture during EBOV infection. Biochemical analysis of recombinant ssGP characterized this protein as a disulfide-linked homodimer that was exclusively N glycosylated. In conclusion, we have identified and characterized a new EBOV nonstructural glycoprotein, which is expressed as a result of transcriptional editing of the GP gene. While ssGP appears to share similar structural properties with sGP, it does not appear to have the same anti-inflammatory function on endothelial cells as sGP.

  15. A novel, broad-range, CTXΦ-derived stable integrative expression vector for functional studies.

    PubMed

    Das, Bhabatosh; Kumari, Reena; Pant, Archana; Sen Gupta, Sourav; Saxena, Shruti; Mehta, Ojasvi; Nair, Gopinath Balakrish

    2014-12-01

    CTXΦ, a filamentous vibriophage encoding cholera toxin, uses a unique strategy for its lysogeny. The single-stranded phage genome forms intramolecular base-pairing interactions between two inversely oriented XerC and XerD binding sites (XBS) and generates a functional phage attachment site, attP(+), for integration. The attP(+) structure is recognized by the host-encoded tyrosine recombinases XerC and XerD (XerCD), which enables irreversible integration of CTXΦ into the chromosome dimer resolution site (dif) of Vibrio cholerae. The dif site and the XerCD recombinases are widely conserved in bacteria. We took advantage of these conserved attributes to develop a broad-host-range integrative expression vector that could irreversibly integrate into the host chromosome using XerCD recombinases without altering the function of any known open reading frame (ORF). In this study, we engineered two different arabinose-inducible expression vectors, pBD62 and pBD66, using XBS of CTXΦ. pBD62 replicates conditionally and integrates efficiently into the dif of the bacterial chromosome by site-specific recombination using host-encoded XerCD recombinases. The expression level of the gene of interest could be controlled through the PBAD promoter by modulating the functions of the vector-encoded transcriptional factor AraC. We validated the irreversible integration of pBD62 into a wide range of pathogenic and nonpathogenic bacteria, such as V. cholerae, Vibrio fluvialis, Vibrio parahaemolyticus, Escherichia coli, Salmonella enterica, and Klebsiella pneumoniae. Gene expression from the PBAD promoter of integrated vectors was confirmed in V. cholerae using the well-studied reporter genes mCherry, eGFP, and lacZ.

  16. Thyroid epithelial cell transformation by a retroviral vector expressing SV40 large T.

    PubMed Central

    Burns, J. S.; Lemoine, L.; Lemoine, N. R.; Williams, E. D.; Wynford-Thomas, D.

    1989-01-01

    A recombinant murine retroviral vector encoding the SV40 virus large T antigen was used to infect stably an immortal line of differentiated rat thyroid epithelial cells, FRTL-5. Expression of SV40 T transformed these cells to anchorage independence and tumorigenicity but did not alter morphology or abolish tissue-specific functions and growth factor requirements. The resulting phenotype provides a model of well-differentiated human thyroid cancer. Images Figure 1 Figure 3 PMID:2544221

  17. Enhanced stability of microRNA expression facilitates classification of FFPE tumour samples exhibiting near total mRNA degradation

    PubMed Central

    Hall, J S; Taylor, J; Valentine, H R; Irlam, J J; Eustace, A; Hoskin, P J; Miller, C J; West, C M L

    2012-01-01

    Background: As degradation of formalin-fixed paraffin-embedded (FFPE) samples limits the ability to profile mRNA expression, we explored factors predicting the success of mRNA expression profiling of FFPE material and investigated an approach to overcome the limitation. Methods: Bladder (n=140, stored 3–8 years) and cervix (n=160, stored 8–23 years) carcinoma FFPE samples were hybridised to Affymetrix Exon 1.0ST arrays. Percentage detection above background (%DABG) measured technical success. Biological signal was assessed by distinguishing cervix squamous cell carcinoma (SCC) and adenocarcinoma (AC) using a gene signature. As miR-205 had been identified as a marker of SCC, precursor mir-205 was measured by Exon array and mature miR-205 by qRT–PCR. Genome-wide microRNA (miRNA) expression (Affymetrix miRNA v2.0 arrays) was compared in eight newer FFPE samples with biological signal and eight older samples without. Results: RNA quality controls (QCs) (e.g., RNA integrity (RIN) number) failed to predict profiling success, but sample age correlated with %DABG in bladder (R=−0.30, P<0.01) and cervix (R=−0.69, P<0.01). Biological signal was lost in older samples and neither a signature nor precursor mir-205 separated samples by histology. miR-205 qRT–PCR discriminated SCC from AC, validated by miRNA profiling (26-fold higher in SCC; P=1.10 × 10−5). Genome-wide miRNA (R=0.95) and small nucleolar RNA (R=0.97) expression correlated well in the eight newer vs older FFPE samples and better than mRNA expression (R=0.72). Conclusion: Sample age is the best predictor of successful mRNA profiling of FFPE material, and miRNA profiling overcomes the limitation of age and copes well with older samples. PMID:22805332

  18. Biological mechanism analysis of acute renal allograft rejection: integrated of mRNA and microRNA expression profiles

    PubMed Central

    Huang, Shi-Ming; Zhao, Xia; Zhao, Xue-Mei; Wang, Xiao-Ying; Li, Shan-Shan; Zhu, Yu-Hui

    2014-01-01

    Objectives: Renal transplantation is the preferred method for most patients with end-stage renal disease, however, acute renal allograft rejection is still a major risk factor for recipients leading to renal injury. To improve the early diagnosis and treatment of acute rejection, study on the molecular mechanism of it is urgent. Methods: MicroRNA (miRNA) expression profile and mRNA expression profile of acute renal allograft rejection and well-functioning allograft downloaded from ArrayExpress database were applied to identify differentially expressed (DE) miRNAs and DE mRNAs. DE miRNAs targets were predicted by combining five algorithm. By overlapping the DE mRNAs and DE miRNAs targets, common genes were obtained. Differentially co-expressed genes (DCGs) were identified by differential co-expression profile (DCp) and differential co-expression enrichment (DCe) methods in Differentially Co-expressed Genes and Links (DCGL) package. Then, co-expression network of DCGs and the cluster analysis were performed. Functional enrichment analysis for DCGs was undergone. Results: A total of 1270 miRNA targets were predicted and 698 DE mRNAs were obtained. While overlapping miRNA targets and DE mRNAs, 59 common genes were gained. We obtained 103 DCGs and 5 transcription factors (TFs) based on regulatory impact factors (RIF), then built the regulation network of miRNA targets and DE mRNAs. By clustering the co-expression network, 5 modules were obtained. Thereinto, module 1 had the highest degree and module 2 showed the most number of DCGs and common genes. TF CEBPB and several common genes, such as RXRA, BASP1 and AKAP10, were mapped on the co-expression network. C1R showed the highest degree in the network. These genes might be associated with human acute renal allograft rejection. Conclusions: We conducted biological analysis on integration of DE mRNA and DE miRNA in acute renal allograft rejection, displayed gene expression patterns and screened out genes and TFs that may

  19. Expression of the human interleukin-2 receptor gamma chain in insect cells using a baculovirus expression vector.

    PubMed

    Raivio, E; Oetken, C; Oker-Blom, C; Engberg, C; Akerman, K; Lindqvist, C

    1995-04-01

    The gene encoding the gamma-chain of the human Interleukin-2 receptor was expressed in lepidopteran insect cells using the baculovirus expression vector system. The corresponding gene was inserted under the polyhedrin promoter of the Autographa californica nuclear polyhedrosis virus and expressed in the Spodoptera frugiperda insect cell line Sf9 during viral infection. The recombinant receptor protein was identified by immunoblotting in cell lysates, prepared from insect cells infected with the recombinant virus. At 40 h post infection the corresponding protein was detected as two major bands with apparent molecular weights of 50-60 kDa using a rabbit anti-human IL-2R gamma-receptor specific antiserum. Metabolic labelling with [35S]-methionine and SDS-PAGE analysis of the recombinant baculovirus infected insect cells verified the immunoblotting data. The expressed IL-2R gamma- protein could also be determined on the surface of infected insect cells by flow cytometer analysis. PMID:7899821

  20. Tolerance and responsive gene expression of Sogatella furcifera under extreme temperature stresses are altered by its vectored plant virus

    PubMed Central

    Xu, Donglin; Zhong, Ting; Feng, Wendi; Zhou, Guohui

    2016-01-01

    Southern rice black-streaked dwarf virus (SRBSDV), a newly emerged fijivirus causing great loss to rice production in eastern and southeastern Asian countries in recent years, is efficiently transmitted by a rice pest, white-backed planthopper (WBPH, Sogatella furcifera) in a persistent, circulative propagative manner and can be considered as an insect virus. In this study, SRBSDV infection in WBPH was found to increase the vector’s death rate under extreme cold stress but improve its survival rate under extreme heat stress. Digital gene expression profiling based on RNA-Seq revealed different gene regulation patterns in WBPH under viral and/or temperature stress. Under cold stress, the virus infection upregulated 1540 genes and downregulated 131 genes in the insect, most of which were related to membrane properties and biological processes of actin and cytoskeleton; whereas under heat stress, it upregulated 363 genes and downregulated 548 genes, most of which were associated to metabolism and intracellular organelles. Several types of stress-responsive genes involving intestinal mucin, cuticle protein, ubiquitin protease, immune response, RNA interference and heat shock response, were largely upregulated under cold stress, but largely downregulated under heat stress, by SRBSDV infection. Our results suggest two distinct mechanisms of virus-altered vector insect tolerance to temperature stress. PMID:27531640

  1. Treatment of canine leukocyte adhesion deficiency by foamy virus vectors expressing CD18 from a PGK promoter.

    PubMed

    Bauer, T R; Olson, E M; Huo, Y; Tuschong, L M; Allen, J M; Li, Y; Burkholder, T H; Russell, D W

    2011-06-01

    Proto-oncogene activation caused by retroviral vector integration can cause malignancies in gene therapy trials. This has led investigators to search for less genotoxic vectors with minimal enhancer activity and a decreased risk of influencing neighboring chromosomal gene expression after integration. We previously showed that foamy virus (FV) vectors expressing the canine CD18 gene from an internal murine stem cell virus (MSCV) promoter could cure canine leukocyte adhesion deficiency (LAD). Here, we have repeated these studies using a FV vector expressing canine CD18 from a phosphoglycerate kinase (PGK) gene promoter. In vitro analysis showed that this vector did not contain an enhancer that activated neighboring genes, and it expressed CD18 efficiently in canine neutrophils and CD34+ cells. However, dogs that received hematopoietic stem cells transduced with the PGK-CD18 vector continued to suffer from LAD, and sometimes died prematurely of the disease. These studies show that the PGK promoter cannot effectively replace the MSCV promoter in CD18-expressing FV vectors, and they suggest that vectors containing a strong promoter-enhancer may be necessary for the treatment of human LAD. PMID:21228879

  2. A Novel and Simple Method for Rapid Generation of Recombinant Porcine Adenoviral Vectors for Transgene Expression

    PubMed Central

    Ma, Jing; Wang, Wenbin; Zhang, Lu; Tikoo, Suresh K.; Yang, Zengqi

    2015-01-01

    Many human (different serotypes) and nonhuman adenovirus vectors are being used for gene delivery. However, the current system for isolating recombinant adenoviral vectors is either time-consuming or expensive, especially for the generation of recombinant non-human adenoviral vectors. We herein report a new and simple cloning approach for the rapid generation of a porcine adenovirus (PAdV-3) vector which shows promise for gene transfer to human cells and evasion of human adenovirus type 5 (HAdV-5) immunity. Based on the final cloning plasmid, pFPAV3-CcdB-Cm, and our modified SLiCE strategy (SLiCE cloning and lethal CcdB screening), the process for generating recombinant PAdV-3 plasmids required only one step in 3 days, with a cloning efficiency as high as 620±49.56 clones/ng and zero background (100% accuracy). The recombinant PAdV-3 plasmids could be successfully rescued in porcine retinal pigment epithelium cells (VR1BL), which constitutively express the HAdV-5 E1 and PAdV-3 E1B 55k genes, and the foreign genes were highly expressed at 24 h after transduction into swine testicle (ST) cells. In conclusion, this strategy for generating recombinant PAdV-3 vectors based on our modified SLiCE cloning system was rapid and cost-efficient, which could be used as universal cloning method for modification the other regions of PAdV-3 genome as well as other adenoviral genomes. PMID:26011074

