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.     

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   

Transgenic RNA interference in ES cell-derived embryos recapitulates a genetic null phenotype

Gene targeting via homologous recombination in murine embryonic stem (ES) cells has been the method of choice for deciphering mammalian gene function in vivo. Despite improvements in this technology, it still remains a laborious method. Recent advances in RNA interference (RNAi) technology have provided a rapid loss-of-function method for assessing gene function in a number of organisms. Studies in mammalian cell lines have shown that introduction of small interfering RNA (siRNA) molecules mediates effective RNA silencing. Plasmid-based systems using RNA polymerase III (RNA pol III) promoters to drive short hairpin RNA (shRNA) molecules were established to stably produce siRNA. Here we report the generation of knockdown ES cell lines with transgenic shRNA. Because of the dominant nature of the knockdown, embryonic phenotypes could be directly assessed in embryos completely derived from ES cells by the tetraploid aggregation method. Such embryos, in which endogenous p120-Ras GTPase-activating protein (RasGAP), encoded by Rasa1 (also known as RasGAP), was silenced, had the same phenotype as did the previously reported Rasa1 null mutation.     

Characterisation and application of a bovine U6 promoter for expression of short hairpin RNAs

Background  

The use of small interfering RNA (siRNA) molecules in animals to achieve double-stranded RNA-mediated interference (RNAi) has recently emerged as a powerful method of sequence-specific gene knockdown. As DNA-based expression of short hairpin RNA (shRNA) for RNAi may offer some advantages over chemical and in vitro synthesised siRNA, a number of vectors for expression of shRNA have been developed. These often feature polymerase III (pol. III) promoters of either mouse or human origin.     

各种RNA干扰库的构建及比较

不同种类的RNA干扰库已经被用于基因功能的研究之中,根据其构建方式和分子形式的不同,可将RNA干扰库分为4种类型,即化学合成的小干扰RNA（siRNA）库、化学合成的小发夹RNA（shRNA）表达库、酶切法构建的siRNA库和酶切法构建的shRNA表达库。简要介绍上述RNA干扰库的构建方法及其特点和局限。     

Suppression of hepatitis C virus replicon by RNA interference directed against the NS3 and NS5B regions of the viral genome

RNA interference (RNAi) is a phenomenon in which small interfering RNA (siRNA), an RNA duplex 21 to 23 nucleotides (nt) long, or short hairpin RNA (shRNA) resembling siRNA, mediates degradation of the target RNA molecule in a sequence-specific manner. RNAi is now expected to be a useful therapeutic strategy for hepatitis C virus (HCV) infection. In the present study we compared the efficacy of a number of shRNAs directed against different target regions of the HCV genome, such as 5'-untranslated region (5'UTR) (nt 286 to 304), Core (nt 371 to 389), NS3-1 (nt 2052 to 2060), NS3-2 (nt 2104 to 2122), and NS5B (nt 7326 to 7344), all of which except for NS5B are conserved among most, if not all, HCV subtype 1b (HCV-1b) isolates in Japan. We utilized two methods to express shRNAs, one utilizing an expression plasmid (pAVU6+27) and the other utilizing a recombinant lentivirus harboring the pAVU6+27-derived expression cassette. Although 5'UTR has been considered to be the most suitable region for therapeutic siRNA and/or shRNA because of its extremely high degree of sequence conservation, we observed only a faint suppression of an HCV subgenomic replicon by shRNA against 5'UTR. In both plasmid-and lentivirus-mediated expression systems, shRNAs against NS3-1 and NS5B suppressed most efficiently the replication of the HCV replicon without suppressing host cellular gene expression. Synthetic siRNA against NS3-1 also inhibited replication of the HCV replicon in a dose-dependent manner. Taken together, the present results imply the possibility that the recombinant lentivirus expressing shRNA against NS3-1 would be a useful tool to inhibit HCV-1b infection.     

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.     

Photosensitizing carrier proteins for photoinducible RNA interference

RNA interference (RNAi) is being widely explored as a tool in functional genomics and tissue engineering, and in the therapy of intractable diseases, including cancer and neurodegenerative diseases. Recently, we developed a photoinducible RNAi method using photosensitizing carrier proteins, named CLIP-RNAi (CPP-linked RBP-mediated RNA internalization and photoinduced RNAi). Novel carrier proteins were designed for this study to establish a highly efficient delivery system for small interfering RNA (siRNA) or short hairpin RNA (shRNA) and to demonstrate light-dependent gene silencing. In addition, the results suggested that the dissociation of the siRNA (or shRNA) from carrier proteins in the cytoplasm is a critical event in CLIP-RNAi-mediated gene silencing.     

