DNA vector-based RNA interference to study gene function in cancer

RNA interference (RNAi) inhibits gene expression by specifically degrading target mRNAs. Since the discovery of double-stranded small interference RNA (siRNA) in gene silencing, RNAi has become a powerful research tool in gene function studies. Compared to genetic deletion, RNAi-mediated gene silencing possesses many advantages, such as the ease with which it is carried out and its suitability to most cell lines. Multiple studies have demonstrated the applications of RNAi technology in cancer research. In particular, the development of the DNA vector-based technology to produce small hairpin RNA (shRNA) driven by the U6 or H1 promoter has made long term and inducible gene silencing possible. Its use in combination with genetically engineered viral vectors, such as lentivirus, facilitates high efficiencies of shRNA delivery and/or integration into genomic DNA for stable shRNA expression. We describe a detailed procedure using the DNA vector-based RNAi technology to determine gene function, including construction of lentiviral vectors expressing shRNA, lentivirus production and cell infection, and functional studies using a mouse xenograft model. Various strategies have been reported in generating shRNA constructs. The protocol described here employing PCR amplification and a 3-fragment ligation can be used to directly and efficiently generate shRNA-containing lentiviral constructs without leaving any extra nucleotide adjacent to a shRNA coding sequence. Since the shRNA-expression cassettes created by this strategy can be cut out by restriction enzymes, they can be easily moved to other vectors with different fluorescent or antibiotic markers. Most commercial transfection reagents can be used in lentivirus production. However, in this report, we provide an economic method using calcium phosphate precipitation that can achieve over 90% transfection efficiency in 293T cells. Compared to constitutive shRNA expression vectors, an inducible shRNA system is particularly suitable to knocking down a gene essential to cell proliferation. We demonstrate the gene silencing of Yin Yang 1 (YY1), a potential oncogene in breast cancer, by a Tet-On inducible shRNA system and its effects on tumor formation. Research using lentivirus requires review and approval of a biosafety protocol by the Biosafety Committee of a researcher's institution. Research using animal models requires review and approval of an animal protocol by the Animal Care and Use Committee (ACUC) of a researcher's institution.     

A new design of a lentiviral shRNA vector with inducible co-expression of ARGONAUTE 2 for enhancing gene silencing efficiency

Background

RNA interference (RNAi) is a robust tool for inhibiting specific gene expression, but it is limited by the uncertain efficiency of siRNA or shRNA constructs. It has been shown that the overexpression of ARGONAUTE 2 (AGO2) protein increases silencing efficiency. However, the key elements required for AGO2-mediated enhancement of gene silencing in lentiviral vector has not been well studied.

Results

To explore the application of AGO2-based shRNA system in mammalian cells, we designed shRNA vectors targeting the EGFP reporter gene and evaluated the effects of various factors on silencing efficiency including stem length, loop sequence, antisense location as well as the ratio between AGO2 and shRNA. We found that 19 ~ 21-bp stem and 6- or 9-nt loop structure in the sense-loop-antisense (S-L-AS) orientation was an optimal design in the AGO2-shRNA system. Then, we constructed a single lentiviral vector co-expressing shRNA and AGO2 and demonstrated that the simultaneous expression of shRNA and AGO2 can achieve robust silencing of exogenous DsRed2 and endogenous ID1 and P65 genes. However, the titers of packaged lentivirus from constitutive expression of AGO2 vector were extremely low, severely limiting its broad application. For the first time, we demonstrated that the problem can be significantly improved by using the inducible expression of AGO2 lentiviral system.

Conclusions

We reported a novel lentiviral vector with an optimal design of shRNA and inducible AGO2 overexpression which provides a new tool for RNAi research.

     