  3. A novel and simple method for rapid generation of recombinant porcine adenoviral vectors for transgene expression.

    PubMed

    Zhang, Peng; Du, Enqi; Ma, Jing; Wang, Wenbin; Zhang, Lu; Tikoo, Suresh K; Yang, Zengqi

    2015-01-01

    Many human (different serotypes) and nonhuman adenovirus vectors are being used for gene delivery. However, the current system for isolating recombinant adenoviral vectors is either time-consuming or expensive, especially for the generation of recombinant non-human adenoviral vectors. We herein report a new and simple cloning approach for the rapid generation of a porcine adenovirus (PAdV-3) vector which shows promise for gene transfer to human cells and evasion of human adenovirus type 5 (HAdV-5) immunity. Based on the final cloning plasmid, pFPAV3-CcdB-Cm, and our modified SLiCE strategy (SLiCE cloning and lethal CcdB screening), the process for generating recombinant PAdV-3 plasmids required only one step in 3 days, with a cloning efficiency as high as 620 ± 49.56 clones/ng and zero background (100% accuracy). The recombinant PAdV-3 plasmids could be successfully rescued in porcine retinal pigment epithelium cells (VR1BL), which constitutively express the HAdV-5 E1 and PAdV-3 E1B 55k genes, and the foreign genes were highly expressed at 24 h after transduction into swine testicle (ST) cells. In conclusion, this strategy for generating recombinant PAdV-3 vectors based on our modified SLiCE cloning system was rapid and cost-efficient, which could be used as universal cloning method for modification the other regions of PAdV-3 genome as well as other adenoviral genomes.

  4. Autoregulation of Inducible Nitric Oxide Synthase Expression by RNA Interference Provides Neuroprotection in Neonatal Rats

    PubMed Central

    Wang, Zhi; Feng, Chenzhuo; Zhao, Huijuan; Ren, Xiaoyan; Peng, Shuling; Zuo, Zhiyi

    2015-01-01

    We have shown that autoregulation of gene expression by RNA interference is achievable in cell cultures. To determine whether this novel concept could be used to produce neuroprotection under in vivo condition, postnatal day (PND) 3 rats received intracerebroventricular injection of lentivirus that carried or did not carry code for short hairpin RNA (shRNA) of inducible nitric oxide synthase (iNOS). The expression of this shRNA was controlled by an iNOS promoter (piNOS-shRNA) or cytomegalovirus promoter (pCMV-shRNA). The rats were subjected to brain hypoxia-ischemia at PND7. Ischemic brain tissues had increased iNOS expression. This increase was attenuated by virus carrying piNOS-shRNA. Virus carrying pCMV-shRNA reduced iNOS to a level that was lower than control. Brain tissue loss and functional impairment after the hypoxia-ischemia were attenuated by the virus carrying piNOS-shRNA but not by pCMV-shRNA. Our results provide proof-of-concept evidence that autoregulation of iNOS expression by RNA interference induces neuroprotection in vivo and that appropriate regulation of gene expression is important. PMID:25767617

  5. Killing of cancer cells through the use of eukaryotic expression vectors harbouring genes encoding nucleases and ribonuclease inhibitor.

    PubMed

    Glinka, Elena M

    2015-05-01

    Cancer gene therapy vectors are promising tools for killing cancer cells with the purpose of eradicating malignant tumours entirely. Different delivery methods of vectors into the cancer cells, including both non-viral and viral, as well as promoters for the targeted expression of genes encoding anticancer proteins were developed for effective and selective killing of cancer cells without harming healthy cells. Many vectors have been created to kill cancer cells, and some vectors suppress malignant tumours with high efficiency. This review is focused on vectors bearing genes for nucleases such as deoxyribonucleases (caspase-activated DNase, deoxyribonuclease I-like 3, endonuclease G) and ribonucleases (human polynucleotide phosphorylase, ribonuclease L, α-sarcin, barnase), as well as vectors harbouring gene encoding ribonuclease inhibitor. The data concerning the functionality and the efficacy of such vectors are presented.

  6. Under-Expression of Chemosensory Genes in Domiciliary Bugs of the Chagas Disease Vector Triatoma brasiliensis

    PubMed Central

    Marchant, Axelle; Mougel, Florence; Jacquin-Joly, Emmanuelle; Costa, Jane; Almeida, Carlos Eduardo; Harry, Myriam

    2016-01-01

    Background In Latin America, the bloodsucking bugs Triatominae are vectors of Trypanosoma cruzi, the parasite that causes Chagas disease. Chemical elimination programs have been launched to control Chagas disease vectors. However, the disease persists because native vectors from sylvatic habitats are able to (re)colonize houses—a process called domiciliation. Triatoma brasiliensis is one example. Because the chemosensory system allows insects to interact with their environment and plays a key role in insect adaption, we conducted a descriptive and comparative study of the chemosensory transcriptome of T. brasiliensis samples from different ecotopes. Methodology/Principal Finding In a reference transcriptome built using de novo assembly, we found transcripts encoding 27 odorant-binding proteins (OBPs), 17 chemosensory proteins (CSPs), 3 odorant receptors (ORs), 5 transient receptor potential channel (TRPs), 1 sensory neuron membrane protein (SNMPs), 25 takeout proteins, 72 cytochrome P450s, 5 gluthatione S-transferases, and 49 cuticular proteins. Using protein phylogenies, we showed that most of the OBPs and CSPs for T. brasiliensis had well supported orthologs in the kissing bug Rhodnius prolixus. We also showed a higher number of these genes within the bloodsucking bugs and more generally within all Hemipterans compared to the other species in the super-order Paraneoptera. Using both DESeq2 and EdgeR software, we performed differential expression analyses between samples of T. brasiliensis, taking into account their environment (sylvatic, peridomiciliary and domiciliary) and sex. We also searched clusters of co-expressed contigs using HTSCluster. Among differentially expressed (DE) contigs, most were under-expressed in the chemosensory organs of the domiciliary bugs compared to the other samples and in females compared to males. We clearly identified DE genes that play a role in the chemosensory system. Conclusion/Significance Chemosensory genes could be good

  7. Pulmonary Delivery of siRNA via Polymeric Vectors as Therapies of Asthma.

    PubMed

    Xie, Yuran; Merkel, Olivia M

    2015-10-01

    Asthma is a chronic inflammatory disease. Despite the fact that current therapies, such as the combination of inhaled corticosteroids and β2-agonists, can control the symptoms of asthma in most patients, there is still an urgent need for an alternative anti-inflammatory therapy for patients who suffer from severe asthma but lack acceptable response to conventional therapies. Many molecular factors are involved in the inflammatory process in asthma, and thus blocking the function of these factors could efficiently alleviate airway inflammation. RNA interference (RNAi) is often thought to be the answer in the search for more efficient and biocompatible treatments. However, difficulties of efficient delivery of small interference RNA (siRNA), the key factor in RNAi, to target cells and tissues have limited its clinical application. In this review, we summarize cytokines and chemokines, transcription factors, tyrosine kinases, and costimulatory factors that have been reported as targets of siRNA-mediated treatment in experimental asthma. Additionally, we conclude several targeted delivery systems of siRNA to specific cells such as T cells, macrophages, and dendritic cells, which could potentially be applied in asthma therapy.

  8. Mesomorphic imidazolium salts: new vectors for efficient siRNA transfection.

    PubMed

    Dobbs, William; Heinrich, Benoît; Bourgogne, Cyril; Donnio, Bertrand; Terazzi, Emmanuel; Bonnet, Marie-Elise; Stock, Fabrice; Erbacher, Patrick; Bolcato-Bellemin, Anne-Laure; Douce, Laurent

    2009-09-23

    The preparation of chloride (1(n)) and bromide (2(n)) derivatives of 1-methyl-3-[3,4-bis(alkoxy)benzyl]-4H-imidazolium with n = 6, 12, 16, 18 is described. The two series of salts possess a rich thermotropic mesomorphism, chain-length dependent. Thus, a lamellar smectic A phase, a bicontinuous cubic Ia3d phase, and a columnar hexagonal liquid crystalline mesophase are induced as a function of increasing chain length. The mesomorphic properties were studied by polarizing optical microscopy, differential scanning calorimetry, and X-ray diffraction, and with the support of dilatometry and molecular dynamics, models for the various supramolecular arrangements of the salts are proposed. Such cationic amphiphiles were expected to be candidate molecules to design a new delivery reagent for nucleic acid transfection, particularly for short interfering RNA (siRNA). The use of an RNA interference mechanism, by introduction into cells by transfection of chemically synthesized siRNAs, is a powerful method for gene silencing studies. To exploit the potential of these amphilic imidazolium salts, these molecules were formulated with cohelper lipids and tested for their efficacy to deliver active siRNAs. Our results show high transfection efficacy of our formulated compounds and high silencing efficiency with more than 80% inhibition of the targeted gene at 10 nM siRNA concentration. Taken together our results show the potency of amphiphilic imidazolium salts as a new generation of transfection reagents for RNA interference. PMID:19715309

  9. Long noncoding RNA #32 contributes to antiviral responses by controlling interferon-stimulated gene expression.

    PubMed

    Nishitsuji, Hironori; Ujino, Saneyuki; Yoshio, Sachiyo; Sugiyama, Masaya; Mizokami, Masashi; Kanto, Tatsuya; Shimotohno, Kunitada

    2016-09-13

    Despite the breadth of knowledge that exists regarding the function of long noncoding RNAs (lncRNAs) in biological phenomena, the role of lncRNAs in host antiviral responses is poorly understood. Here, we report that lncRNA#32 is associated with type I IFN signaling. The silencing of lncRNA#32 dramatically reduced the level of IFN-stimulated gene (ISG) expression, resulting in sensitivity to encephalomyocarditis virus (EMCV) infection. In contrast, the ectopic expression of lncRNA#32 significantly suppressed EMCV replication, suggesting that lncRNA#32 positively regulates the host antiviral response. We further demonstrated the suppressive function of lncRNA#32 in hepatitis B virus and hepatitis C virus infection. lncRNA#32 bound to activating transcription factor 2 (ATF2) and regulated ISG expression. Our results reveal a role for lncRNA#32 in host antiviral responses. PMID:27582466

  10. microRNA regulation of molecular networks mapped by global microRNA, mRNA, and protein expression in activated T-lymphocytes

    PubMed Central

    Grigoryev, Yevgeniy A.; Kurian, Sunil M.; Hart, Traver; Nakorchevsky, Aleksey A.; Chen, Caifu; Campbell, Daniel; Head, Steven R.; Yates, John R.; Salomon, Daniel. R

    2011-01-01

    MicroRNAs (miRNAs) regulate specific immune mechanisms but their genome-wide regulation of T-lymphocyte activation is largely unknown. We performed a multidimensional functional genomics analysis to integrate genome-wide differential mRNA, miRNA, and protein expression as a function of human T-lymphocyte activation and time. We surveyed expression of 420 human miRNAs in parallel with genome-wide mRNA expression. We identified a unique signature of 71 differentially expressed miRNAs, 57 of which were previously not known as regulators of immune activation. The majority of miRNAs are upregulated, mRNA expression of these target genes is downregulated and this is a function of binding multiple miRNAs (combinatorial targeting). Our data reveal that consideration of this complex signature, rather than single miRNAs, is necessary to construct a full picture of miRNA-mediated regulation. Molecular network mapping of miRNA targets revealed the regulation of activation-induced immune signaling. In contrast, pathways populated by genes that are not miRNA targets are enriched for metabolism and biosynthesis. Finally, we specifically validated miR-155 (known) and miR-221 (novel in T-lymphocytes) using locked nucleic acid inhibitors. Inhibition of these 2 highly upregulated miRNAs in CD4+ T cells were shown to increase proliferation by removing suppression of 4 target genes linked to proliferation and survival. Thus, multiple lines of evidence link top functional networks directly to T-lymphocyte immunity underlining the value of mapping global gene, protein and miRNA expression. PMID:21788445

  11. Development of Plant Gene Vectors for Tissue-Specific Expression Using GFP as a Reporter Gene

    NASA Technical Reports Server (NTRS)

    Jackson, Jacquelyn; Egnin, Marceline; Xue, Qi-Han; Prakash, C. S.

