Effective suppression of fibronectin synthesis by retrovirus delivered shRNA in rat cardiac fibroblasts

Extracellular matrix overexpression is a common final pathway that leads to ventricular remodeling. Fibronectin plays a pivotal role in this progress. In the work presented here, we explored the possibility of direct inhibition of fibronectin synthesis in rat cardiac fibroblasts by small interference RNA (siRNA). We found that siRNA could successfully suppress the fibronectin overexpression stimulated by angiotensin II. To overcome the limitations of plasmid-based siRNA, we subcloned the H1 promoter into pLXIN, a commercially available retroviral vector. We found that H1 promoter worked very well to form the small hairpin RNA (shRNA) on the retroviral vector, and the fibronectin expression was dramatically down regulated by shRNA. We think the retroviral shRNA delivery system that we have constructed may have potential roles in treating ventricular remodeling.     

Knockdown of human p53 gene expression in 293-T cells by retroviral vector-mediated short hairpin RNA

RNA interference （RNAi） is an evolutionarily conserved process of gene silencing in multiple organisms, which has become a powerful tool for investigating gene function by reverse genetics. Recently, many groups have reported to use synthesized oligonucleotides or siRNA encoding plasmids to induce RNAi in mammalian cells by transfection, but this is still limited in its application, especially when it is necessary to generate long-term gene silencing in vivo. To circumvent this problem, retrovirus- or lentivirus-delivered RNAi has been developed. Here, we described two retroviral systems for delivering short hairpin RNA （shRNA） transcribed from the H1 promoter. The results showed that retroviral vector-mediated RNAi can substantially downregulate the expression of human p53 in 293-T cells. Furthermore, the retroviral vectormediated RNAi in our transduction system can stably inactivate the p53 gene for a long time. Compared to shRNAs transcribed from the U6 promoter, HI-driven shRNA also dramatically reduced the expression of p53. The p53 downregulation efficiencies of H1- and U6-driven shRNAs were almost identical. The results indicate that retroviral vector-delivered RNAi would be a useful tool in functional genomics and gene therapy.     

A retroviral vector for siRNA expression in mammalian cells

Utilization of RNA interference (RNAi) for knockdown of gene expression has become a standard tool for the study of gene function. Short hairpin RNAs (shRNAs) expressed from RNA polymerase III promoters are widely used to achieve stable knockdown of gene expression by RNAi. We have constructed a retroviral-based shRNA expression vector, pSiRPG, as a tool for shRNA-based functional genomic studies. This vector is based on a widely used shRNA expression system and was modified to harbor an enhanced green fluorescent protein (EGFP) and a puromycin selection marker. The functionality of the elements in the pSiRPG vector was validated. The H1(TetO₂) promoter in the vector facilitates doxycycline-inducible shRNA expression, which was demonstrated in cells expressing the Tet repressor (TetR). However, we also demonstrated limited efficiency of the inhibition of shRNA expression in an uninduced TetR-expressing cell line. This observation strongly indicates that the H1(TetO₂) promoter, which is used in a wide range of vectors, is not optimal for tightly regulated shRNA expression. Stable repression of the NDRG1 protein level was observed when introducing pSiRPG constructs expressing shRNAs targeting NDRG1 into two mammary epithelial cell lines by retroviral delivery. This vector should therefore facilitate functional studies in breast cell lines that are hard to transfect with conventional plasmid-based methods.     

Primer extension-based method for the generation of a siRNA/miRNA expression vector.

RNA interference (RNAi) has become a powerful technique for studying gene function, biological pathways, and the physiology of diseases. Typically, the RNAi response in mammalian cells is mediated by small interfering RNA (siRNA). The use of synthesized siRNA to silence gene is relatively quick and easy, but it is costly with transient effects. A short hairpin RNA (shRNA) with complementary sense and antisense sequences of a target gene separated by a loop structure results in gene silencing that is as effective as chemically synthesized siRNA with fewer limitations. However, current methods for constructing shRNA vectors require the synthesis of long oligonucleotides, which is costly and often suffers from mutation problems during synthesis. Here, we report an alternative approach to generate a shRNA expression vector with high efficacy. We utilized shorter (相似文献   

pHYPER, a shRNA vector for high-efficiency RNA interference in embryonic stem cells