猪Nanog基因四环素诱导干扰载体的构建和鉴定

Nanog基因是在早期胚胎和干细胞等多能性细胞中特异表达的重要基因,但有关猪Nanog基因功能的相关研究甚少。四环素诱导干扰载体是一种可通过四环素等药物条件性诱导干扰目的基因的载体,尤其适用于在发育过程中起着关键作用的基因沉默。常规的四环素干扰系统为二元载体,与一元载体相比获得针对特定基因干扰的稳定细胞系所需周期更长。首先通过构建pGenesil 1.0-shRNA重组干扰载体,瞬时转染稳定过表达猪Nanog基因的猪胎儿成纤维细胞后通过Realtime-PCR筛选出干扰效率可达80%以上的干扰片段。之后将筛选得到的干扰片段插入到改造的一元四环素诱导干扰载体TREsilencer,对稳定表达猪Nanog基因的猪胎儿成纤维细胞进行了瞬时转染。实验分别通过光密度检测以及Realtime-PCR检测了不同浓度doxycycline的诱导效率和干扰效率。结果表明,所构建的四环素诱导干扰载体TREsilencer-shRNA5随着四环素浓度的增加,诱导Nanog基因的干扰效率增加,在处理浓度为1μg/ml时干扰效率可达70%以上,为后续得到可诱导的稳定干扰猪Nanog基因的细胞系和进一步研究猪Nanog基因功能奠定了基础。     

An inducible system for expression and validation of the specificity of short hairpin RNA in mammalian cells

RNA interference (RNAi) by means of short hairpin RNA (shRNA) has developed into a powerful tool for loss-of-function analysis in mammalian cells. The principal problem in RNAi experiments is off-target effects, and the most vigorous demonstration of the specificity of shRNA is the rescue of the RNAi effects with a shRNA-resistant target gene. This presents its own problems, including the unpredictable relative expression of shRNA and rescue cDNA in individual cells, and the difficulty in generating stable cell lines. In this report, we evaluated the plausibility of combining the expression of shRNA and rescue cDNA in the same vector. In addition to facilitate the validation of shRNA specificity, this system also considerably simplifies the generation of shRNA-expressing cell lines. Since the compensatory cDNA is under the control of an inducible promoter, stable shRNA-expressing cells can be generated before the knockdown phenotypes are studied by conditionally turning off the rescue protein. Conversely, the rescue protein can be activated after the endogenous protein is completely repressed. This approach is particularly suitable when prolonged expression of either the shRNA or the compensatory cDNA is detrimental to cell growth. This system allows a convenient one-step validation of shRNA and generation of stable shRNA-expressing cells.     

Reproducible and inducible knockdown of gene expression in mice

RNA interference (RNAi) has emerged as an efficient approach for rapid analysis of gene function. In mammalian cells, vector-based expression of small hairpin RNAs (shRNA) produces potent and stable gene knockdown effects. An inducible RNAi system with reproducible levels of siRNA expression will extend the usefulness of this methodology to the identification of gene functions within the developing or adult mouse. We present evidence that an RNA polymerase III-driven U6 promoter with stuffer sequences flanked by loxP sites inserted at three different sites within the promoter drives shRNA expression in a Cre recombinase-dependent manner. We utilized this approach to develop a generic strategy for the reproducible knockdown of gene expression in mice. By placing the inducible shRNA cassette into the ROSA26 locus of the mouse, we were able to generate reproducible levels of controlled expression of shRNA to produce discernable phenotypes in vitro and in vivo. This approach circumvents the prescreening of random integration in embryonic stem cell clones and further enables conditional gene knockdown with temporal and/or tissue specificity. This methodology should expedite large-scale functional studies.     

Gene knockdown by ecdysone-based inducible RNAi in stable mammalian cell lines

RNA interference (RNAi) is a powerful tool for the functional analysis of essential genes in the mammalian genome. Here, we present a simple ecdysone-based inducible RNAi approach that allows high induction and adjustable control of short hairpin RNA (shRNA) expression for silencing gene expression in a wide range of mammalian cells. This protocol describes the following: the design and cloning of inducible shRNA; testing and validation of gene knockdown; and methodology for establishing stable cell lines. This step-by-step protocol offers a quick and cost-effective approach for addressing the function of genes essential for cell cycle regulation and development and can be completed in less than 6 weeks.     

SW620细胞RACK1稳定RNA干扰细胞系的构建与鉴定

目的:为研究RACK1在结肠癌发生发展中的作用,构建结肠癌RACK1基因稳定RNA干扰(RNAi)细胞系。方法:根据人Gnb211 c DNA序列,运用干扰原则选择5个干扰位点并合成相应干扰片段,定向克隆入p Lentilox3.7干扰载体鼠U6启动子后并测序验证。用干扰及对照质粒分别转染HEK293T细胞48小时后,RT-PCR鉴定干扰效率,选出干扰效率较高的质粒包装慢病毒感染人结肠癌细胞SW620,流式无菌分选出荧光阳性的细胞扩增培养,RT-PCR及Western blotting鉴定慢病毒干扰效率。使用慢病毒构建的SW620 RACK1稳定RNAi细胞系及对照组进行MTT实验初步研究RACK1对SW620增殖的影响。结果:酶切和测序证实RACK1sh RNA质粒构建正确,产生能同时表达绿色荧光蛋白(EGFP)和RACK1 sh RNA的慢病毒载体质粒。慢病毒转导SW620并流式无菌分选扩增培养后,与空载体组相比,2个RNAi组均不同程度抑制RACK1表达,RACK1sh RNA5抑制作用最明显,RACK1干扰组细胞增殖得到了抑制。结论:SW620细胞RACK1稳定RNAi细胞系构建成功,为深入研究RACK1在结直肠癌发生发展中的作用奠定了基础。     