    1997-01-01

    Reporter genes are widely employed in plant molecular biology research to analyze gene expression and to identify promoters. Gus (UidA) is currently the most popular reporter gene but its detection requires a destructive assay. The use of jellyfish green fluorescent protein (GFP) gene from Aequorea Victoria holds promise for noninvasive detection of in vivo gene expression. To study how various plant promoters are expressed in sweet potato (Ipomoea batatas), we are transcriptionally fusing the intron-modified (mGFP) or synthetic (modified for codon-usage) GFP coding regions to these promoters: double cauliflower mosaic virus 35S (CaMV 35S) with AMV translational enhancer, ubiquitin7-intron-ubiquitin coding region (ubi7-intron-UQ) and sporaminA. A few of these vectors have been constructed and introduced into E. coli DH5a and Agrobacterium tumefaciens EHA105. Transient expression studies are underway using protoplast-electroporation and particle bombardment of leaf tissues.

  12. Vector Development for the Expression of Foreign Proteins in the Vaccine Strain Brucella abortus S19

    PubMed Central

    Comerci, Diego J.; Pollevick, Guido D.; Vigliocco, Ana M.; Frasch, Alberto C. C.; Ugalde, Rodolfo A.

    1998-01-01

    A vector for the expression of foreign antigens in the vaccine strain Brucella abortus S19 was developed by using a DNA fragment containing the regulatory sequences and the signal peptide of the Brucella bcsp31 gene. This fragment was cloned in broad-host-range plasmid pBBR4MCS, resulting in plasmid pBEV. As a reporter protein, a repetitive antigen of Trypanosoma cruzi was used. The recombinant fusion protein is stably expressed and secreted into the Brucella periplasmic space, inducing a good antibody response against the T. cruzi antigen. The expression of the repetitive antigen in Brucella neither altered its growth pattern nor generated a toxic or lethal effect during experimental infection. The application of this strategy for the generation of live recombinant vaccines and the tagging of B. abortus S19 vaccine is discussed. This is the first time that a recombinant protein has been expressed in the periplasm of brucellae. PMID:9673273

  13. A new series of vectors for constitutive, inducible or repressible gene expression in Candida guilliermondii.

    PubMed

    Defosse, Tatiana A; Melin, Céline; Obando Montoya, Erika J; Lanoue, Arnaud; Foureau, Emilien; Glévarec, Gaëlle; Oudin, Audrey; Simkin, Andrew J; Crèche, Joël; Atehortùa, Lucia; Giglioli-Guivarc'h, Nathalie; Clastre, Marc; Courdavault, Vincent; Papon, Nicolas

    2014-06-20

    The biotechnological potential of C. guilliermondii is now well established. This yeast species currently benefits from the availability of a convenient molecular toolbox including recipient strains, selectable markers and optimized transformation protocols. However, the number of expression systems for biotechnological applications in C. guilliermondii remains limited. We have therefore developed and characterized a new series of versatile controllable expression vectors for this yeast. While previous studies firmly demonstrated that knock-out systems represent efficient genetic strategies to interrupt yeast biochemical pathways at a specific step in C. guilliermondii, the set of expression plasmids described in this study will provide new powerful opportunities to boost homologous or heterologous biosynthetic routes by fine controlled over-expression approaches. PMID:24709398

  14. Combination of the somatic cell nuclear transfer method and RNAi technology for the production of a prion gene-knockdown calf using plasmid vectors harboring the U6 or tRNA promoter.

    PubMed

    Wongsrikeao, Pimprapar; Sutou, Shizuyo; Kunishi, Miho; Dong, Ya Juan; Bai, Xuejin; Otoi, Takeshige

    2011-01-01

    By combining RNAi technology with SCNT method, we attempted to produce transgenic calves with knocked down bPRNP for technological assessments. The respective utilities of type II (tRNA) and type III (hU6) Pol III promoters in mediating plasmid vector-based RNAi for the production of a bPRNP-knockdown calf were compared. Plasmid harboring DNA for siRNA expression was introduced stably into the genome of primary cultured bovine cells. By inserting the transgenic cell into an enucleated bovine egg, SCNT embryos were produced. The ability for SCNT embryos to develop to blastocysts was higher in hU6 based vector groups (44-53%) than in a tRNA group (32%). In all, 30 hU6-embryos and 12 tRNA-embryos were transferred to 11 recipients. Only tRNA-embryos were able to impregnate recipients (6 out of 11 transfers), resulting in four aborted fetuses, one stillbirth, and one live-born calf. The expression of EGFP, a marker, was detected in all six. The bPRNP transcript levels in the nervous tissues (brain, cerebellum, spinal bulb, and spinal cord) from the calf, which was killed 20 days after birth, were reduced to 35% of those of the control calf on average, as determined by qRT-PCR. The PrPC levels, as estimated by western blot were reduced to 86% on average in the nervous tissues. These findings suggest that SCNT technology remains immature, that the tRNA promoter is useful, and that RNAi can significantly reduce PRNP mRNA levels, but insufficient reduction of PrPC levels exists in cattle under these conditions.

  15. Genome-Wide Patterns of Gene Expression during Aging in the African Malaria Vector Anopheles gambiae

    PubMed Central

    Wang, Mei-Hui; Marinotti, Osvaldo; James, Anthony A.; Walker, Edward; Githure, John; Yan, Guiyun

    2010-01-01

    The primary means of reducing malaria transmission is through reduction in longevity in days of the adult female stage of the Anopheles vector. However, assessing chronological age is limited to crude physiologic methods which categorize the females binomially as either very young (nulliparous) or not very young (parous). Yet the epidemiologically relevant reduction in life span falls within the latter category. Age-grading methods that delineate chronological age, using accurate molecular surrogates based upon gene expression profiles, will allow quantification of the longevity-reducing effects of vector control tools aimed at the adult, female mosquito. In this study, microarray analyses of gene expression profiles in the African malaria vector Anopheles gambiae were conducted during natural senescence of females in laboratory conditions. Results showed that detoxification-related and stress-responsive genes were up-regulated as mosquitoes aged. A total of 276 transcripts had age-dependent expression, independently of blood feeding and egg laying events. Expression of 112 (40.6%) of these transcripts increased or decreased monotonically with increasing chronologic age. Seven candidate genes for practical age assessment were tested by quantitative gene amplification in the An. gambiae G3 strain in a laboratory experiment and the Mbita strain in field enclosures set up in western Kenya under conditions closely resembling natural ones. Results were similar between experiments, indicating that senescence is marked by changes in gene expression and that chronological age can be gauged accurately and repeatedly with this method. These results indicate that the method may be suitable for accurate gauging of the age in days of field-caught, female An. gambiae. PMID:20967211

  16. Systemic spread of an RNA insect virus in plants expressing plant viral movement protein genes

    PubMed Central

    Dasgupta, Ranjit; Garcia, Bradley H.; Goodman, Robert M.

    2001-01-01

    Flock house virus (FHV), a single-stranded RNA insect virus, has previously been reported to cross the kingdom barrier and replicate in barley protoplasts and in inoculated leaves of several plant species [Selling, B. H., Allison, R. F. & Kaesberg, P. (1990) Proc. Natl. Acad. Sci. USA 87, 434–438]. There was no systemic movement of FHV in plants. We tested the ability of movement proteins (MPs) of plant viruses to provide movement functions and cause systemic spread of FHV in plants. We compared the growth of FHV in leaves of nontransgenic and transgenic plants expressing the MP of tobacco mosaic virus or red clover necrotic mosaic virus (RCNMV). Both MPs mobilized cell-to-cell and systemic movement of FHV in Nicotiana benthamiana plants. The yield of FHV was more than 100-fold higher in the inoculated leaves of transgenic plants than in the inoculated leaves of nontransgenic plants. In addition, FHV accumulated in the noninoculated upper leaves of both MP-transgenic plants. RCNMV MP was more efficient in mobilizing FHV to noninoculated upper leaves. We also report here that FHV replicates in inoculated leaves of six additional plant species: alfalfa, Arabidopsis, Brassica, cucumber, maize, and rice. Our results demonstrate that plant viral MPs cause cell-to-cell and long-distance movement of an animal virus in plants and offer approaches to the study of the evolution of viruses and mechanisms governing mRNA trafficking in plants as well as to the development of promising vectors for transient expression of foreign genes in plants. PMID:11296259

  17. Targeted RNA Sequencing Assay to Characterize Gene Expression and Genomic Alterations.

    PubMed

    Martin, Dorrelyn P; Miya, Jharna; Reeser, Julie W; Roychowdhury, Sameek

    2016-01-01

    RNA sequencing (RNAseq) is a versatile method that can be utilized to detect and characterize gene expression, mutations, gene fusions, and noncoding RNAs. Standard RNAseq requires 30 - 100 million sequencing reads and can include multiple RNA products such as mRNA and noncoding RNAs. We demonstrate how targeted RNAseq (capture) permits a focused study on selected RNA products using a desktop sequencer. RNAseq capture can characterize unannotated, low, or transiently expressed transcripts that may otherwise be missed using traditional RNAseq methods. Here we describe the extraction of RNA from cell lines, ribosomal RNA depletion, cDNA synthesis, preparation of barcoded libraries, hybridization and capture of targeted transcripts and multiplex sequencing on a desktop sequencer. We also outline the computational analysis pipeline, which includes quality control assessment, alignment, fusion detection, gene expression quantification and identification of single nucleotide variants. This assay allows for targeted transcript sequencing to characterize gene expression, gene fusions, and mutations. PMID:27585245

  18. Regulation of microRNA expression and function by nuclear receptor signaling

    PubMed Central

    2011-01-01

    MicroRNAs (miRNAs) are small non-coding RNA transcripts that affect various cellular pathways by serving as regulators of gene expression at the translational and transcriptional level. Nuclear receptors (NRs) are ligand-activated transcription factors that regulate gene transcription by binding to the promoter region or by interacting with other transcription factors. NRs can regulate miRNA expression either at the transcriptional level, or through posttranscriptional maturation by interacting with miRNA processing factors. This review will summarize recent advances in knowledge of the modulation of miRNA expression by NRs. Increased understanding of the molecular basis of miRNA expression may enable new therapeutic interventions that modulate miRNA activities through NR-mediated signaling. PMID:21936947

  19. Retinoic Acid Induces Embryonic Stem Cell Differentiation by Altering Both Encoding RNA and microRNA Expression.

    PubMed

    Zhang, Jingcheng; Gao, Yang; Yu, Mengying; Wu, Haibo; Ai, Zhiying; Wu, Yongyan; Liu, Hongliang; Du, Juan; Guo, Zekun; Zhang, Yong

    2015-01-01

    Retinoic acid (RA) is a vitamin A metabolite that is essential for early embryonic development and promotes stem cell neural lineage specification; however, little is known regarding the impact of RA on mRNA transcription and microRNA levels on embryonic stem cell differentiation. Here, we present mRNA microarray and microRNA high-output sequencing to clarify how RA regulates gene expression. Using mRNA microarray analysis, we showed that RA repressed pluripotency-associated genes while activating ectoderm markers in mouse embryonic stem cells (mESCs). Moreover, RA modulated the DNA methylation of mESCs by altering the expression of epigenetic-associated genes such as Dnmt3b and Dnmt3l. Furthermore, H3K4me2, a pluripotent histone modification, was repressed by RA stimulation. From microRNA sequence data, we identified two downregulated microRNAs, namely, miR-200b and miR-200c, which regulated the pluripotency of stem cells. We found that miR-200b or miR-200c deficiency suppressed the expression of pluripotent genes, including Oct4 and Nanog, and activated the expression of the ectodermal marker gene Nestin. These results demonstrate that retinoid induces mESCs to differentiate by regulating miR-200b/200c. Our findings provide the landscapes of mRNA and microRNA gene networks and indicate the crucial role of miR-200b/200c in the RA-induced differentiation of mESCs.

