miRNA cassettes in viral vectors: problems and solutions

The discovery of RNA interference (RNAi), an evolutionary conserved gene silencing mechanism that is triggered by double stranded RNA, has led to tremendous efforts to use this technology for basic research and new RNA therapeutics. RNAi can be induced via transfection of synthetic small interfering RNAs (siRNAs), which results in a transient knockdown of the targeted mRNA. For stable gene silencing, short hairpin RNA (shRNA) or microRNA (miRNA) constructs have been developed. In mammals and humans, the natural RNAi pathway is triggered via endogenously expressed miRNAs. The use of modified miRNA expression cassettes to elucidate fundamental biological questions or to develop therapeutic strategies has received much attention. Viral vectors are particularly useful for the delivery of miRNA genes to specific target cells. To date, many viral vectors have been developed, each with distinct characteristics that make one vector more suitable for a certain purpose than others. This review covers the recent progress in miRNA-based gene-silencing approaches that use viral vectors, with a focus on their unique properties, respective limitations and possible solutions. Furthermore, we discuss a related topic that involves the insertion of miRNA-target sequences in viral vector systems to restrict their cellular range of gene expression. This article is part of a Special Issue entitled: MicroRNAs in viral gene regulation.     

Enzymatic production and expression of shRNAmir30 from cDNAs

RNA interference (RNAi) is an important tool for studying gene function and genetic networks. Double-stranded RNA (dsRNA) triggers RNAi that selectively silences gene expression mainly by degrading target mRNA sequences. Short interfering RNA, short hairpin RNA (shRNA), long dsRNA, and microRNA-based shRNA (shRNAmir) are four different types of dsRNA that have been widely used to silence gene expression in cultured cells, tissues, organs, and organisms. Long dsRNAs are usually 200–500 nucleotides in length and can selectively suppress expression of target genes in Caenorhabditis elegans and Drosophila but not in mammals due to unwanted non-specific knockdown. Thus, multiple attempts have been made to synthesize, express, and deliver short dsRNAs that specifically silence target genes in mammals. We describe a method for constructing an RNAi library by converting cDNAs into shRNAmir30 sequences by sequential treatment with different enzymes and affinity purification of biotin- or digoxygenin-labeled DNA fragments. We also developed a system to generate stable cell lines that uniformly express shRNAmir30s and fluorescence reporters by Cre recombinase-dependent site-specific recombination. Thus, combined with the RNAi library, this system facilitates screening for potent RNAi sequences that strongly suppress expression of target genes.     

Development of methods for RNA interference in the sheep gastrointestinal parasite, Trichostrongylus colubriformis

The efficiency of RNA interference (RNAi) delivery to L1 through L3 stage worms of the sheep parasitic nematode Trichostrongylus colubriformis was investigated using several techniques. These were: (i) feeding of Escherichia coli expressing double stranded RNA (dsRNA); (ii) soaking of short interfering (synthetic) RNA oligonucleotides (siRNA) or in vitro transcribed dsRNA molecules; and (iii) electroporation of siRNA or in vitro transcribed dsRNA molecules. Ubiquitin and tropomyosin were used as a target gene because they are well conserved genes whose DNA sequences are available for several nematode parasite species. Ubiquitin siRNA or dsRNA delivered by soaking or electroporation inhibited development in T. colubriformis but with feeding as a delivery method, RNAi of ubiquitin was not successful. Feeding was, however, successful with tropomyosin as a target, suggesting that mode of delivery is an important parameter of RNAi. Electroporation is a particularly efficient means of inducing RNA in nematodes with either short dsRNA oligonucleotides or with long in vitro transcribed dsRNA molecules. These methods permit routine delivery of dsRNA for RNAi in T. colubriformis larval stage parasites and should be applicable to moderate to high-throughput screening.     

组织特异RNAi转基因小鼠模型的构建

RNAi是一种行之有效的基因沉默的新方法,被广泛地应用于基因功能的研究、疾病的治疗以及新型疫苗的研制等领域.本研究通过原核显微注射干扰载体的方法制备转基因小鼠.选用皮肤组织特异表达的人源角蛋白14（K14）基因启动子（2000bp）作为表达载体启动子,成功地驱动融合表达载体EGFP-shRNA进行干扰片段前体的转录,进而生成成熟的干扰片段,靶向小鼠BMP4基因使其发生沉默.所得到的转基因小鼠及其杂交后代经PCR和Southern杂交鉴定,结果表明外源基因准确无误地整合到小鼠基因组.Northern杂交结果证明,小干扰RNA在皮肤组织中有较高水平的表达,在肺和肠组织中有较低水平的表达.研究结果表明,利用PolⅡ型（K14）启动子驱动shRNA融合转录本的表达,在特定组织高表达siRNA,从而达到抑制特定组织目的基因表达的技术路线是可行的.同时为利用K14启动子进行毛囊相关基因干扰研究积累了基础数据,为制备组织特异抑制基因表达的转基因大家畜提供了一个参考方法.     

UP-TORR: Online Tool for Accurate and Up-to-Date Annotation of RNAi Reagents

RNA interference (RNAi) is a widely adopted tool for loss-of-function studies but RNAi results only have biological relevance if the reagents are appropriately mapped to genes. Several groups have designed and generated RNAi reagent libraries for studies in cells or in vivo for Drosophila and other species. At first glance, matching RNAi reagents to genes appears to be a simple problem, as each reagent is typically designed to target a single gene. In practice, however, the reagent–gene relationship is complex. Although the sequences of oligonucleotides used to generate most types of RNAi reagents are static, the reference genome and gene annotations are regularly updated. Thus, at the time a researcher chooses an RNAi reagent or analyzes RNAi data, the most current interpretation of the RNAi reagent–gene relationship, as well as related information regarding specificity (e.g., predicted off-target effects), can be different from the original interpretation. Here, we describe a set of strategies and an accompanying online tool, UP-TORR (for Updated Targets of RNAi Reagents; www.flyrnai.org/up-torr), useful for accurate and up-to-date annotation of cell-based and in vivo RNAi reagents. Importantly, UP-TORR automatically synchronizes with gene annotations daily, retrieving the most current information available, and for Drosophila, also synchronizes with the major reagent collections. Thus, UP-TORR allows users to choose the most appropriate RNAi reagents at the onset of a study, as well as to perform the most appropriate analyses of results of RNAi-based studies.     

