RNA interference in mammalian cells using siRNAs synthesized with T7 RNA polymerase

Methods that allow the specific silencing of a desired gene are invaluable tools for research. One of these is based on RNA interference (RNAi), a process by which double-stranded RNA (dsRNA) specifically suppresses the expression of a target mRNA. Recently, it has been reported that RNAi also works in mammalian cells if small interfering RNAs (siRNAs) are used to avoid activation of the interferon system by long dsRNA. Thus, RNAi could become a major tool for reverse genetics in mammalian systems. However, the high cost and the limited availability of the short synthetic RNAs and the lack of certainty that a designed siRNA will work present major drawbacks of the siRNA technology. Here we present an alternative method to obtain cheap and large amounts of siRNAs using T7 RNA polymerase. With multiple transfection procedures, including calcium phosphate co-precipitation, we demonstrate silencing of both exogenous and endogenous genes.     

RNA干扰与抗病毒感染

RNA干扰(RNAi)是由小干扰RNA(siRNA)引发的生物细胞内同源基因的转录后基因沉默(PTGS)现象,是一种古老的生物抵抗外在感染的防御机制。RNAi因其在维持基因组稳定、调控基因表达和保护基因组免受外源核酸侵入等方面发挥的重要作用,已被广泛用于探索基因功能、基因治疗和新药的研发。外源导入siRNA引发的RNAi可以特异性抑制病毒的复制与感染,为抗病毒感染治疗开辟了一条新的途径。     

RNAi in mice: a promising approach to decipher gene functions in vivo

RNA interference (RNAi) is a simple and powerful tool widely used to study gene functions in many species. Vector-based systems using RNA polymerase III promoters have been developed to achieve stable expression of small interfering RNA (siRNA) or small hairpin RNA (shRNA) in mammalian cells. Recent investigations demonstrated that when, combined with the Cre-loxP system, the vector-based RNAi can be used to achieve conditional or tissue specific knockdown of endogenous genes with high efficiency in mice. Here, we review these recent progresses and discuss the advantages, limitations and future development of this emerging technology.     

RNA干涉（RNAi）技术应用于哺乳动物细胞的研究策略

RNAi作为新近发展起来的基因功能分析技术,近年来在哺乳动物细胞中的研究已取得了长足进展,且有着广泛的应用前景。对RNAi作用机制及RNAi实验操作技术的探讨是目前研究的热点。研究表明,哺乳动物细胞中的RNAi作用模式与植物有所不同。文章对RNAi作用机制、哺乳动物细胞RNAi实验的一般策略（包括靶siRNA序列选择、siRNA获取方法、siRNA转染、RNAi效果检测等）以及最新研究进展进行简述,以供类似工作的参考。     

Structural diversity repertoire of gene silencing small interfering RNAs

Since the discovery of double-stranded (ds) RNA-mediated RNA interference (RNAi) phenomenon in Caenorhabditis elegans, specific gene silencing based upon RNAi mechanism has become a novel biomedical tool that has extended our understanding of cell biology and opened the door to an innovative class of therapeutic agents. To silence genes in mammalian cells, short dsRNA referred to as small interfering RNA (siRNA) is used as an RNAi trigger to avoid nonspecific interferon responses induced by long dsRNAs. An early structure-activity relationship study performed in Drosophila melanogaster embryonic extract suggested the existence of strict siRNA structural design rules to achieve optimal gene silencing. These rules include the presence of a 3' overhang, a fixed duplex length, and structural symmetry, which defined the structure of a classical siRNA. However, several recent studies performed in mammalian cells have hinted that the gene silencing siRNA structure could be much more flexible than that originally proposed. Moreover, many of the nonclassical siRNA structural variants reported improved features over the classical siRNAs, including increased potency, reduced nonspecific responses, and enhanced cellular delivery. In this review, we summarize the recent progress in the development of gene silencing siRNA structural variants and discuss these in light of the flexibility of the RNAi machinery in mammalian cells.     