  20. Oscillatory kinetics of gene expression: Protein conversion and slow mRNA transport

    SciTech Connect

    Zhdanov, V. P.

    2009-06-15

    The negative feedback between mRNA and regulatory-protein production may result in oscillations in the kinetics of gene expression if the mRNA-protein interplay includes protein conversion. Using a mean-field kinetic model, we show that such oscillations can be amplified due to limitations of the mRNA transport between the nucleus and cytoplasm. This effect may be dramatic for the mRNA population in the nucleus.

  1. MicroRNA expression profiling studies on bronchopulmonary dysplasia: a systematic review and meta-analysis.

    PubMed

    Yang, Y; Qiu, J; Kan, Q; Zhou, X-G; Zhou, X-Y

    2013-01-01

    Over the past several years, several microRNA (miRNA) expression profiling studies have been carried out on bronchopulmonary dysplasia (BPD) in mammalian lung tissues. The most effective way to identify these important miRNAs is to systematically search for similar signatures identified in multiple independent studies. Accordingly, a meta-analysis was conducted to review published miRNA expression profiling studies that compared miRNA expression profiles between BPD lung tissues and normal lung tissues. A vote-counting strategy that considered the total number of studies and time points reporting differential expression was applied. Furthermore, cut-off criteria of statistically significant differentially expressed miRNAs as defined by the author and their predicted target genes, if available, as well as the list of up- and down-regulated miRNA features, were collected and recorded. Results of the meta-analysis revealed that four up-regulated miRNAs (miRNA-21, miRNA-34a, miRNA-431, and Let-7f) and one down-regulated miRNA (miRNA-335) were differentially expressed in BPD lung tissues compared with normal groups. In addition, eight miRNAs (miRNA-146b, miRNA-29a, miRNA-503, miRNA-411, miRNA-214, miRNA-130b, miRNA-382, and miRNA-181a-1*) were found to show differential expression not only in the process of normal lung development, but also during the progress of BPD. Finally, several meaningful target genes (such as the HPGD and NTRK genes) of common miRNAs (such as miRNA-21 and miRNA-141) were systematically predicted. These specific miRNAs may provide clues of the potential mechanisms involved in BPD. Further mechanistic and external validation studies are needed to confirm the clinical significance of these miRNAs in the development of BPD. PMID:24301780

  2. Formaldehyde exposure alters miRNA expression profiles in the olfactory bulb.

    PubMed

    Li, Guifa; Yang, Jing; Ling, Shucai

    2015-01-01

    It has been reported that inhaling formaldehyde (FA) causes damage to the central nervous system. However, it is unclear whether FA can disturb the function of the olfactory bulb. Using a microarray, we found that FA inhalation altered the miRNA expression profile. Functional enrichment analysis of the predicted targets of the changed miRNA showed that the enrichment canonical pathways and networks associated with cancer and transcriptional regulation. FA exposure disrupts miRNA expression profiles within the olfactory bulb.

  3. Japanese Encephalitis Virus exploits the microRNA-432 to regulate the expression of Suppressor of Cytokine Signaling (SOCS) 5

    PubMed Central

    Sharma, Nikhil; Kumawat, Kanhaiya L.; Rastogi, Meghana; Basu, Anirban; Singh, Sunit K.

    2016-01-01

    Japanese encephalitis virus (JEV) is a plus strand RNA virus, which infects brain. MicroRNAs are regulatory non-coding RNAs which regulate the expression of various genes in cells. Viruses modulate the expression of various microRNAs to suppress anti-viral signaling and evade the immune response. SOCS (Suppressor of cytokine signalling) family of proteins are negative regulators of anti-viral Jak-STAT pathway. In this study, we demonstrated the regulatory role of SOCS5 in Jak-STAT signaling and its exploitation by JEV through a microRNA mediated mechanism. JEV infection in human brain microglial cells (CHME3) downregulated the expression of miR-432, and upregulated SOCS5 levels. SOCS5 was validated as a target of miR-432 by using 3′UTR clone of SOCS5 in luciferase vector along with miR-432 mimic. The overexpression of miR-432 prior to JEV infection enhanced the phosphorylation of STAT1 resulting into increased ISRE activity and cellular inflammatory response resulting into diminished viral replication. The knockdown of SOCS5 resulted into increased STAT1 phosphorylation and suppressed viral replication. JEV infection mediated downregulation of miR-432 leads to SOCS5 upregulation, which helps the virus to evade cellular anti-viral response. This study demonstrated that JEV utilizes this microRNA mediated strategy to manipulate cellular immune response promoting JEV pathogenesis. PMID:27282499

  4. Identification of miRNA-Mediated Core Gene Module for Glioma Patient Prediction by Integrating High-Throughput miRNA, mRNA Expression and Pathway Structure

    PubMed Central

    Han, Junwei; Shang, Desi; Zhang, Yunpeng; Zhang, Wei; Yao, Qianlan; Han, Lei; Xu, Yanjun; Yan, Wei; Bao, Zhaoshi; You, Gan; Jiang, Tao; Kang, Chunsheng; Li, Xia

    2014-01-01

    The prognosis of glioma patients is usually poor, especially in patients with glioblastoma (World Health Organization (WHO) grade IV). The regulatory functions of microRNA (miRNA) on genes have important implications in glioma cell survival. However, there are not many studies that have investigated glioma survival by integrating miRNAs and genes while also considering pathway structure. In this study, we performed sample-matched miRNA and mRNA expression profilings to systematically analyze glioma patient survival. During this analytical process, we developed pathway-based random walk to identify a glioma core miRNA-gene module, simultaneously considering pathway structure information and multi-level involvement of miRNAs and genes. The core miRNA-gene module we identified was comprised of four apparent sub-modules; all four sub-modules displayed a significant correlation with patient survival in the testing set (P-values≤0.001). Notably, one sub-module that consisted of 6 miRNAs and 26 genes also correlated with survival time in the high-grade subgroup (WHO grade III and IV), P-value = 0.0062. Furthermore, the 26-gene expression signature from this sub-module had robust predictive power in four independent, publicly available glioma datasets. Our findings suggested that the expression signatures, which were identified by integration of miRNA and gene level, were closely associated with overall survival among the glioma patients with various grades. PMID:24809850

  5. Antennal-Expressed Ammonium Transporters in the Malaria Vector Mosquito Anopheles gambiae

    PubMed Central

    Pulous, Fadi E.; Zwiebel, Laurence J.

    2014-01-01

    The principal Afrotropical malaria vector mosquito, Anopheles gambiae remains a significant threat to human health. In this anthropophagic species, females detect and respond to a range of human-derived volatile kairomones such as ammonia, lactic acid, and other carboxylic acids in their quest for blood meals. While the molecular underpinnings of mosquito olfaction and host seeking are becoming better understood, many questions remain unanswered. In this study, we have identified and characterized two candidate ammonium transporter genes, AgAmt and AgRh50 that are expressed in the mosquito antenna and may contribute to physiological and behavioral responses to ammonia, which is an important host kairomone for vector mosquitoes. AgAmt transcripts are highly enhanced in female antennae while a splice variant of AgRh50 appears to be antennal-specific. Functional expression of AgAmt in Xenopus laevis oocytes facilitates inward currents in response to both ammonium and methylammonium, while AgRh50 is able to partially complement a yeast ammonium transporter mutant strain, validating their conserved roles as ammonium transporters. We present evidence to suggest that both AgAmt and AgRh50 are in vivo ammonium transporters that are important for ammonia sensitivity in An. gambiae antennae, either by clearing ammonia from the sensillar lymph or by facilitating sensory neuron responses to environmental exposure. Accordingly, AgAmt and AgRh50 represent new and potentially important targets for the development of novel vector control strategies. PMID:25360676

  6. Gateway Vectors for Efficient Artificial Gene Assembly In Vitro and Expression in Yeast Saccharomyces cerevisiae

    PubMed Central

    Giuraniuc, Claudiu V.; MacPherson, Murray; Saka, Yasushi

    2013-01-01

    Construction of synthetic genetic networks requires the assembly of DNA fragments encoding functional biological parts in a defined order. Yet this may become a time-consuming procedure. To address this technical bottleneck, we have created a series of Gateway shuttle vectors and an integration vector, which facilitate the assembly of artificial genes and their expression in the budding yeast Saccharomyces cerevisiae. Our method enables the rapid construction of an artificial gene from a promoter and an open reading frame (ORF) cassette by one-step recombination reaction in vitro. Furthermore, the plasmid thus created can readily be introduced into yeast cells to test the assembled gene’s functionality. As flexible regulatory components of a synthetic genetic network, we also created new versions of the tetracycline-regulated transactivators tTA and rtTA by fusing them to the auxin-inducible degron (AID). Using our gene assembly approach, we made yeast expression vectors of these engineered transactivators, AIDtTA and AIDrtTA and then tested their functions in yeast. We showed that these factors can be regulated by doxycycline and degraded rapidly after addition of auxin to the medium. Taken together, the method for combinatorial gene assembly described here is versatile and would be a valuable tool for yeast synthetic biology. PMID:23675537

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

    PubMed

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

    2013-01-01

    In order to realise the full potential of cancer suicide gene therapy that allows the precise expression of suicide gene in cancer cells, we used a tissue specific Epithelial cell adhesion molecule (EpCAM) promoter (EGP-2) that directs transgene Herpes simplex virus-thymidine kinase (HSV-TK) expression preferentially in EpCAM over expressing cancer cells. EpCAM levels are considerably higher in retinoblastoma (RB), a childhood eye cancer with limited expression in normal cells. Use of miRNA regulation, adjacent to the use of the tissue-specific promoter, would provide the second layer of control to the transgene expression only in the tumor cells while sparing the normal cells. To test this hypothesis we cloned let-7b miRNA targets in the 3'UTR region of HSV-TK suicide gene driven by EpCAM promoter because let-7 family miRNAs, including let-7b, were found to be down regulated in the RB tumors and cell lines. We used EpCAM over expressing and let-7 down regulated RB cell lines Y79, WERI-Rb1 (EpCAM (+ve)/let-7b(down-regulated)), EpCAM down regulated, let-7 over expressing normal retinal Müller glial cell line MIO-M1(EpCAM (-ve)/let-7b(up-regulated)), and EpCAM up regulated, let-7b up-regulated normal thyroid cell line N-Thy-Ori-3.1(EpCAM (+ve)/let-7b(up-regulated)) in the study. The cell proliferation was measured by MTT assay, apoptosis was measured by probing cleaved Caspase3, EpCAM and TK expression were quantified by Western blot. Our results showed that the EGP2-promoter HSV-TK (EGP2-TK) construct with 2 or 4 copies of let-7b miRNA targets expressed TK gene only in Y79, WERI-Rb-1, while the TK gene did not express in MIO-M1. In summary, we have developed a tissue-specific, miRNA-regulated dual control vector, which selectively expresses the suicide gene in EpCAM over expressing cells. PMID:24391761

  8. Integrative analyses of RNA editing, alternative splicing, and expression of young genes in human brain transcriptome by deep RNA sequencing.

