High-throughput RNAi screening in mammalian cells with esiRNAs

The development of advanced functional genomic tools has paved the way for systematic investigations of biological processes in health and disease. In particular, the implementation of RNA interference (RNAi) as a genome-wide, loss-of-function screening tool has enabled scientists to probe the role for every gene in cellular assays and many new factors for various processes have been discovered employing RNAi screens in recent years. However, the results also demonstrate the complexity of biological systems and indicate that we are still a long way from understanding functional networks in depth. Nevertheless, RNAi screens present a powerful method to interrogate gene function in high-throughput and different methods to elicit RNAi in mammalian cells have been developed. Here, we describe steps that should be considered when planning an RNAi screen employing endoribonuclease prepared (e)siRNAs. We provide useful information on how to implement the screen and analyze the results. Furthermore, we discuss strategies for hit validation and present an outline on how to follow-up on verified hits to gain a molecular understanding of the underlying phenotypes.     

RNA干扰文库及其在功能基因组学研究中的应用

随着人类基因组大规模测序的完成,下一步的挑战是了解每一个基因的功能 . RNA 干扰文库为大规模基因功能筛选提供了可能 . 虽然用于线虫等模式生物的 RNAi 文库,已经证明是大规模基因功能筛选的有效方法,但这些文库不能用于高等动物的细胞 . 自 2003 年以来,用于人的细胞和哺乳动物细胞的 RNAi 文库取得了突破,相继出现构建已知基因 RNAi 文库和构建随机 RNAi 文库的报道,并成功地应用于大规模基因功能的筛选 . RNAi 文库作为一种简单、高效、大规模、高通量的功能基因组学研究的工具,将在基因功能研究、发现新的药物靶基因、发现疾病相关基因等方面有广阔的应用前景 .     

A novel class of temperature-sensitive mutants generated by RNAi-mediated knockdown

Temperature-sensitive (ts) mutants are powerful tools with which to investigate gene function, but it has been difficult to generate ts mutants in mammalian cells. Recently, RNA interference (RNAi) has been widely used for loss of function analyses. In addition, in various organisms, hypothermic-temperature-sensitive RNAi has been reported. By using this characteristic of RNAi, we attempted to generate ts mutants in mammalian cells and were able to successfully generate ts mutants of cell cycle regulator cdc2 and ubiquitin-activating enzyme E1. We compared ts mutants previously isolated by mutagenesis with those generated by RNAi knockdown, and observed similar phenotypes. This method enabled us to generate ts mutants (KDts, knockdown temperature-sensitive mutants) of the genes of interest and will be utilized to facilitate understanding of the biological processes regulated by an essential gene in mammalian cells.     

RNAi knock-down mice: an emerging technology for post-genomic functional genetics

RNA interference (RNAi) has been extensively used for sequence-specific silencing of gene function in mammalian cells. The latest major breakthrough in the application of RNAi technology came from experiments demonstrating RNAi-mediated gene repression in mice and rats. After more than two decades of functional mouse research aimed at developing and continuously improving transgenic and knock-out technology, the advent of RNAi knock-down mice represents a valuable new alternative for studying gene function in vivo. In this review we provide some basic insight as to how RNAi can induce gene silencing to then focus on recent findings concerning the applicability of RNAi for regulating gene function in the mouse. Reviewed topics will include delivery methods for RNAi-mediating molecules, a comparison between traditional knock-out and innovative transgenic RNAi technology and the generation of graded RNAi knock-down phenotypes. Apart from the exciting possibilities RNAi provides for studying gene function in mice, we discuss several caveats and limitations to be considered. Finally, we present prospective strategies as to how RNAi technology might be applied for generating conditional and tissue-restricted knock-down mice.     

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.     

