RNA干扰文库在功能基因组学研究中的发展及应用

RNA干扰（RNAi）是双链RNA分子在mRNA水平上诱发的序列特异性转录后基因表达沉默,从基因组水平设计针对多个靶基因的RNAi序列,建立RNAi文库进行系统性、大规模的筛选工作是功能基因组学研究的有力工具。目前RNAi文库主要包括质粒（或病毒）文库、siRNA表达盒文库、寡核苷酸文库和随机RNAi文库,已经被成功应用于基因功能鉴别、信号转导途径解析和药物靶标筛选等研究领域。近年来,这一领域发展迅速,本文就RNAi文库的发展应用以及存在的问题与展望进行综述。     

cDNA文库及其在原虫研究中的应用

cDNA文库构建和筛选是基因克隆的重要方法之一,它是目前发现新基因和研究基因功能的基本工具.从cDNA文库中可以筛选到目的基因,并直接用于该基因的表达.由于cDNA文库在基因分离和克隆中具有重要作用,因此其应用也日益广泛.简要介绍自cDNA文库创建以来,发展起来的各类文库及其构建cDNA文库的方法.作者重点阐述了弓形虫、利什曼原虫、阴道毛滴虫、疟原虫等原虫cDNA文库的构建及其应用.     

RNA干扰与基因敲除

RNAi是指通过双链RNA介导特异性降解靶mRNA,导致转录后水平基因沉默的现象。其作用途径有RdRP依赖的RNAi的途径与非RdRP依赖的RNAi途径2种。利用RNAi的基因敲除技术在dsRNA序列选择、质粒或病毒为载体的dsRNA体内合成、发夹样siRNA的转录、dsRNA的导入方法等方面取得了很大进展,在研究人类或其他生物基因组中未知基因及蛋白质的功能等领域具有诱人的应用前景。     

RNAi在抗病毒防治领域的应用

RNAi是由双链RNA(dsRNA)所诱发的转录后水平上的基因沉默.由于对靶基因沉默作用的高度特异性和高效性,因此近年来用于肿瘤性疾病、感染性疾病、遗传性疾病等疾病的基因治疗研究,特别是在抗病毒领域的研究更是成为其应用热点之一.虽然目前RNAi已经较为广泛地应用于动物病毒及各种疾病病毒的基因治疗研究中,但其在应用过程中还有许多亟待解决的问题.本文就RNAi及其在抗病毒领域的应用研究和其存在的问题展开综述.     

RNA干扰作用(RNAi)研究进展

RNA干扰作用 (RNAi)是生物界一种古老而且进化上高度保守的现象 ,是基因转录后沉默作用 (PTGS)的重要机制之一 .RNAi主要通过dsRNA被核酸酶切割成 2 1～ 2 5nt的干扰性小RNA即siRNA ,由siRNA介导识别并靶向切割同源性靶mRNA分子而实现 .RNAi要有多种蛋白因子以及ATP参与 ,而且具有生物催化反应特征 .RNAi是新发现的一种通过dsRNA介导的特异性高效抑制基因表达途径 ,在后基因组时代的基因功能研究和药物开发中具有广阔应用前景     

小鼠胚胎干细胞中RNA干涉现象

报道了不同品系小鼠胚胎干细胞 (ES细胞 )系MESPU13、B3和R1中存在的RNA干涉 (RNAi)现象。应用脂质体法 ,将转录绿色荧光蛋白 (GFP)基因双链RNA(dsRNA)的载体 (pdsGFP)转染GFP标记的ES细胞 ,dsRNA的瞬时表达可引起ES细胞中的RNAi效应 ,即质粒转录的GFP基因的dsRNA能够显著降低ES细胞内相应的外源GFP基因的表达 ;同时 ,用电穿孔转染法将线性化的pdsGFP puro导入ES细胞中 ,筛选后 ,在 3 0 %左右的抗性克隆中GFP表达量明显降低 ,少数细胞内的干涉效率达到RT PCR检测不到的程度。在此基础上 ,构建了可转录ES细胞特异标记基因OCT 4基因片断dsRNA的载体 ,经基因打靶和抗性筛选得到了稳定整合的ES细胞克隆 ,随机扩增了 5 1个克隆 ,并对其中的 48个阳性克隆进行了PCR半定量检测 ,结果显示 :在 11个ES细胞克隆中具有显著的RNAi效应 ,干涉效率达到RT PCR检测不到的程度。这一结果表明 ,应用RNAi在不同品系ES细胞中研究哺乳动物及人的基因功能是可行的     