    PubMed

    Wu, Dong-Dong; Ye, Ling-Qun; Li, Yan; Sun, Yan-Bo; Shao, Yi; Chen, Chunyan; Zhu, Zhu; Zhong, Li; Wang, Lu; Irwin, David M; Zhang, Yong E; Zhang, Ya-Ping

    2015-08-01

    Next-generation RNA sequencing has been successfully used for identification of transcript assembly, evaluation of gene expression levels, and detection of post-transcriptional modifications. Despite these large-scale studies, additional comprehensive RNA-seq data from different subregions of the human brain are required to fully evaluate the evolutionary patterns experienced by the human brain transcriptome. Here, we provide a total of 6.5 billion RNA-seq reads from different subregions of the human brain. A significant correlation was observed between the levels of alternative splicing and RNA editing, which might be explained by a competition between the molecular machineries responsible for the splicing and editing of RNA. Young human protein-coding genes demonstrate biased expression to the neocortical and non-neocortical regions during evolution on the lineage leading to humans. We also found that a significantly greater number of young human protein-coding genes are expressed in the putamen, a tissue that was also observed to have the highest level of RNA-editing activity. The putamen, which previously received little attention, plays an important role in cognitive ability, and our data suggest a potential contribution of the putamen to human evolution.

  9. Ingestion of bacterially expressed double-stranded RNA inhibits gene expression in planarians

    PubMed Central

    Newmark, Phillip A.; Reddien, Peter W.; Cebrià, Francesc; Alvarado, Alejandro Sánchez

    2003-01-01

    Freshwater planarian flatworms are capable of regenerating complete organisms from tiny fragments of their bodies; the basis for this regenerative prowess is an experimentally accessible stem cell population that is present in the adult planarian. The study of these organisms, classic experimental models for investigating metazoan regeneration, has been revitalized by the application of modern molecular biological approaches. The identification of thousands of unique planarian ESTs, coupled with large-scale whole-mount in situ hybridization screens, and the ability to inhibit planarian gene expression through double-stranded RNA-mediated genetic interference, provide a wealth of tools for studying the molecular mechanisms that regulate tissue regeneration and stem cell biology in these organisms. Here we show that, as in Caenorhabditis elegans, ingestion of bacterially expressed double-stranded RNA can inhibit gene expression in planarians. This inhibition persists throughout the process of regeneration, allowing phenotypes with disrupted regenerative patterning to be identified. These results pave the way for large-scale screens for genes involved in regenerative processes. PMID:12917490

  10. Breast cancer metastasis suppressor 1 coordinately regulates metastasis-associated microRNA expression

    PubMed Central

    Edmonds, Mick D.; Hurst, Douglas R.; Vaidya, Kedar S.; Stafford, Lewis J.; Chen, Dongquan; Welch, Danny R.

    2009-01-01

    Breast cancer metastasis suppressor 1 (BRMS1) suppresses metastasis of multiple tumor types without blocking tumorigenesis. BRMS1 forms complexes with SIN3, histone deacetylases and selected transcription factors that modify metastasis-associated gene expression (e.g., EGFR, OPN, PI4P5K1A, PLAU). microRNA (miRNA) are a recently discovered class of regulatory, noncoding RNA, some of which are involved in neoplastic progression. Based on these data, we hypothesized that BRMS1 may also exert some of its antimetastatic effects by regulating miRNA expression. Micro-RNA arrays were done comparing small RNAs that were purified from metastatic MDA-MB-231 and MDA-MB-435 and their non-metastatic BRMS1-transfected counterparts. miRNA expression changed by BRMS1 were validated using SYBR Green RT-PCR. BRMS1 decreased metastasis-promoting (miR-10b, -373 and -520c) miRNA, with corresponding reduction of their downstream targets (e.g., RhoC which is downstream of miR-10b). Concurrently, BRMS1 increased expression of metastasis suppressing miRNA (miR-146a, -146b and -335). Collectively, these data show that BRMS1 coordinately regulates expression of multiple metastasis-associated miRNA and suggests that recruitment of BRMS1-containing SIN3:HDAC complexes to, as yet undefined, miRNA promoters might be involved in the regulation of cancer metastasis. PMID:19585508

  11. Integrated Analysis of Dysregulated ncRNA and mRNA Expression Profiles in Humans Exposed to Carbon Nanotubes

    PubMed Central

    Shvedova, Anna A.; Yanamala, Naveena; Kisin, Elena R.; Khailullin, Timur O.; Birch, M. Eileen; Fatkhutdinova, Liliya M.

    2016-01-01

    Background As the application of carbon nanotubes (CNT) in consumer products continues to rise, studies have expanded to determine the associated risks of exposure on human and environmental health. In particular, several lines of evidence indicate that exposure to multi-walled carbon nanotubes (MWCNT) could pose a carcinogenic risk similar to asbestos fibers. However, to date the potential markers of MWCNT exposure are not yet explored in humans. Methods In the present study, global mRNA and ncRNA expression profiles in the blood of exposed workers, having direct contact with MWCNT aerosol for at least 6 months (n = 8), were compared with expression profiles of non-exposed (n = 7) workers (e.g., professional and/or technical staff) from the same manufacturing facility. Results Significant changes in the ncRNA and mRNA expression profiles were observed between exposed and non-exposed worker groups. An integrative analysis of ncRNA-mRNA correlations was performed to identify target genes, functional relationships, and regulatory networks in MWCNT-exposed workers. The coordinated changes in ncRNA and mRNA expression profiles revealed a set of miRNAs and their target genes with roles in cell cycle regulation/progression/control, apoptosis and proliferation. Further, the identified pathways and signaling networks also revealed MWCNT potential to trigger pulmonary and cardiovascular effects as well as carcinogenic outcomes in humans, similar to those previously described in rodents exposed to MWCNTs. Conclusion This study is the first to investigate aberrant changes in mRNA and ncRNA expression profiles in the blood of humans exposed to MWCNT. The significant changes in several miRNAs and mRNAs expression as well as their regulatory networks are important for getting molecular insights into the MWCNT-induced toxicity and pathogenesis in humans. Further large-scale prospective studies are necessary to validate the potential applicability of such changes in mRNAs and mi

  12. Dietary glycerol for quail: association between productive performance and COX III mRNA expression.

    PubMed

    Silva, S C C; Gasparino, E; Batista, E; Tanamati, F; Vesco, A P D; Lala, B; de Oliveira, D P

    2016-01-01

    This study was carry out to evaluate mRNA expression of mitochondrial cytochrome c oxidase III in the Pectoralis superficialis muscle of 28-day-old quails fed diets containing 0, 8, and 12% glycerol. Total RNA was extracted (N = 10) and cDNA was amplified using specifics primers for qRT-PCR. Feed efficiency and feed intake were evaluated. COX III mRNA expression in breast muscle was higher in the group fed with 12% glycerol (0.863 AU); no differences were observed in the expression of this gene between the muscle of animals fed diets without glycerol (0.357 AU) and 8% glycerol (0.415 AU). Quails that showed greater COX III mRNA expression also showed the lowest feed efficiency. These results show that there is a difference in COX III mRNA expression in breast muscle of 28-day-old quail fed diets different concentrations of glycerol. PMID:27323091

  13. In-gel imaging of RNA processing using Broccoli reveals optimal aptamer expression strategies

    PubMed Central

    Filonov, Grigory S.; Kam, Christina W.; Song, Wenjiao; Jaffrey, Samie R.

    2015-01-01

    SUMMARY RNA aptamers can be expressed in cells to influence and image cellular processes. Aptamer folding is maintained by inserting the aptamers into highly structured RNA scaffolds. Here we show that commonly used RNA scaffolds exhibit unexpected instability and cleavage in bacterial and mammalian cells. Using an in-gel staining approach for rapid and simple detection of Spinach- or Broccoli-tagged RNAs in cells, we monitored the processing of RNAs tagged with scaffolded aptamers, revealing endonucleolytic cleavage, RNA instability and poor expression. We reengineered a natural three-way junction structure to generate an alternative scaffold that enables stable aptamer expression in cells. This scaffold was used to create cassettes containing up to four Broccoli units, markedly enhancing the brightness of mammalian cells expressing cassette-tagged RNAs. These experiments describe methods for screening RNA cleavage events in cells, and identify cell-compatible scaffolds that enable efficient tagging of RNAs with aptamers for cellular expression. PMID:26000751

  14. Positive-selection and ligation-independent cloning vectors for large scale in planta expression for plant functional genomics.

    PubMed

    Oh, Sang-Keun; Kim, Saet-Byul; Yeom, Seon-In; Lee, Hyun-Ah; Choi, Doil

    2010-12-01

    Transient expression is an easy, rapid and powerful technique for producing proteins of interest in plants. Recombinational cloning is highly efficient but has disadvantages, including complicated, time consuming cloning procedures and expensive enzymes for large-scale gene cloning. To overcome these limitations, we developed new ligation-independent cloning (LIC) vectors derived from binary vectors including tobacco mosaic virus (pJL-TRBO), potato virus X (pGR106) and the pBI121 vector-based pMBP1. LIC vectors were modified to enable directional cloning of PCR products without restriction enzyme digestion or ligation reactions. In addition, the ccdB gene, which encodes a potent cell-killing protein, was introduced between the two LIC adapter sites in the pJL-LIC, pGR-LIC, and pMBP-LIC vectors for the efficient selection of recombinant clones. This new vector does not require restriction enzymes, alkaline phosphatase, or DNA ligase for cloning. To clone, the three LIC vectors are digested with SnaBI and treated with T4 DNA polymerase, which includes 3' to 5' exonuclease activity in the presence of only one dNTP (dGTP for the inserts and dCTP for the vector). To make recombinants, the vector plasmid and the insert PCR fragment were annealed at room temperature for 20 min prior to transformation into the host. Bacterial transformation was accomplished with 100% efficiency. To validate the new LIC vector systems, we were used to coexpressed the Phytophthora AVR and potato resistance (R) genes in N. benthamiana by infiltration of Agrobacterium. Coexpressed AVR and R genes in N. benthamiana induced the typical hypersensitive cell death resulting from in vivo interaction of the two proteins. These LIC vectors could be efficiently used for high-throughput cloning and laboratory-scale in planta expression. These vectors could provide a powerful tool for high-throughput transient expression assays for functional genomic studies in plants. PMID:21340673

  15. Lac-regulated system for generating adenovirus 5 vaccine vectors expressing cytolytic human immunodeficiency virus 1 genes

    PubMed Central

    Zhao, Chunxia; Crews, Charles Jefferson; Derdeyn, Cynthia A.; Blackwell, Jerry L.