RNA interference (RNAi) is a powerful method to generate loss-of-function phenotypes. Plasmid vectors with RNA polymerase III promoters have been developed to express short hairpin RNAs (shRNAs) in mammalian cells. In order to optimize the efficiency of these vectors in embryonic stem (ES) cells, we have constructed and tested several plasmids, based on the H1 promoter; that direct the expression of shRNAs. The original pSUPER vector was used as a reference in this study. This vector drives the expression of shRNAs from a basic 0.2-kb H1 promoter; which exhibits a variable expression when integrated into the genome of ES cells. We used a 2.5-kb mouse genomic fragment containing the H1 promoter to construct a new H1 shRNA vector pHYPER. A comparison of this vector with the basic 0.2-kb H1 vector showed that pHYPER directs the synthesis of higher amounts of shRNAs. Using epifluorescence and fluorescent-activated cell sorting (FACS) analysis, we demonstrated that pHYPER is 4-fold more active than the 0.2-kb H1-based vector after integration into the genome of mouse ES cells. We provide a new, improved H1 shRNA vector that is optimized for both transient transfection studies and the generation of stable ES cell lines.     

Silencing of antiapoptotic survivin gene by multiple approaches of RNA interference technology

Silencing of mammalian gene expression by RNA interference (RNAi) technology can be achieved using small interfering RNA (siRNA) or short hairpin RNA (shRNA). However, the relative effectiveness of these two approaches is not known. It is also not clear whether gene-specific shRNA transcribed from an RNA polymerase II (Pol II)-directed promoter in a fusion form can disrupt the targeted gene expression. Here, we report that using both luciferase and antiapoptotic survivin genes as targets, both siRNA and shRNA approaches significantly silenced the targeted gene expression in cancer cells. We further demonstrated that shRNAs transcribed from an RNA Pol II-mediated promoter in a green fluorescent protein (GFP) fusion form at the 3'-untranslated region silenced luciferase and survivin expression as well, suggesting that the extra RNA sequence outside of the shRNA hairpin does not disrupt shRNA function. We also showed that silencing of survivin expression selectively induces apoptosis in transfected cells. Together, we have validated multiple approaches of RNAi technology using both survivin and luciferase genes as targets and demonstrated for the first time that GFP-shRNAs transcribed from an RNA Pol II-mediated promoter could mediate gene silencing, which may lead to new directions for the application of RNAi technology.     

Characterisation and comparison of the chicken H1 RNA polymerase III promoter for short hairpin RNA expression

The U6 and 7SK RNA polymerase III promoters are widely used in RNAi research for the expression of shRNAs. However, with their increasing use in vitro and in vivo, issues associated with cytotoxicity have become apparent with their use. Therefore, alternative promoters such as the weaker H1 promoter are becoming a popular choice. With interest in the chicken as a model organism, we aimed to identify and characterise the chicken H1 promoter for the expression of shRNAs for the purpose of RNAi. The chicken H1 promoter was isolated and sequence analysis identified conserved RNA polymerase III promoter elements. A shRNA expression cassette containing the chicken H1 promoter and shRNA targeting enhanced green fluorescent protein (EGFP) was developed. An RNAse protection assay confirmed activity of the promoter determined by the detection of expressed shRNAs. Comparison of the H1 promoter to the chicken RNA polymerase III 7SK and U6 promoters demonstrated that expressed shRNAs from the H1 promoter induced gene specific silencing, albeit to lower levels in comparison to both 7SK and U6 promoters. Here we have identified a new tool for RNAi research with specific applications to the chicken. The availability of a RNA polymerase III promoter that drives shRNA expression to reduced levels will greatly benefit in ovo/in vivo applications where there are concerns of cytotoxicity resulting from overexpression of an shRNA.     