U6 promoter-driven siRNAs with four uridine 3' overhangs efficiently suppress targeted gene expression in mammalian cells

The first evidence for gene disruption by double-stranded RNA (dsRNA) came from careful analysis in Caenorhabditis elegans. This phenomenon, called RNA interference (RNAi), was observed subsequently in various organisms, including plants, nematodes, Drosophila, and protozoans. Very recently, it has been reported that in mammalian cells, 21- or 22-nucleotide (nt) RNAs with 2-nt 3' overhangs (small inhibitory RNAs, siRNAs) exhibit an RNAi effect. This is because siRNAs are not recognized by the well-characterized host defense system against viral infections, involving dsRNA-dependent inhibition of protein synthesis. However, the current method for introducing synthetic siRNA into cells by lipofection restricts the range of applications of RNAi as a result of the low transfection efficiencies in some cell types and/or short-term persistence of silencing effects. Here, we report a vector-based siRNA expression system that can induce RNAi in mammalian cells. This technical advance for silencing gene expression not only facilitates a wide range of functional analysis of mammalian genes but might also allow therapeutic applications by means of vector-mediated RNAi.     

A simple strategy for generation of gene knockdown constructs with convergent H1 and U6 promoters

RNA interference (RNAi) is a powerful tool for functional genetic studies in model organisms and mammalian cells. To facilitate rapid construction of gene knockdown constructs and RNAi libraries for known genes of mammalian cells, a new and simple strategy to produce small interfering RNA (siRNA) expression vectors with two opposing polymerase III promoters was developed. The design involved a one-step PCR amplification and single cloning procedure to construct a dual promoter siRNA expression vector. The forward primer is identical for all PCR reactions, only a single reverse primer that contains the siRNA targeting sequence has to be synthesized in the construction of each individual vector. This single primer design is cost-effective and it reduces the risk of sequence errors during synthesis of long oligos. Sense and antisense strands of siRNA duplexes were transcribed from the same template and this eliminated the need to synthesize long hairpin-forming oligonucleotides. Our study demonstrated that this vector design could mediate potent inhibition of expression of both exogenous and endogenous genes in mammalian cells.     

Mammalian RNAi: a practical guide

Silencing of gene expression by RNA interference (RNAi) has become a powerful tool for the functional annotation of the Caenorhabditis elegans and Drosophila melanogaster genomes. Recent advances in the design and delivery of targeting molecules now permit efficient and highly specific gene silencing in mammalian systems as well. RNAi offers a simple, fast, and cost-effective alternative to existing gene targeting technologies both in cell-based and in vivo settings. Synthetic small interfering RNA (siRNA) and retroviral short hairpin RNA (shRNA) libraries targeting thousands of human and mouse genes are publicly available for high-throughput genetic screens, and knockdown animals can be rapidly generated by lentivirus-mediated transgenesis. RNAi also holds great promise as a novel therapeutic approach. This review provides insight into the current gene silencing techniques in mammalian systems.     

A peptidomimetic siRNA transfection reagent for highly effective gene silencing

RNA interference (RNAi) techniques hold forth great promise for therapeutic silencing of deleterious genes. However, clinical applications of RNAi require the development of safe and efficient methods for intracellular delivery of small interfering RNA (siRNA) oligonucleotides specific to targeted genes. We describe the use of a lipitoid, a cationic oligopeptoid-phospholipid conjugate, for non-viral transfection of synthetic siRNA oligos in cell culture. This peptidomimetic delivery vehicle allows for efficient siRNA transfection in a variety of human cell lines with negligible toxicity and promotes extensive downregulation of the targeted genes at both the protein and the mRNA level. We compare the lipitoid reagent to a standard commercial transfection reagent. The lipitoid is highly efficient even in primary IMR-90 human lung fibroblasts in which other commercial reagents are typically ineffective.     

Optimization procedure for small interfering RNA transfection in a 384-well format