    2009-01-01

    Adenovirus (Ad) vectors have been developed as human immunodeficiency-1 (HIV-1) vaccine vectors because they consistently induce immune responses in preclinical animal models and human trials. Strong promoters and codon-optimization are often used to enhance vaccine-induced HIV-1 gene expression and immunogenicity. However, if the transgene is inherently cytotoxic in the cell line used to produce the vector, and is expressed at high levels, it is difficult to rescue a stable Ad HIV-1 vaccine vector. Therefore we hypothesized that generation of Ad vaccine vectors expressing cytotoxic genes, such as HIV-1 env, would be more efficient if expression of the transgene was down regulated during Ad rescue. To test this hypothesis, a Lac repressor-operator system was applied to regulate expression of reporter luciferase and HIV-1 env transgenes during Ad rescue. The results demonstrate that during Ad rescue, constitutive expression of the Lac repressor in 293 cells reduced transgene expression levels to approximately 5% of that observed in the absence of regulation. Furthermore, Lag-regulation translated into more efficient Ad rescue compared to traditional 293 cells. Importantly, Ad vectors rescued with this system showed high levels of transgene expression when transduced into cells that lack the Lac repressor protein. The Lac-regulated system also facilitated the rescue of modified Ad vectors that have non-native receptor tropism. These tropism-modified Ad vectors infect a broader range of cell types than the unmodified Ad, which could increase their effectiveness as a vaccine vector. Overall, the Lac-regulated system described here (i) is backwards compatible with Ad vector methods that employ bacterial-mediated homologous recombination (ii) is adaptable for the engineering of tropism-modified Ad vectors and (iii) does not require co-expression of regulatory genes from the vector or the addition of exogenous chemicals to induce or repress transgene expression. This

  16. SNP Regulation of microRNA Expression and Subsequent Colon Cancer Risk

    PubMed Central

    Mullany, Lila E.; Wolff, Roger K.; Herrick, Jennifer S.; Buas, Matthew F.; Slattery, Martha L.

    2015-01-01

    Introduction MicroRNAs (miRNAs) regulate messenger RNAs (mRNAs) and as such have been implicated in a variety of diseases, including cancer. MiRNAs regulate mRNAs through binding of the miRNA 5’ seed sequence (~7–8 nucleotides) to the mRNA 3’ UTRs; polymorphisms in these regions have the potential to alter miRNA-mRNA target associations. SNPs in miRNA genes as well as miRNA-target genes have been proposed to influence cancer risk through altered miRNA expression levels. Methods MiRNA-SNPs and miRNA-target gene-SNPs were identified through the literature. We used SNPs from Genome-Wide Association Study (GWAS) data that were matched to individuals with miRNA expression data generated from an Agilent platform for colon tumor and non-tumor paired tissues. These samples were used to evaluate 327 miRNA-SNP pairs for associations between SNPs and miRNA expression levels as well as for SNP associations with colon cancer. Results Twenty-two miRNAs expressed in non-tumor tissue were significantly different by genotype and 21 SNPs were associated with altered tumor/non-tumor differential miRNA expression across genotypes. Two miRNAs were associated with SNP genotype for both non-tumor and tumor/non-tumor differential expression. Of the 41 miRNAs significantly associated with SNPs all but seven were significantly differentially expressed in colon tumor tissue. Two of the 41 SNPs significantly associated with miRNA expression levels were associated with colon cancer risk: rs8176318 (BRCA1), ORAA 1.31 95% CI 1.01, 1.78, and rs8905 (PRKAR1A), ORGG 2.31 95% CI 1.11, 4.77. Conclusion Of the 327 SNPs identified in the literature as being important because of their potential regulation of miRNA expression levels, 12.5% had statistically significantly associations with miRNA expression. However, only two of these SNPs were significantly associated with colon cancer. PMID:26630397

  17. Mouse Endometrium Temporal and Spatial Expression mRNA and MicroRNA Associated With Embryo Implantation.

    PubMed

    Chen, Ke; Chen, Xuemei; He, Junlin; Ding, Yubin; Geng, Yanqing; Liu, Shangjing; Liu, Xueqing; Wang, Yingxiong

    2015-11-01

    Embryo implantation is a dynamic physiological process involving morphological and molecular changes in the endometrium during the pre-receptivity, receptivity, and implantation phases. A comprehensive analysis of messenger RNA (mRNA) and microRNA (miRNA) profiles during implantation will likely provide new clues to elucidate the underlying mechanisms governing embryo implantation. We characterized the mRNA and miRNA transcriptomes using next generation sequencing (NGS) of the endometrium 1 day postcoitum (dpc) and 4dpc and the implantation site (IMS) and inter-implantation (IIM) site of the endometrium on 5dpc. Real-time quantitative polymerase chain reaction was performed on selected miRNAs and their predicted target mRNAs to validate their negatively correlated expression. Statistical analysis of the data based on Gene Ontology (GO) group annotation and Kyoto Encyclopedia of Genes and Genomes pathway analysis demonstrated that the genes with significant expression at the IIM site were primarily involved in glucose, protein, and lipoprotein metabolism to provide energy for embryo implantation, while the genes identified at the IMS were involved in RNA functions to produce proteins in support of embryo development and trophoblast invasion. Extracellular matrix (ECM)-receptor interactions between cells and the ECM was the most remarkable event during implantation. The miRNA-mRNA interaction network unraveled the regulatory relationship between miRNAs and mRNAs. Hub miRNAs (mmu-miR-96 and mmu-miR-200b) were identified to target B-cell lymphoma 2 (Bcl-2), Kruppel-like factor 13 (Klf13), and Progesterone receptor (PGR), which are associated with the preparation of the receptive condition or the maintenance of early pregnancy. PMID:25862677

  18. The Selenocysteine tRNA STAF-Binding Region is Essential for Adequate Selenocysteine tRNA Status, Selenoprotein Expression and Early Age Survival of Mice

    Technology Transfer Automated Retrieval System (TEKTRAN)

    STAF is a transcription activating factor for a number of RNA Pol III-and RNA Pol II-dependent genes including the selenocysteine (Sec) tRNA gene. Here, the role of STAF in regulating expression of Sec tRNA and selenoproteins was examined in an invivo model. Heterozygous inactivation of the Staf gen...

  19. The Development of a Viral Mediated CRISPR/Cas9 System with Doxycycline Dependent gRNA Expression for Inducible In vitro and In vivo Genome Editing.

    PubMed

    de Solis, Christopher A; Ho, Anthony; Holehonnur, Roopashri; Ploski, Jonathan E

    2016-01-01

    The RNA-guided Cas9 nuclease, from the type II prokaryotic Clustered Regularly Interspersed Short Palindromic Repeats (CRISPR) adaptive immune system, has been adapted and utilized by scientists to edit the genomes of eukaryotic cells. Here, we report the development of a viral mediated CRISPR/Cas9 system that can be rendered inducible utilizing doxycycline (Dox) and can be delivered to cells in vitro and in vivo utilizing adeno-associated virus (AAV). Specifically, we developed an inducible gRNA (gRNAi) AAV vector that is designed to express the gRNA from a H1/TO promoter. This AAV vector is also designed to express the Tet repressor (TetR) to regulate the expression of the gRNAi in a Dox dependent manner. We show that H1/TO promoters of varying length and a U6/TO promoter can edit DNA with similar efficiency in vitro, in a Dox dependent manner. We also demonstrate that our inducible gRNAi vector can be used to edit the genomes of neurons in vivo within the mouse brain in a Dox dependent manner. Genome editing can be induced in vivo with this system by supplying animals Dox containing food for as little as 1 day. This system might be cross compatible with many existing S. pyogenes Cas9 systems (i.e., Cas9 mouse, CRISPRi, etc.), and therefore it likely can be used to render these systems inducible as well. PMID:27587996

  20. The Development of a Viral Mediated CRISPR/Cas9 System with Doxycycline Dependent gRNA Expression for Inducible In vitro and In vivo Genome Editing

    PubMed Central

    de Solis, Christopher A.; Ho, Anthony; Holehonnur, Roopashri; Ploski, Jonathan E.

    2016-01-01

    The RNA-guided Cas9 nuclease, from the type II prokaryotic Clustered Regularly Interspersed Short Palindromic Repeats (CRISPR) adaptive immune system, has been adapted and utilized by scientists to edit the genomes of eukaryotic cells. Here, we report the development of a viral mediated CRISPR/Cas9 system that can be rendered inducible utilizing doxycycline (Dox) and can be delivered to cells in vitro and in vivo utilizing adeno-associated virus (AAV). Specifically, we developed an inducible gRNA (gRNAi) AAV vector that is designed to express the gRNA from a H1/TO promoter. This AAV vector is also designed to express the Tet repressor (TetR) to regulate the expression of the gRNAi in a Dox dependent manner. We show that H1/TO promoters of varying length and a U6/TO promoter can edit DNA with similar efficiency in vitro, in a Dox dependent manner. We also demonstrate that our inducible gRNAi vector can be used to edit the genomes of neurons in vivo within the mouse brain in a Dox dependent manner. Genome editing can be induced in vivo with this system by supplying animals Dox containing food for as little as 1 day. This system might be cross compatible with many existing S. pyogenes Cas9 systems (i.e., Cas9 mouse, CRISPRi, etc.), and therefore it likely can be used to render these systems inducible as well. PMID:27587996

  1. MicroRNA expression and its association with DNA repair in preimplantation embryos

    PubMed Central

    TULAY, Pinar; SENGUPTA, Sioban B.

    2016-01-01

    Active DNA repair pathways are crucial for preserving genomic integrity and are likely among the complex mechanisms involved in the normal development of preimplantation embryos. MicroRNAs (miRNA), short non-coding RNAs, are key regulators of gene expression through the post-transcriptional and post-translational modification of mRNA. The association of miRNA expression with infertility or polycystic ovarian syndrome has been widely investigated; however, there are limited data regarding the importance of miRNA regulation in DNA repair during preimplantation embryo development. In this article, we review normal miRNA biogenesis and consequences of aberrant miRNA expression in the regulation of DNA repair in gametes and preimplantation embryos. PMID:26853522

  2. Cloning and expression of autogenes encoding RNA polymerases of T7-like bacteriophages

    DOEpatents

    Studier, F.W.; Dubendorff, J.W.

    1998-11-03

    This invention relates to the cloning and expression of autogenes encoding RNA polymerases of T7 and T7-like bacteriophages, in which the RNA polymerase gene is transcribed from a promoter which is recognized by the encoded RNA polymerase. Cloning of T7 autogenes was achieved by reducing the activity of the RNA polymerase sufficiently to permit host cell growth. T7 RNA polymerase activity was controlled by combining two independent methods: lac-repression of the recombinant lac operator-T7 promoter in the autogene and inhibition of the polymerase by T7 lysozyme. Expression systems for producing the RNA polymerases of T7 and other T7-like bacteriophages, and expression systems for producing selected gene products are described, as well as other related materials and methods. 12 figs.

  3. Cloning and expression of autogenes encoding RNA polymerases of T7-like bacteriophages

    DOEpatents

    Studier, F.W.; Dubendorff, J.W.

    1998-10-20

    This invention relates to the cloning and expression of autogenes encoding RNA polymerases of T7 and T7-like bacteriophages, in which the RNA polymerase gene is transcribed from a promoter which is recognized by the encoded RNA polymerase. Cloning of T7 autogenes was achieved by reducing the activity of the RNA polymerase sufficiently to permit host cell growth. T7 RNA polymerase activity was controlled by combining two independent methods: lac-repression of the recombinant lac operator-T7 promoter in the autogene and inhibition of the polymerase by T7 lysozyme. Expression systems for producing the RNA polymerases of T7 and other T7-like bacteriophages, and expression systems for producing selected gene products are described, as well as other related materials and methods. 12 figs.

  4. Cloning and expression of autogenes encoding RNA poly,erases of T7-like bacteriophages

    DOEpatents

    Studier, F. William; Dubendorff, John W.

    1998-01-01

    This invention relates to the cloning and expression of autogenes encoding RNA polymerases of T7 and T7-like bacteriophages, in which the RNA polymerase gene is transcribed from a promoter which is recognized by the encoded RNA polymerase. Cloning of T7 autogenes was achieved by reducing the activity of the RNA polymerase sufficiently to permit host cell growth. T7 RNA polymerase activity was controlled by combining two independent methods: lac-repression of the recombinant lac operator-T7 promoter in the autogene and inhibition of the polymerase by T7 lysozyme. Expression systems for producing the RNA polymerases of T7 and other T7-like bacteriophages, and expression systems for producing selected gene products are described, as well as other related materials and methods.

  5. Cloning and expression of autogenes encoding RNA polymerases of T7-like bacteriophages

    DOEpatents

    Studier, F. William; Dubendorff, John W.