Properties of gene knockdown system by vector‐based siRNA in zebrafish

RNA interference (RNAi) has emerged as a powerful tool to silence specific genes. Vector‐based RNAi systems have been developed to downregulate targeted genes in a spatially and temporally regulated fashion both in vitro and in vivo. The zebrafish (Danio rerio) is a model animal that has been examined based on a wide variety of biological techniques, including embryonic manipulations, forward and reverse genetics, and molecular biology. However, a heritable and tissue‐specific knockdown of gene expression has not yet been developed in zebrafish. We examined two types of vector, which produce small interfering RNA (siRNA), the direct effector in RNAi system; microRNA (miRNA) process mimicking vectors with a promoter for RNA polymerase II and short hairpin RNA (shRNA) expressing vector through a promoter for RNA polymerase III. Though gene‐silencing phenotypes were not observed in the miRNA process mimicking vectors, the transgenic embryos of the second vector (Tg(zU6‐shGFP)), shRNA expressing vector for enhanced green fluorescence protein, revealed knockdown of the targeted gene. Interestingly, only the embryos from Tg(zU6‐shGFP) female but not from the male fish showed the downregulation. Comparison of the quantity of siRNA produced by each vector indicates that the vectors tested here induced siRNA, but at low levels barely sufficient to silence the targeted gene.     

Specific inhibition of gene expression using a stably integrated,inducible small-interfering-RNA vector

We have designed a doxycycline-regulated form of the H1 promoter of RNA polymerase III that allows the inducible knockdown of gene expression by small interfering RNAs (siRNAs). As a proof-of-principle, we have targeted β-catenin in colorectal cancer (CRC) cells. T-cell factor (TCF) target-gene expression is induced by accumulated β-catenin, and is the main transforming event in these cells. We have shown previously that the disruption of β-catenin/TCF4 activity in CRC cells by the overexpression of dominant-negative TCF induces rapid G1 arrest and differentiation. Stable integration of our inducible siRNA vector allowed the rapid production of siRNAs on doxycycline induction, followed by specific downregulation of β-catenin. In these CRC cells, TCF reporter-gene activity was inhibited, and G1 arrest and differentiation occurred. The inhibition of two other genes using this vector system shows that it should be useful for the inducible knockdown of gene expression.     

Promoter choice affects the potency of HIV-1 specific RNA interference

RNA interference (RNAi) is mediated by small interfering (si) RNAs that target and degrade mRNA in a sequence-specific manner. Cellular expression of siRNA can be achieved by the use of expression cassettes driven by RNA polymerase III (pol III) promoters. Here, we demonstrate that a modified tRNA^met-derived (MTD) promoter effectively drives the cellular expression of HIV-1-specific siRNA. We observed up to 56% greater inhibition of virus production when the MTD promoter was used to drive the expression of short hairpin (sh) RNA targeting the HIV-1 transactivator protein tat compared to cassettes containing other pol III promoters such as H1, U6+1 and U6+27. We conclude that the MTD promoter is ideally suited to drive intracellular expression of HIV-1 specific siRNA and may serve as an important component of future RNAi vector delivery systems.     

A miR-21 hairpin structure-based gene knockdown vector

RNA interference (RNAi) is widely used to study gene functions as a reverse genetic means from first-generation siRNA to second-generation short hairpin RNA (shRNA) or the newly developed microRNA (shRNA-miR). Here we report a gene knockdown vector system based on the mouse miR-21 hairpin structure. In this system, the pre-miRNA hairpin of the miR-21 gene was modified by replacing the 22-nucleotide mature sequence with shRNA sequences that target genes of interest, flanked by 160-bp upstream and 65-bp downstream sequences of the mouse pre-miR-21. We tested this system by knocking down the enhanced green fluorescence protein (EGFP) reporter gene using different vectors, in which shRNA-miR was driven by the polymerase II (pol II) promoter. We found that miR-21 hairpin-based shRNA-miR can be directly placed under pol II promoter, like UbC or CMV promoter to knockdown the gene of interest. To facilitate the wide application of the miR-21 hairpin-based gene knockdown system, we further knocked down the endogenous gene lamin (A/C), which showed that endogenous lamin A/C expression can be efficiently silenced using the miR-21 hairpin-based lentiviral vector. The miR-21 hairpin-based gene knockdown vector will provide a new genetic tool for gene functional studies in vitro and in vivo.