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。     

Towards <Emphasis Type="Italic">in vivo</Emphasis>application of RNA interference – new toys,old problems

RNA interference (RNAi) is the sequence-specific degradation of mRNA by short double-stranded RNA molecules. The technology, introduced only 5 years ago, has stimulated many fantasies regarding the future of functional gene analysis and gene therapy. Given its ease of application, its high efficiency and remarkable specificity, RNAi holds great promise for broad in vitro and in vivo application in all areas of biomedicine. Despite its potential, the major obstacle to the use of RNAi (as for all previous gene silencing approaches) is the need for efficient and sustained delivery of small interfering RNA into primary mammalian cells, and specific targeting of particular cell types in vivo.     

High-throughput RNAi screening to dissect cellular pathways: A how-to guide

RNA interference (RNAi) has become a powerful tool to dissect cellular pathways and characterize gene functions. The availability of genome-wide RNAi libraries for various model organisms and mammalian cells has enabled high-throughput RNAi screenings. These RNAi screens successfully identified key components that had previously been missed in classical forward genetic screening approaches and allowed the assessment of combined loss-of-function phenotypes. Crucially, the quality of RNAi screening results depends on quantitative assays and the choice of the right biological context. In this review, we provide an overview on the design and application of high-throughput RNAi screens as well as data analysis and candidate validation strategies.     

Genome-wide screening for gene function using RNAi in mammalian cells

Mammalian genome sequencing has identified numerous genes requiring functional annotation. The discovery that dsRNA can direct gene-specific silencing in both model organisms and mammalian cells through RNA interference (RNAi) has provided a platform for dissecting the function of independent genes. The generation of large-scale RNAi libraries targeting all predicted genes within mouse, rat and human cells, combined with the large number of cell-based assays, provides a unique opportunity to perform high-throughput genetics in these complex cell systems. Many different formats exist for the generation of genome-wide RNAi libraries for use in mammalian cells. Furthermore, the use of these libraries in either genetic screens or genetic selections allows for the identification of known and novel genes involved in complex cellular phenotypes and biological processes, some of which underpin human disease. In this review, we examine genome-wide RNAi libraries used in model organisms and mammalian cells and provide examples of how these information rich reagents can be used for determining gene function, discovering novel therapeutic targets and dissecting signalling pathways, cellular processes and complex phenotypes.     

RNA 干扰技术抑制病毒复制的研究进展

RNA干扰是指dsRNA抑制细胞内同源基因表达的现象,RNA干扰现象自发现以来在短短的几年里,已成功地用于不同种属的生物研究。dsRNA不仅参与内源基因表达调控,而且能够抑制宿主内病原微生物基因的表达,参与构筑生物体的防御机制。近年来,运用RNAi技术在哺乳动物中的研究不断深入,尤其是抑制病毒复制的研究成果令人欣喜,这为人类动物抗病毒治疗提供了新的思路。     

Antisense-RNA regulation and RNA interference

For a long time, RNA has been merely regarded as a molecule that can either function as a messenger (mRNA) or as part of the translational machinery (tRNA, rRNA). Meanwhile, it became clear that RNAs are versatile molecules that do not only play key roles in many important biological processes like splicing, editing, protein export and others, but can also--like enzymes--act catalytically. Two important aspects of RNA function--antisense-RNA control and RNA interference (RNAi)--are emphasized in this review. Antisense-RNA control functions in all three kingdoms of life--although the majority of examples are known from bacteria. In contrast, RNAi, gene silencing triggered by double-stranded RNA, the oldest and most ubiquitous antiviral system, is exclusively found in eukaryotes. Our current knowledge about occurrence, biological roles and mechanisms of action of antisense RNAs as well as the recent findings about involved genes/enzymes and the putative mechanism of RNAi are summarized. An interesting intersection between both regulatory mechanisms is briefly discussed.     