    1998-01-01

    This invention relates to the cloning and expression of autogenes encoding RNA polymerases of T7 and T7-like bacteriophages, in which the RNA polymerase gene is transcribed from a promoter which is recognized by the encoded RNA polymerase. Cloning of T7 autogenes was achieved by reducing the activity of the RNA polymerase sufficiently to permit host cell growth. T7 RNA polymerase activity was controlled by combining two independent methods: lac-repression of the recombinant lac operator-T7 promoter in the autogene and inhibition of the polymerase by T7 lysozyme. Expression systems for producing the RNA polymerases of T7 and other T7-like bacteriophages, and expression systems for producing selected gene products are described, as well as other related materials and methods.

  6. Quantitative visualization of alternative exon expression from RNA-seq data

    PubMed Central

    Katz, Yarden; Wang, Eric T.; Silterra, Jacob; Schwartz, Schraga; Wong, Bang; Thorvaldsdóttir, Helga; Robinson, James T.; Mesirov, Jill P.; Airoldi, Edoardo M.; Burge, Christopher B.

    2015-01-01

    Motivation: Analysis of RNA sequencing (RNA-Seq) data revealed that the vast majority of human genes express multiple mRNA isoforms, produced by alternative pre-mRNA splicing and other mechanisms, and that most alternative isoforms vary in expression between human tissues. As RNA-Seq datasets grow in size, it remains challenging to visualize isoform expression across multiple samples. Results: To help address this problem, we present Sashimi plots, a quantitative visualization of aligned RNA-Seq reads that enables quantitative comparison of exon usage across samples or experimental conditions. Sashimi plots can be made using the Broad Integrated Genome Viewer or with a stand-alone command line program. Availability and implementation: Software code and documentation freely available here: http://miso.readthedocs.org/en/fastmiso/sashimi.html Contact: mesirov@broadinstitute.org, airoldi@fas.harvard.edu or cburge@mit.edu Supplementary information: Supplementary data are available at Bioinformatics online. PMID:25617416

  7. Dynamic signal processing by ribozyme-mediated RNA circuits to control gene expression.

    PubMed

    Shen, Shensi; Rodrigo, Guillermo; Prakash, Satya; Majer, Eszter; Landrain, Thomas E; Kirov, Boris; Daròs, José-Antonio; Jaramillo, Alfonso

    2015-05-26

    Organisms have different circuitries that allow converting signal molecule levels to changes in gene expression. An important challenge in synthetic biology involves the de novo design of RNA modules enabling dynamic signal processing in live cells. This requires a scalable methodology for sensing, transmission, and actuation, which could be assembled into larger signaling networks. Here, we present a biochemical strategy to design RNA-mediated signal transduction cascades able to sense small molecules and small RNAs. We design switchable functional RNA domains by using strand-displacement techniques. We experimentally characterize the molecular mechanism underlying our synthetic RNA signaling cascades, show the ability to regulate gene expression with transduced RNA signals, and describe the signal processing response of our systems to periodic forcing in single live cells. The engineered systems integrate RNA-RNA interaction with available ribozyme and aptamer elements, providing new ways to engineer arbitrary complex gene circuits.

  8. Calpain expression in lymphoid cells. Increased mRNA and protein levels after cell activation.

    PubMed

    Deshpande, R V; Goust, J M; Chakrabarti, A K; Barbosa, E; Hogan, E L; Banik, N L

    1995-02-10

    Although calpain is ubiquitously present in human tissues and is thought to play a role in demyelination, its activity is very low in resting normal lymphocytes. To determine the nature of calpain expression at the mRNA and protein levels in human lymphoid cells, we studied human T lymphocytic, B lymphocytic, and monocytic lines as well as peripheral blood mononuclear cells. Stimulation of cells with the phorbol ester phorbol myristate acetate and the calcium ionophore A23187 resulted in increased calpain mRNA and protein expression. Calpain mRNA expression is also increased in human T cells stimulated with anti-CD3. A dissociation between the increases of RNA and protein suggested that calpain could be released from the cells; the subsequent experiments showed its presence in the extracellular environment. 5,6-Dichloro-1b-D-ribofuranosylbenzimidazole, a reversible inhibitor of mRNA synthesis, reduced calpain mRNA levels by 50-67% and protein levels by 72-91%. Its removal resulted in resumption of both calpain mRNA and protein synthesis. Cycloheximide, a translational inhibitor, reduced calpain protein levels by 77-81% and calpain mRNA levels by 96% in activated THP-1 cells. Interferon-gamma induced calpain mRNA and protein in U-937 and THP-1 cells. Dexamethasone increased mRNA expression in THP-1 cells. Our results indicate that activation of lymphoid cells results in de novo synthesis and secretion of calpain. PMID:7852311

  9. Synthesis of Poly Linear shRNA Expression Cassettes Through Branch-PCR.

    PubMed

    Liu, Jianbing; Xi, Zhen

    2016-01-01

    A facile and universal strategy to construct the poly linear small hairpin RNA (shRNA) expression cassettes with multiple shRNA transcription templates through polymerase chain reaction with flexible branched primers (branch-PCR) is described in this protocol. Double-stranded RNA (dsRNA) is not stable enough for the study of RNA interference (RNAi) delivery in mammalian cells. Therefore, the more stable shRNA transcription template is employed to produce the endogenous transcribed dsRNA. Then, the covalent crosslinked linear shRNA expression cassettes are constructed through the branch-PCR for the long-lasting RNAi effect in this protocol. The branched primer pair is efficiently synthesized through classic click chemistry. In one step of PCR, the much more stable poly linear shRNA expression cassettes can be produced in large scale. This strategy of efficient synthesis of the poly linear gene expression cassettes can also be applied in the field for other target gene delivery. © 2016 by John Wiley & Sons, Inc. PMID:27584702

  10. Construction of a host-independent T7 expression system with small RNA regulation.

    PubMed

    Wang, Gang; Li, Qiang; Xu, Dikai; Cui, Mingxin; Sun, Xiao; Xu, Yanyan; Wang, Wenya

    2014-11-10

    It is desirable to build a universal and efficient protein expression system for wild-type prokaryotic strains in biotechnology industry and the outstanding T7 expression system could be a good candidate. However, the current utilization of T7 system depends on the specific DE3 lysogenic hosts, which severely limits its application in wild-type strains. In this study, a host-independent T7 expression system without relying on DE3 lysogenic hosts to provide T7 RNA Polymerase was developed. T7 RNA Polymerase gene (Gene1) and T7 Promoter were successfully integrated into a single plasmid with the regulation of proper antisense RNA to limit T7 RNA Polymerase expression at a non-lethal level. This host-independent T7 expression system realized efficient protein expression in 4 non-DE3 Escherichia coli strains and a wild-type Sinorhizobium strain TH572. PMID:25193711

  11. A custom microarray platform for analysis of microRNA gene expression.

    PubMed

    Thomson, J Michael; Parker, Joel; Perou, Charles M; Hammond, Scott M

    2004-10-01

    MicroRNAs are short, noncoding RNA transcripts that post-transcriptionally regulate gene expression. Several hundred microRNA genes have been identified in Caenorhabditis elegans, Drosophila, plants and mammals. MicroRNAs have been linked to developmental processes in C. elegans, plants and humans and to cell growth and apoptosis in Drosophila. A major impediment in the study of microRNA function is the lack of quantitative expression profiling methods. To close this technological gap, we have designed dual-channel microarrays that monitor expression levels of 124 mammalian microRNAs. Using these tools, we observed distinct patterns of expression among adult mouse tissues and embryonic stem cells. Expression profiles of staged embryos demonstrate temporal regulation of a large class of microRNAs, including members of the let-7 family. This microarray technology enables comprehensive investigation of microRNA expression, and furthers our understanding of this class of recently discovered noncoding RNAs.

  12. Lentiviral vector-mediated over-expression of Sox9 protected chondrocytes from IL-1β induced degeneration and apoptosis

    PubMed Central

    Lu, Huading; Zeng, Chun; Chen, Mingwei; Lian, Liyi; Dai, Yuhu; Zhao, Huiqing

    2015-01-01

    To explore whether the over-expression of Sry-related HMG box (Sox9) in degenerative chondrocytes is able to improve cell regeneration and protects cells from inflammation induced apoptosis, we generated a Sox9 over-expressing vector delivery system in which the Sox9 gene was inserted into a lentiviral vector. After infecting mouse chondrocytes with the Sox9-encoding vector, we observed a high level of gene transduction efficiency and achieved a high level of Sox9 expression in the infected chondrocytes. To explore whether over-expression of Sox9 is able to induce cell regeneration and improve cell survival, we induced Sox9 over-expression by lentiviral vector infection 48 hours before IL-1β treatment. The cells were infected with the reporter gene GFP-encoded lentiviral vector as a negative control or left uninfected. 48-hours after IL-1β treatment, the chrondrocytes treated with IL-1β alone, underwent a degenerative process, with elevated expression of MMP-3, MMP-13, ADAMTS-5 and ALP, but the cell specific anabolic proteins collagen II and aggrecan were significantly suppressed. The cells infected with the GFP reporter vector had no increased regeneration after IL-1β treatment. The results indicated that Sox9 is an important chondrocyte transcription factor, promoting chondrocyte regeneration and cell survival, which were mediated through affecting multiple cell differentiation as well as anti-apoptotic signaling pathways. PMID:26617711

  13. Integrative Approaches for microRNA Target Prediction: Combining Sequence Information and the Paired mRNA and miRNA Expression Profiles

    PubMed Central

    Naifang, Su; Minping, Qian; Minghua, Deng

    2013-01-01

    Gene regulation is a key factor in gaining a full understanding of molecular biology. microRNA (miRNA), a novel class of non-coding RNA, has recently been found to be one crucial class of post-transactional regulators, and play important roles in cancer. One essential step to understand the regulatory effect of miRNAs is the reliable prediction of their target mRNAs. Typically, the predictions are solely based on the sequence information, which unavoidably have high false detection rates. Recently, some novel approaches are developed to predict miRNA targets by integrating the typical algorithm with the paired expression profiles of miRNA and mRNA. Here we review and discuss these integrative approaches and propose a new algorithm called HCTarget. Applying HCtarget to the expression data in multiple myeloma, we predict target genes for ten specific miRNAs. The experimental verification and a loss of function study validate our predictions. Therefore, the integrative approach is a reliable and effective way to predict miRNA targets, and could improve our comprehensive understanding of gene regulation. PMID:23467572

  14. EBV noncoding RNA EBER2 interacts with host RNA-binding proteins to regulate viral gene expression.

    PubMed

    Lee, Nara; Yario, Therese A; Gao, Jessica S; Steitz, Joan A

    2016-03-22

    Epstein-Barr virus (EBV) produces a highly abundant noncoding RNA called EBV-encoded RNA 2 (EBER2) that interacts indirectly with the host transcription factor paired box protein 5 (PAX5) to regulate viral latent membrane protein 1/2 (LMP1/2) gene expression as well as EBV lytic replication. To identify intermediary proteins, we isolated EBER2-PAX5-containing complexes and analyzed the protein components by mass spectrometry. The top candidates include three host proteins splicing factor proline and glutamine rich (SFPQ), non-POU domain-containing octamer-binding protein (NONO), and RNA binding motif protein 14 (RBM14), all reported to be components of nuclear bodies called paraspeckles. In vivo RNA-protein crosslinking indicates that SFPQ and RBM14 contact EBER2 directly. Binding studies using recombinant proteins demonstrate that SFPQ and NONO associate with PAX5, potentially bridging its interaction with EBER2. Similar to EBER2 or PAX5 depletion, knockdown of any of the three host RNA-binding proteins results in the up-regulation of viral LMP2A mRNA levels, supporting a physiologically relevant interaction of these newly identified factors with EBER2 and PAX5. Identification of these EBER2-interacting proteins enables the search for cellular noncoding RNAs that regulate host gene expression in a manner similar to EBER2. PMID:26951683

  15. EBV noncoding RNA EBER2 interacts with host RNA-binding proteins to regulate viral gene expression.

    PubMed

    Lee, Nara; Yario, Therese A; Gao, Jessica S; Steitz, Joan A

    2016-03-22

    Epstein-Barr virus (EBV) produces a highly abundant noncoding RNA called EBV-encoded RNA 2 (EBER2) that interacts indirectly with the host transcription factor paired box protein 5 (PAX5) to regulate viral latent membrane protein 1/2 (LMP1/2) gene expression as well as EBV lytic replication. To identify intermediary proteins, we isolated EBER2-PAX5-containing complexes and analyzed the protein components by mass spectrometry. The top candidates include three host proteins splicing factor proline and glutamine rich (SFPQ), non-POU domain-containing octamer-binding protein (NONO), and RNA binding motif protein 14 (RBM14), all reported to be components of nuclear bodies called paraspeckles. In vivo RNA-protein crosslinking indicates that SFPQ and RBM14 contact EBER2 directly. Binding studies using recombinant proteins demonstrate that SFPQ and NONO associate with PAX5, potentially bridging its interaction with EBER2. Similar to EBER2 or PAX5 depletion, knockdown of any of the three host RNA-binding proteins results in the up-regulation of viral LMP2A mRNA levels, supporting a physiologically relevant interaction of these newly identified factors with EBER2 and PAX5. Identification of these EBER2-interacting proteins enables the search for cellular noncoding RNAs that regulate host gene expression in a manner similar to EBER2.