LRP16短发夹RNA慢病毒载体的构建及LRP16稳定抑制细胞的建立简

目的：构建靶向LRPl6基因的短发夹RNA（shRNA）慢病毒表达载体,鉴定其在HeLa细胞中对LRP16的抑制效果。方法：构建pWPT-U6-LRPl6shRNA-CMV-GFP慢病毒载体,通过病毒感染、细胞筛选、Western印迹等步骤,获得LRP16基因稳定抑制的细胞株。结果：构建了具有LRP16干扰效果的慢病毒载体,感染HeLa细胞后获得了稳定沉默LRP16及对照的细胞株;经克隆筛选,在荧光显微镜下观察到近似100％感染细胞发出绿色荧光;Western印迹证实pWPT-U6-L374-CMV-GFP和pWPT-U6-L668-CMV-GFP均可显著抑制HeLa细胞株中LRP16蛋白的表达,其中pWPT-Gsi-L374-GFP的抑制效果更好。结论：构建了靶向人LRP16基因shRNA慢病毒载体及LRP16稳定抑制的HeLa细胞系。     

New technology for an old favorite: lentiviral transgenesis and RNAi in rats

The ability to produce targeted deletions in the mouse genome via homologous recombination has been a hallmark of mouse genetics, and has lead to the production of thousands of gene knockouts. New technologies are making it possible to disrupt gene function in many other species. This article reviews some of these methods, highlighting the powerful combination of lentiviral vectors with RNA interference (RNAi), which allows one to produce transgenic animals expressing short hairpin RNA (shRNA) to “knock down” specific gene expression. Lentiviral transduction of embryos has been shown to be a highly efficient means of transgenesis, and is particularly promising for animals that are considered difficult to genetically modify by DNA pronuclear injection. This technique has been popular for introducing transgenes for shRNA expression into rodents and its utility for creating new genetic models has already been demonstrated. One of the purported advantages of in vivo RNAi is that shRNA expressing transgenes would be expected to act in a dominant nature, resulting in a phenotype in founder animals. However, one possible concern with lentiviral-mediated transgenesis is the potential for mosaicism in founders, and the data for this phenomenon and the potential causes and solutions are discussed. Emphasis is placed on the application of in vivo RNAi, and other reverse genetic methods, for creating new genetic models in the rat.     

慢病毒载体介导的层黏连蛋白受体稳定抑制细胞株的建立

目的:构建靶向层黏连蛋白受体(LR)基因的小发卡RNA(sh RNA)慢病毒表达载体,鉴定其对LR的抑制效果,并筛选LR稳定抑制的He La细胞株。方法:设计针对LR的sh RNA序列,将此序列和H1启动子克隆入含有EGFP报告基因的p Lenti6/v5慢病毒表达载体,通过病毒包装、细胞感染、抗生素筛选获得稳定细胞株,用real-time PCR和Western印迹检测筛选得到的稳定细胞株中LR的表达水平。结果和结论:构建了含有LR靶向sh RNA的慢病毒表达载体,包装成病毒后感染He La细胞,经抗生素筛选后获得了稳定抑制LR的细胞株;筛选后的细胞均可观察到报告基因EGFP的表达;经m RNA和蛋白水平检测,LR-sh6和LR-sh7均可显著抑制He La细胞株中LR的表达。     