High-throughput screening of RNAi libraries has become an essential part of functional analysis in academic and industrial settings. The transition of a cell-based RNAi assay into a 384-well format requires several optimization steps to ensure the phenotype being screened is appropriately measured and that the signal-to-background ratio is above a certain quantifiable threshold. Methods currently used to assess small interfering RNA (siRNA) efficacy after transfection, including quantitative PCR or branch DNA analysis, face several technical limitations preventing the accurate measurement of mRNA levels in a 384-well format. To overcome these difficulties, the authors developed an approach using a viral-based transfection system that measures siRNA efficacy in a standardized 384-well assay. This method allows measurement of siRNA activity in a phenotypically neutral manner by quantifying the knockdown of an exogenous luciferase gene delivered by a lentiviral vector. In this assay, the efficacy of a luciferase siRNA is compared to a negative control siRNA across many distinct assay parameters including cell type, cell number, lipid type, lipid volume, time of the assay, and concentration of siRNA. Once the siRNA transfection is optimized as a 384-well luciferase knockdown, the biologically relevant phenotypic analysis can proceed using the best siRNA transfection conditions. This approach provides a key technology for 384-well assay development when direct measurement of mRNA knockdown is not possible. It also allows for direct comparison of siRNA activity across cell lines from almost any mammalian species. Defining optimal conditions for siRNA delivery into mammalian cells will greatly increase the speed and quality of large-scale siRNA screening campaigns.     

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.     

哺乳动物细胞小干扰RNA库的建立和应用

双链小干扰RNA（siRNA）在多种类型细胞中介导特异性的基因沉默,这一现象的发现为深入研究单个基因的功能提供了重要的方法学基础,从而得到了广泛的应用.最近的文献报道了全基因组siRNA库的建立,为高通量基因功能分析和研究提供了新的方法,成为新的研究热点.小干扰RNA库可以用来筛选和研究介导细胞复杂表型和生物学过程的关键基因,通过建立一系列具有目的表型的细胞系,有可能对特定细胞信号调节通路进行更为全面的解析.本文综述了目前在siRNA建库方法方面的进展,并探讨了建立小干扰RNA库中的关键问题.     

Predicting siRNA activity based on back-propagation neural network

RNA interference (RNAi) is a phenomenon of gene silence induced by a double-stranded RNA (dsRNA) homologous to a target gene.RNAi can be used to identify the function of genes or to knock down the targeted genes.In RNAi technology,19 bp double-stranded short interfering RNAs (siRNA) with characteristic 3' overhangs are usually used.The effects of siRNAs are quite varied due to the different choices in the sites of target mRNA.Moreover,there are many factors influencing siRNA activity and these factors are usually nonlinear.To find the motif features and the effect on siRNA activity,we carried out a feature extraction on some published experimental data and used these features to train a backpropagation neural network (BP NN).Then,we used the trained BP NN to predict siRNA activity.     

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.     

针对leader序列的siRNA能够有效抑制SARS冠状病毒多个mRNA的表达

小干扰RNAs(siRNAs)能够有效降解具有互补序列的RNA.在SARS-CoV的基因组RNA和所有亚基因组RNA的5′端均有一段共同的leader序列,而且该leader序列在不同的病毒分离物中高度保守,因此leader序列可作为一个用于抑制SARS-CoV复制的有效靶点.研究表明,针对leader序列化学合成的siRNA和DNA载体表达的shRNA都可以有效抑制SARS-CoV mRNA的表达.Leader序列特异的siRNA或shRNA不仅可以有效抑制leader与报告基因EGFP融合基因的表达,而且还可以有效抑制leader与刺突蛋白(spikeprotein)、膜蛋白(membrane protein)和核衣壳蛋白(nucleocapsid protein)基因的融合转录产物的表达.结果表明,针对leader序列的RNA干扰可以发展成为一种抗SARS-CoV治疗的有效策略.     

Combination of active and inactive siRNA targeting the mitotic kinesin Eg5 impairs silencing efficiency in several cancer cell lines

Gene silencing by RNA interference (RNAi) has proven to be a powerful tool for investigating gene function in mammalian cells. Combination of several short interfering RNA (siRNA) targeting the same gene is commonly used to improve RNA interference. However, in contrary to the well-described mechanism of RNAi, efficiency of single siRNA compared to pool remains poorly documented. We addressed this issue using several active and inactive siRNA targeting Eg5, a kinesin-related motor involved in mitotic spindle assembly. These siRNA, used alone or in combination, were tested for their silencing efficiency in several cancer cell lines. Here we show that presence of inactive Eg5 siRNA in a pool dramatically decreases knockdown efficacy in a cell line- and dose-dependent manner. Lack of inhibition by unrelated siRNA suggests that a competition may occur during siRNA incorporation into RNA-induced silencing complexes (RISCs) along with the target mRNA. Altogether, our results, which need to be confirmed with additional inactive siRNA, indicate that combination of siRNA may not increase but instead decrease silencing efficiency.