Development of strategies for conditional RNA interference

BACKGROUND: RNA interference (RNAi) represents a powerful tool with which to undertake sequence-dependent suppression of gene expression. Synthesized double-stranded RNA (dsRNA) or dsRNA generated endogenously from plasmid or viral vectors can be used for RNAi. For the latter, polymerase III promoters which drive ubiquitous expression in all tissues have typically been adopted. Given that dsRNA molecules must contain few 5' and 3' over-hanging bases to maintain potency, employing polymerase II promoters to drive tissue-specific expression of RNAi may be problematic due to potential inclusion of nucleotides 5' and 3' of siRNA sequences. METHODS: To circumvent this, polymerase II promoters in combination with cis-acting hammerhead ribozymes and short-hairpin RNA sequences have been explored as a means to generate potent dsRNA molecules in tissues defined by the promoter in use. RESULTS: The novel constructs evaluated in this study produced functional siRNA which suppressed the enhanced green fluorescent protein (eGFP) both in vitro and in vivo (in mice). Additionally, the constructs did not appear to elicit a significant type-1 interferon response compared to traditional H1-transcribed shRNA. CONCLUSIONS: Given the potential 'off-target' effects of dsRNAs, it would be preferable in many cases to limit expression of dsRNA to the tissue of interest and moreover would significantly augment the resolution of RNAi technologies. Notably, the system under evaluation in this study could readily be adapted to achieve this objective.     

Absence of transitive and systemic pathways allows cell-specific and isoform-specific RNAi in Drosophila

RNA interference (RNAi) designates the multistep process by which double-stranded RNA induces the silencing of homologous endogenous genes. Some aspects of RNAi appear to be conserved throughout evolution, including the processing of trigger dsRNAs into small 21-23-bp siRNAs and their use to guide the degradation of complementary mRNAs. Two remarkable features of RNAi were uncovered in plants and Caenorhabditid elegans. First, RNA-dependent RNA polymerase activities allow the synthesis of siRNA complementary to sequences upstream of or downstream from the initial trigger region in the target mRNA, leading to a transitive RNAi with sequences that had not been initially targeted. Secondly, systemic RNAi may cause the targeting of gene silencing in one tissue to spread to other tissues. Using transgenes expressing dsRNA, we investigated whether transitive and systemic RNAi occur in DROSOPHILA: DsRNA-producing transgenes targeted RNAi to specific regions of alternative mRNA species of one gene without transitive effect directed to sequences downstream from or upstream of the initial trigger region. Moreover, specific expression of a dsRNA, using either cell-specific GAL4 drivers or random clonal activation of a GAL4 driver, mediated a cell-autonomous RNAi. Together, our results provide evidence that transitive and systemic aspects of RNAi are not conserved in Drosophila and demonstrate that dsRNA-producing transgenes allow powerful reverse genetic approaches to be conducted in this model organism, by knocking down gene functions at the resolution of a single-cell type and of a single isoform.     

siRNA delivery technologies for mammalian systems

Inhibition of gene expression using the RNA interference (RNAi) pathway is rapidly becoming the method of choice for studying gene function in mammalian cells. However, successful knockdown of the target gene requires efficient delivery of short interfering RNAs (siRNAs). Several technologies have been developed that enable effective delivery of siRNAs to both cells in culture and whole animals. These technologies will allow the use of RNAi to study gene function in mammalian model systems in which classical methods are often limited and costly.     

pSAT RNA interference vectors: a modular series for multiple gene down-regulation in plants

RNA interference (RNAi) is a powerful tool for functional gene analysis, which has been successfully used to down-regulate the levels of specific target genes, enabling loss-of-function studies in living cells. Hairpin (hp) RNA expression cassettes are typically constructed on binary plasmids and delivered into plant cells by Agrobacterium-mediated genetic transformation. Realizing the importance of RNAi for basic plant research, various vectors have been developed for RNAi-mediated gene silencing, allowing the silencing of single target genes in plant cells. To further expand the collection of available tools for functional genomics in plant species, we constructed a set of modular vectors suitable for hpRNA expression under various constitutive promoters. Our system allows simple cloning of the target gene sequences into two distinct multicloning sites and its modular design provides a straightforward route for replacement of the expression cassette's regulatory elements. More importantly, our system was designed to facilitate the assembly of several hpRNA expression cassettes on a single plasmid, thereby enabling the simultaneous suppression of several target genes from a single vector. We tested the functionality of our new vector system by silencing overexpressed marker genes (green fluorescent protein, DsRed2, and nptII) in transgenic plants. Various combinations of hpRNA expression cassettes were assembled in binary plasmids; all showed strong down-regulation of the reporter genes in transgenic plants. Furthermore, assembly of all three hpRNA expression cassettes, combined with a fourth cassette for the expression of a selectable marker, resulted in down-regulation of all three different marker genes in transgenic plants. This vector system provides an important addition to the plant molecular biologist's toolbox, which will significantly facilitate the use of RNAi technology for analyses of multiple gene function in plant cells.