AKT2基因短发夹结构RNA慢病毒载体的构建及鉴定

目的：构建丝/苏氨酸蛋白激酶2（AKT2）基因RNA干扰（RNAi）慢病毒载体。方法：利用公用网站按照RNAi序列设计原则,设计RNAi靶点序列并合成靶序列的Oligo DNA,退火形成双链DNA,与经MluI和ClaI进行酶切后的PLVTHM载体连接产生shRNA慢病毒载体。应用shRNA慢病毒载体转染293T细胞及U87细胞,测定病毒滴度,流式细胞仪测定U87细胞的转染效率,PCR及Western blot鉴定AKT2基因在U87细胞中的下调作用。结果：成功构建了shRNA-AKT2慢病毒载体,经测序与设计合成的靶向链完全一致。荧光显微镜下观察293T细胞感染效率大于90%,病毒滴度为3.59×107TU/ml;流式细胞仪测定对AKT2细胞的转染效率为86.93%。PCR测定shRNA载体感染U87细胞后AKT2的干扰效率为68%。Western Blot结果显示该慢病毒载体对AKT2的表达有较为显著的敲减作用。结论：成功构建了人胶质瘤细胞株AKT2基因RNAi慢病毒载体,为后续的体内外功能学试验创造了条件。     

Silencing of the Pink1 gene expression by conditional RNAi does not induce dopaminergic neuron death in mice

Transgenic RNAi, an alternative to the gene knockout approach, can induce hypomorphic phenotypes that resemble those of the gene knockout in mice. Conditional transgenic RNAi is an attractive choice of method for reverse genetics in vivo because it can achieve temporal and spatial silencing of targeted genes. Pol III promoters such as U6 are widely used to drive the expression of RNAi transgenes in animals. Tested in transgenic mice, a Cre-loxP inducible U6 promoter drove the broad expression of an shRNA against the Pink1 gene whose loss-of-functional mutations cause one form of familial Parkinson's disease. The expression of the shRNA was tightly regulated and, when induced, silenced the Pink1 gene product by more than 95% in mouse brain. However, these mice did not develop dopaminergic neurodegeneration, suggesting that silencing of the Pink1 gene expression from embryo in mice is insufficient to cause similar biochemical or morphological changes that are observed in Parkinson's disease. The results demonstrate that silencing of the PINK1 gene does not induce a reliable mouse model for Parkinson's disease, but that technically the inducible U6 promoter is useful for conditional RNAi in vivo.     

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.     

Development of a Multipurpose GATEWAY-Based Lentiviral Tetracycline-Regulated Conditional RNAi System (GLTR)

RNA interference (RNAi) has become an essential technology for functional gene analysis. Its success, however, depends on the effective expression of RNAi-inducing small double-stranded interfering RNA molecules (siRNAs) in target cells. In many cell types, RNAi can be achieved by transfection of chemically synthesised siRNAs, which results in transient knockdown of protein expression. Expression of double-stranded short hairpin RNA (shRNA) provides another means to induce RNAi in cells that are hard to transfect. To facilitate the generation of stable, conditional RNAi cell lines, we have developed novel one- and two-component vector GATEWAY-compatible lentiviral tetracycline-regulated RNAi (GLTR) systems. The combination of a modified RNA-polymerase-III-dependent H1 RNA promoter (designated ‘THT’) for conditional shRNA expression with different lentiviral delivery vectors allows (1) the use of fluorescent proteins for colour-coded combinatorial RNAi or for monitoring RNAi induction (pGLTR-FP), (2) selection of transduced cells (pGLTR-S), and (3) the generation of conditional cell lines using a one vector system (pGLTR-X). All three systems were found to be suitable for the analysis of essential genes, such as CDC27, a component of the mitotic ubiquitin ligase APC/C, in cell lines and primary human cells.     

发夹RNA(shRNA)在哺乳动物RNAi研究中的应用

在哺乳动物的RNAi研究中,载体表达的shRNA分子比细胞同时表达的siRNA分子的正义链与反义链对靶基因的抑制效率要高。shRNA可由PolⅢ的启动子在体内表达产生,酶切cDNA和shRNA芯片是产生shRNA的最新方法。对shRNA的设计应注意靶基因序列、环序列以及载体酶切位点的选择。诱导表达shRNA的载体系统的表达效率有所差异,质粒载体转染效率尚不稳定,且持续时间短,通过病毒载体介导是目前进行基因敲除最有效的工具。     

In vivo knockdown of antisense non‐coding mitochondrial RNAs by a lentiviral‐encoded shRNA inhibits melanoma tumor growth and lung colonization