  16. Glucocorticoids modulate BDNF mRNA expression in the rat hippocampus after traumatic brain injury.

    PubMed

    Grundy, P L; Patel, N; Harbuz, M S; Lightman, S L; Sharples, P M

    2000-10-20

    Brain-derived neurotrophic factor (BDNF) expression in rat hippocampus is increased after experimental traumatic brain injury (TBI) and may be neuroprotective. Glucocorticoids are important regulators of brain neurotrophin levels and are often prescribed following TBI. The effect of adrenalectomy (ADX) on the expression of BDNF mRNA in the hippocampus after TBI has not been investigated to date. We used fluid percussion injury (FPI) and in situ hybridization to evaluate the expression of BDNF mRNA in the hippocampus 4 h after TBI in adrenal-intact or adrenalectomized rats (with or without corticosterone replacement). FPI and ADX independently increased expression of BDNF mRNA. In animals undergoing FPI, prior ADX caused further elevation of BDNF mRNA and this upregulation was prevented by corticosterone replacement in ADX rats. These findings suggest that glucocorticoids are involved in the modulation of the BDNF mRNA response to TBI.

  17. MiRNA-21 Expression Decreases from Primary Tumors to Liver Metastases in Colorectal Carcinoma

    PubMed Central

    Feiersinger, Fabian; Nolte, Elke; Wach, Sven; Rau, Tilman T.; Vassos, Nikolaos; Geppert, Carol; Konrad, Andreas; Merkel, Susanne; Taubert, Helge; Stürzl, Michael; Croner, Roland S.

    2016-01-01

    Objective Metastasis is the major cause of death in colorectal cancer patients. Expression of certain miRNAs in the primary tumors has been shown to be associated with progression of colorectal cancer and the initiation of metastasis. In this study, we compared miRNA expression in primary colorectal cancer and corresponding liver metastases in order to get an idea of the oncogenic importance of the miRNAs in established metastases. Methods We analyzed the expression of miRNA-21, miRNA-31 and miRNA-373 in corresponding formalin-fixed paraffin-embedded (FFPE) tissue samples of primary colorectal cancer, liver metastasis and healthy tissues of 29 patients by quantitative real-time PCR. Results All three miRNAs were significantly up-regulated in the primary tumor tissues as compared to healthy colon mucosa of the respective patients (p < 0.01). MiRNA-21 and miRNA-31 were also higher expressed in liver metastases as compared to healthy liver tissues (p < 0.01). No significant difference of expression of miRNA-31 and miRNA-373 was observed between primary tumors and metastases. Of note, miRNA-21 expression was significantly reduced in liver metastases as compared to the primary colorectal tumors (p < 0.01). Conclusion In the context of previous studies demonstrating increased miRNA-21 expression in metastatic primary tumors, our findings raise the question whether miRNA-21 might be involved in the initiation but not in the perpetuation and growth of metastases. PMID:26845148

  18. Expression of human choriogonadotropin in monkey cells using a single simian virus 40 vector.

    PubMed Central

    Reddy, V B; Beck, A K; Garramone, A J; Vellucci, V; Lustbader, J; Bernstine, E G

    1985-01-01

    We have inserted the cDNAs coding for both polypeptide subunits, alpha and beta, of human choriogonadotropin (hCG) into a single simian virus 40 expression vector in such a way that they replace the viral VP2 and VP1 coding sequences, respectively. Monkey cells infected with this virus and the appropriate helper virus produce dimeric hCG. hCG produced in this system was shown to chromatograph identically to standard hCG preparations on gel filtration and to be biologically active in the mouse uterine weight assay. Images PMID:2987938

  19. Concurrent micro-RNA mediated silencing of tick-borne flavivirus replication in tick vector and in the brain of vertebrate host

    PubMed Central

    Tsetsarkin, Konstantin A.; Liu, Guangping; Kenney, Heather; Hermance, Meghan; Thangamani, Saravanan; Pletnev, Alexander G.

    2016-01-01

    Tick-borne viruses include medically important zoonotic pathogens that can cause life-threatening diseases. Unlike mosquito-borne viruses, whose impact can be restrained via mosquito population control programs, for tick-borne viruses only vaccination remains the reliable means of disease prevention. For live vaccine viruses a concern exists, that spillovers from viremic vaccinees could result in introduction of genetically modified viruses into sustainable tick-vertebrate host transmission cycle in nature. To restrict tick-borne flavivirus (Langat virus, LGTV) vector tropism, we inserted target sequences for tick-specific microRNAs (mir-1, mir-275 and mir-279) individually or in combination into several distant regions of LGTV genome. This caused selective attenuation of viral replication in tick-derived cells. LGTV expressing combinations of target sequences for tick- and vertebrate CNS-specific miRNAs were developed. The resulting viruses replicated efficiently and remained stable in simian Vero cells, which do not express these miRNAs, however were severely restricted to replicate in tick-derived cells. In addition, simultaneous dual miRNA targeting led to silencing of virus replication in live Ixodes ricinus ticks and abolished virus neurotropism in highly permissive newborn mice. The concurrent restriction of adverse replication events in vertebrate and invertebrate hosts will, therefore, ensure the environmental safety of live tick-borne virus vaccine candidates. PMID:27620807

  20. Concurrent micro-RNA mediated silencing of tick-borne flavivirus replication in tick vector and in the brain of vertebrate host.

    PubMed

    Tsetsarkin, Konstantin A; Liu, Guangping; Kenney, Heather; Hermance, Meghan; Thangamani, Saravanan; Pletnev, Alexander G

    2016-01-01

    Tick-borne viruses include medically important zoonotic pathogens that can cause life-threatening diseases. Unlike mosquito-borne viruses, whose impact can be restrained via mosquito population control programs, for tick-borne viruses only vaccination remains the reliable means of disease prevention. For live vaccine viruses a concern exists, that spillovers from viremic vaccinees could result in introduction of genetically modified viruses into sustainable tick-vertebrate host transmission cycle in nature. To restrict tick-borne flavivirus (Langat virus, LGTV) vector tropism, we inserted target sequences for tick-specific microRNAs (mir-1, mir-275 and mir-279) individually or in combination into several distant regions of LGTV genome. This caused selective attenuation of viral replication in tick-derived cells. LGTV expressing combinations of target sequences for tick- and vertebrate CNS-specific miRNAs were developed. The resulting viruses replicated efficiently and remained stable in simian Vero cells, which do not express these miRNAs, however were severely restricted to replicate in tick-derived cells. In addition, simultaneous dual miRNA targeting led to silencing of virus replication in live Ixodes ricinus ticks and abolished virus neurotropism in highly permissive newborn mice. The concurrent restriction of adverse replication events in vertebrate and invertebrate hosts will, therefore, ensure the environmental safety of live tick-borne virus vaccine candidates. PMID:27620807

  1. Long Noncoding RNA and mRNA Expression Profiles in the Thyroid Gland of Two Phenotypically Extreme Pig Breeds Using Ribo-Zero RNA Sequencing.

    PubMed

    Shen, Yifei; Mao, Haiguang; Huang, Minjie; Chen, Lixing; Chen, Jiucheng; Cai, Zhaowei; Wang, Ying; Xu, Ningying

    2016-01-01

    The thyroid gland is an important endocrine organ modulating development, growth, and metabolism, mainly by controlling the synthesis and secretion of thyroid hormones (THs). However, little is known about the pig thyroid transcriptome. Long non-coding RNAs (lncRNAs) regulate gene expression and play critical roles in many cellular processes. Yorkshire pigs have a higher growth rate but lower fat deposition than that of Jinhua pigs, and thus, these species are ideal models for studying growth and lipid metabolism. This study revealed higher levels of THs in the serum of Yorkshire pigs than in the serum of Jinhua pigs. By using Ribo-zero RNA sequencing-which can capture both polyA and non-polyA transcripts-the thyroid transcriptome of both breeds were analyzed and 22,435 known mRNAs were found to be expressed in the pig thyroid. In addition, 1189 novel mRNAs and 1018 candidate lncRNA transcripts were detected. Multiple TH-synthesis-related genes were identified among the 455 differentially-expressed known mRNAs, 37 novel mRNAs, and 52 lncRNA transcripts. Bioinformatics analysis revealed that differentially-expressed genes were enriched in the microtubule-based process, which contributes to THs secretion. Moreover, integrating analysis predicted 13 potential lncRNA-mRNA gene pairs. These data expanded the repertoire of porcine lncRNAs and mRNAs and contribute to understanding the possible molecular mechanisms involved in animal growth and lipid metabolism.

  2. RNA Extraction from Xenopus Auditory and Vestibular Organs for Molecular Cloning and Expression Profiling with RNA-Seq and Microarrays.

    PubMed

    Trujillo-Provencio, Casilda; Powers, TuShun R; Sultemeier, David R; Ramirez-Gordillo, Daniel; Serrano, Elba E

    2016-01-01

    The amphibian Xenopus offers a unique model system for uncovering the genetic basis of auditory and vestibular function in an organism that is well-suited for experimental manipulation during animal development. However, many procedures for analyzing gene expression in the peripheral auditory and vestibular systems mandate the ability to isolate intact RNA from inner ear tissue. Methods presented here facilitate preparation of high-quality inner ear RNA from larval and post-metamorphic Xenopus specimens that can be used for a variety of purposes. We demonstrate that RNA isolated with these protocols is suitable for microarray analysis and Illumina-Solexa sequencing (RNA-Seq) of inner ear organs, and for cloning of large transcripts, such as those for ion channels. Genetic sequences cloned with these procedures can be used for transient transfection of Xenopus kidney cell lines with fluorescent protein fusion constructs. PMID:27259922

  3. RNA Extraction from Xenopus Auditory and Vestibular Organs for Molecular Cloning and Expression Profiling with RNA-Seq and Microarrays.

    PubMed

    Trujillo-Provencio, Casilda; Powers, TuShun R; Sultemeier, David R; Ramirez-Gordillo, Daniel; Serrano, Elba E

    2016-01-01

    The amphibian Xenopus offers a unique model system for uncovering the genetic basis of auditory and vestibular function in an organism that is well-suited for experimental manipulation during animal development. However, many procedures for analyzing gene expression in the peripheral auditory and vestibular systems mandate the ability to isolate intact RNA from