RNA干扰(RNAi)文库研究进展

RNAi是由双链RNA(dsRNA)引发的转录后基因沉默现象,由dsRNA产生的小分子siRNA会导致生物体内同源转录产物特异性降解,是基因表达调控的重要方式之一。目前RNAi技术已发展成为遗传分析强有力的工具,在基因功能分析鉴定方面发挥越来越大的作用。构建大规模的RNAi文库进而转变成RNAi突变体库是功能基因组学研究的重要手段,因此如何利用简单经济的方法构建特定物种的高效RNAi文库就成为关键问题。综述了目前构建RNAi文库的不同方法以及每种构建方法的优点和存在的不足,为不同研究目的的RNAi文库的构建提供参考。     

Adipocyte differentiation induced using nonspecific siRNA controls in cultured human mesenchymal stem cells

RNA interference (RNAi) is gene silencing induced by double-stranded RNA of 21-23 nucleotides in length, termed small interfering RNA, or siRNA. RNAi-based techniques have been widely applied to elucidate gene function, identify drug targets, and used in trials as a promising adjunct to silence disease-causing genes. However, emerging evidence suggests unexpected changes in expression of untargeted genes as a consequence of an off-target effect by RNAi in mammalian cells. To date, our understanding of such effects on stem cells is limited. We transfected human fetal femur-derived mesenchymal stem cells using commercially available nonspecific siRNA controls and examined adipocyte differentiation in the cells using morphology, histochemistry, and quantitative real-time PCR to examine the expression of key genes for adipogenic or osteogenic differentiation. We report here the induction of adipocyte differentiation in human mesenchymal stem cells using nonspecific siRNAs raising concerns as to the specificity of RNAi in stem cells and, critically, a need to understand and delineate the rules governing the specificity of RNAi.     

Lentivirus-delivered stable gene silencing by RNAi in primary cells

Genome-wide genetic approaches have proven useful for examining pathways of biological significance in model organisms such as Saccharomyces cerevisiae, Drosophila melanogastor, and Caenorhabditis elegans, but similar techniques have proven difficult to apply to mammalian systems. Although manipulation of the murine genome has led to identification of genes and their function, this approach is laborious, expensive, and often leads to lethal phenotypes. RNA interference (RNAi) is an evolutionarily conserved process of gene silencing that has become a powerful tool for investigating gene function by reverse genetics. Here we describe the delivery of cassettes expressing hairpin RNA targeting green fluorescent protein (GFP) using Moloney leukemia virus-based and lentivirus-based retroviral vectors. Both transformed cell lines and primary dendritic cells, normally refractory to transfection-based gene transfer, demonstrated stable silencing of targeted genes, including the tumor suppressor gene TP53 in normal human fibroblasts. This report demonstrates that both Moloney leukemia virus and lentivirus vector-mediated expression of RNAi can achieve effective, stable gene silencing in diverse biological systems and will assist in elucidating gene functions in numerous cell types including primary cells.     

Therapeutic potential of RNAi in metabolic diseases

Over the past years RNA interference (RNAi) has exploded as a new approach to manipulate gene expression in mammalian systems. More recently, RNAi has acquired interest as a potential therapeutic strategy. This review focuses on the potential therapeutic use of RNAi for metabolic diseases, the current understanding of RNAi biology, and how RNAi has been utilized to study the role of different genes in the pathogenesis of diabetes and obesity. Also reviewed are the in vivo proof-of-principle experiments that provide the preclinical justification for the development of RNAi-based therapeutics for diabetes and the key challenges that currently limit its application in the clinical setting.     

Advances in CRISPR-Cas9 genome engineering: lessons learned from RNA interference

The discovery that the machinery of the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-Cas9 bacterial immune system can be re-purposed to easily create deletions, insertions and replacements in the mammalian genome has revolutionized the field of genome engineering and re-invigorated the field of gene therapy. Many parallels have been drawn between the newly discovered CRISPR-Cas9 system and the RNA interference (RNAi) pathway in terms of their utility for understanding and interrogating gene function in mammalian cells. Given this similarity, the CRISPR-Cas9 field stands to benefit immensely from lessons learned during the development of RNAi technology. We examine how the history of RNAi can inform today''s challenges in CRISPR-Cas9 genome engineering such as efficiency, specificity, high-throughput screening and delivery for in vivo and therapeutic applications.