The family of non‐coding mitochondrial RNAs (ncmtRNA) is differentially expressed according to proliferative status. Normal proliferating cells express sense (SncmtRNA) and antisense ncmtRNAs (ASncmtRNAs), whereas tumor cells express SncmtRNA and downregulate ASncmtRNAs. Knockdown of ASncmtRNAs with oligonucleotides induces apoptotic cell death of tumor cells, leaving normal cells unaffected, suggesting a potential application for developing a novel cancer therapy. In this study, we knocked down the ASncmtRNAs in melanoma cell lines with a lentiviral‐encoded shRNA approach. Transduction with lentiviral constructs targeted to the ASncmtRNAs induced apoptosis in murine B16F10 and human A375 melanoma cells in vitro and significantly retarded B16F10 primary tumor growth in vivo. Moreover, the treatment drastically reduced the number of lung metastatic foci in a tail vein injection assay, compared to controls. These results provide additional proof of concept to the knockdown of ncmtRNAs for cancer therapy and validate lentiviral–shRNA vectors for gene therapy.     

Titers of lentiviral vectors encoding shRNAs and miRNAs are reduced by different mechanisms that require distinct repair strategies

RNAi-based gene therapy is a powerful approach to treat viral infections because of its high efficiency and sequence specificity. The HIV-1-based lentiviral vector system is suitable for the delivery of RNAi inducers to HIV-1 susceptible cells due to its ability to transduce nondividing cells, including hematopoietic stem cells, and its ability for stable transgene delivery into the host cell genome. However, the presence of anti-HIV short hairpin RNA (shRNA) and microRNA (miRNA) cassettes can negatively affect the lentiviral vector titers. We show that shRNAs, which target the vector genomic RNA, strongly reduced lentiviral vector titers but inhibition of the RNAi pathway via saturation could rescue vector production. The presence of miRNAs in the vector RNA genome (sense orientation) results in a minor titer reduction due to Drosha processing. A major cause for titer reduction of miRNA vectors is due to incompatibility of the cytomegalovirus promoter with the lentiviral vector system. Replacement of this promoter with an inducible promoter resulted in an almost complete restoration of the vector titer. We also showed that antisense poly(A) signal sequences can have a dramatic effect on the vector titer. These results show that not all sequences are compatible with the lentiviral vector system and that care should be taken in the design of lentiviral vectors encoding RNAi inducers.     

Intensive RNAi with lentiviral vectors in mammalian cells

RNAi is a powerful technology for analyzing gene function in human cells. However, its utility can be compromised by inadequate knockdown of the target mRNA or by interpretation of effects without rigorous controls. We review lentiviral vector-based methods that enable transient or stable knockdowns to trace mRNA levels in human CD4+ T cell lines and other targets. Critical controls are reviewed, including rescue of the pre-knockdown phenotype by re-expression of the targeted gene. The time from thinking about a potential knockdown target to analysis of phenotypes can be as short as a few weeks.     

Lentiviral vectors that carry anti-HIV shRNAs: problems and solutions

BACKGROUND: HIV-1 replication can be inhibited with RNA interference (RNAi) by expression of short hairpin RNA (shRNA) from a lentiviral vector. Because lentiviral vectors are based on HIV-1, viral sequences in the vector system are potential targets for the antiviral shRNAs. Here, we investigated all possible routes by which shRNAs can target the lentiviral vector system. METHODS: Expression cassettes for validated shRNAs with targets within HIV-1 Leader, Gag-Pol, Tat/Rev and Nef sequences were inserted in the lentiviral vector genome. Third-generation self-inactivating HIV-1-based lentiviral vectors were produced and lentiviral vector capsid production and transduction titer determined. RESULTS: RNAi against HIV-1 sequences within the vector backbone results in a reduced transduction titer while capsid production was unaffected. The notable exception is self-targeting of the shRNA encoding sequence, which does not affect transduction titer. This is due to folding of the stable shRNA hairpin structure, which masks the target for the RNAi machinery. Targeting of Gag-Pol mRNA reduces both capsid production and transduction titer, which was improved with a human codon-optimized Gag-Pol construct. When Rev mRNA was targeted, no reduction in capsid production and transduction titer was observed. CONCLUSIONS: Lentiviral vector titers can be negatively affected when shRNAs against the vector backbone and the Gag-Pol mRNA are expressed during lentiviral vector production. Titer reductions due to targeting of the Gag-Pol mRNA can be avoided with a human codon-optimized Gag-Pol packaging plasmid. The remaining targets in the vector backbone may be modified by point mutations to resist RNAi-mediated degradation during vector production.