New paradigms on siRNA local application

Small interfering RNA (siRNA) functions through pairing with specific mRNA sequences and results in the mRNA’s degradation. It is a potential therapeutic approach for many diseases caused by altered gene expression. The delivery of siRNA is still a major problem due to its rapid degradation in the circulation. Various strategies have been proposed to help with the cellular uptake of siRNA and short or small hairpin RNA (shRNA). Here, we reviewed recently published data regarding local applications of siRNA. Compared with systemic delivery methods, local delivery of siRNA/shRNA has many advantages, such as targeting the specific tissues or organs, mimicking a gene knockout effect, or developing certain diseases models. The eye, brain, and tumor tissues are ‘hot’ target tissues/organs for local siRNA delivery. The siRNA can be delivered locally, in naked form, with chemical modifications, or in formulations with viral or non-viral vectors, such as liposomes and nanoparticles. This review provides a comprehensive overview of RNAi local administration and potential future applications in clinical treatment. [BMB Reports 2015; 48(3): 147-152]     

Gene silencing in chick embryos with a vector-based small interfering RNA system

In this paper, the use of vector-based RNA interference (RNAi) to specifically interfere with gene expression in chick embryos is reported. In ovo electroporation was carried out to transfer a small interfering RNA (siRNA) expression vector into chick embryos. En2 was chosen for the target gene because the family gene, En1, is expressed in a similar pattern. Four sets of 19-mer sequences were designed with the En2 open reading frame region connected to a sequence of short hairpin RNA (shRNA), which exerts siRNA effects after being transcribed, and inserted into pSilencer U6-1.0 vector. En2 and En1 expression were suppressed by the siRNA whose sequence completely matched En2 and En1. Suppression occurred when the siRNA sequence differed by up to two nucleotides from the target sequence. The sequence that differed by four nucleotides from the target gene did not show siRNA effects. One set that completely matched the En2 target did not show siRNA effects, which may be due to location of the siRNA in the target gene. Thus, multiple sets of shRNA must be prepared if we are to consider. This system will greatly contribute to the analysis of function of genes of interest, because the target gene can be silenced in a locally and temporally desired manner.     

Adenovirus-delivered antisense RNA and shRNA exhibit different silencing efficiencies for the endogenous transforming growth factor-beta (TGF-beta) type II receptor

Gene silencing is an essential tool in gene discovery and gene therapy. Traditionally, viral delivery of antisense RNA and, more recently, small interfering RNA (siRNA) molecules in the form of small hairpin RNAs (shRNA) has been used as a strategy to achieve gene silencing. Nevertheless, the enduring challenge is to identify molecules that specifically and optimally silence a given target gene. In this study, we tested a set of adenovirus-delivered antisense RNA fragments and adenovirus-delivered shRNA molecules for their ability to target human transforming growth factor-beta type II receptor (TGFbetaRII). We used a dicistronic reporter, consisting of the coding sequences for TGFbetaRII and green fluorescent protein (GFP) to screen for optimal silencing agents targeting TGFbetaRII. Our results show, for both antisense RNA and shRNA molecules, that their effectiveness in the GFP screen correlated directly with their ability to reduce exogenously expressed TGFbetaRII. Unexpectedly, the antisense RNAs were unable to silence endogenous TGFbetaRII. In contrast, the shRNAs were able to silence endogenous TGFbetaRII. The shRNA that demonstrated the most pronounced effect on the dicistronic TGFbetaRII/GFP reporter reduced endogenous TGFbetaRII protein expression by 70% in A549 cells and reduced TGFbeta signaling by >80% in HeLa cells.     

Strand antagonism in RNAi: an explanation of differences in potency between intracellularly expressed siRNA and shRNA

Strategies to regulate gene function frequently use small interfering RNAs (siRNAs) that can be made from their shRNA precursors via Dicer. However, when the duplex components of these siRNA effectors are expressed from their respective coding genes, the RNA interference (RNAi) activity is much reduced. Here, we explored the mechanisms of action of shRNA and siRNA and found the expressed siRNA, in contrast to short hairpin RNA (shRNA), exhibits strong strand antagonism, with the sense RNA negatively and unexpectedly regulating RNAi. Therefore, we altered the relative levels of strands of siRNA duplexes during their expression, increasing the level of the antisense component, reducing the level of the sense component, or both and, in this way we were able to enhance the potency of the siRNA. Such vector-delivered siRNA attacked its target effectively. These findings provide new insight into RNAi and, in particular, they demonstrate that strand antagonism is responsible for making siRNA far less potent than shRNA.     

Short-hairpin RNAs delivered by lentiviral vector transduction trigger RIG-I-mediated IFN activation

Activation of the type I interferon (IFN) pathway by small interfering RNA (siRNA) is a major contributor to the off-target effects of RNA interference in mammalian cells. While IFN induction complicates gene function studies, immunostimulation by siRNAs may be beneficial in certain therapeutic settings. Various forms of siRNA, meeting different compositional and structural requirements, have been reported to trigger IFN activation. The consensus is that intracellularly expressed short-hairpin RNAs (shRNAs) are less prone to IFN activation because they are not detected by the cell-surface receptors. In particular, lentiviral vector-mediated transduction of shRNAs has been reported to avoid IFN response. Here we identify a shRNA that potently activates the IFN pathway in human cells in a sequence- and 5′-triphosphate-dependent manner. In addition to suppressing its intended mRNA target, expression of the shRNA results in dimerization of interferon regulatory factor-3, activation of IFN promoters and secretion of biologically active IFNs into the extracellular medium. Delivery by lentiviral vector transduction did not avoid IFN activation by this and another, unrelated shRNA. We also demonstrated that retinoic-acid-inducible gene I, and not melanoma differentiation associated gene 5 or toll-like receptor 3, is the cytoplasmic sensor for intracellularly expressed shRNAs that trigger IFN activation.