RNA干涉及其应用前景

RNA干涉是指由特定双链RNA(dsRNA)引起的转录后基因沉默现象。研究表明,Dicer断裂dsRNA产生的小干涉RNA可以抑制哺乳动物体细胞和胚胎中的基因的表达。RdRP在扩增RNAi中起着关键性的作用,RdRP活性复制较长的触发性dsRNA或以一种非引物的方式复制短的siRNA,即以siRNA为引物的RdRP反应使靶mRNA转变为dsRNA,同时复制触发性dsRNA。所有的产物又可作为Dicer的底物,起始RdRP级联反应。本文综述了RNAi可能的作用机制,并对RNAi在分析功能基因组、药物治疗等方面的应用前景进行了展望。     

RNA干扰:脑学习和记忆的可能机制

dsRNA介导同源靶基因沉默的RNA干扰 (RNAi)是转录后基因水平沉默的主要作用方式 ,具有普遍的生物学意义。RNAi是dsRNA介导的核酸酶作用于dsRNA(>2 6nt)同源不成熟mR NA的酶解过程 ,mRNA降解为 2 1 2 3nt的dsRNA而使基因表达沉默。RNAi所具有的特性和脑学习和记忆的特征 ,提示RNAi可能是RNA介导的脑记忆移转的潜在机制     

H1启动子siRNA载体的构建及应用

利用双链RNA(dsRNA)调控基因表达已经成为研究基因功能的有力工具。用人H1启动子构建了pBS／H1PS小干扰RNA(siRNA)表达载体,用于在哺乳动物细胞中产生特异性dsRNA转录产物。通过对293细胞中的PSMA7分子进行表达抑制,证明该siRNA载体能够有效产生针对靶基因的RNA干扰(RNAi)效应。     

Genome-wide RNAi screening in Caenorhabditis elegans

In Caenorhabditis elegans, introduction of double-stranded RNA (dsRNA) results in the specific inactivation of an endogenous gene with corresponding sequence; this technique is known as RNA interference (RNAi). It has previously been shown that RNAi can be performed by direct microinjection of dsRNA into adult hermaphrodite worms, by soaking worms in a solution of dsRNA, or by feeding worms Escherichia coli expressing target-gene dsRNA. We have developed a simple optimized protocol exploiting this third mode of dsRNA introduction, RNAi by feeding, which allows rapid and effective analysis of gene function in C. elegans. Furthermore, we have constructed a library of bacterial strains corresponding to roughly 86% of the estimated 19,000 predicted genes in C. elegans, and we have used it to perform genome-wide analyses of gene function. This library is publicly available, reusable resource allowing for rapid large-scale RNAi experiments. We have used this library to perform genome-wide analyses of gene function in C. elegans. Here, we describe the protocols used for bacterial library construction and for high-throughput screening in C. elegans using RNAi by feeding.     

Genome-wide RNAi screening in Caenorhabditis elegans

In Caenorhabditis elegans, introduction of double-stranded RNA (dsRNA) results in the specific inactivation of an endogenous gene with corresponding sequence; this technique is known as RNA interference (RNAi). It has previously been shown that RNAi can be performed by direct microinjection of dsRNA into adult hermaphrodite worms, by soaking worms in a solution of dsRNA, or by feeding worms Escherichia coli expressing target-gene dsRNA. We have developed a simple optimized protocol exploiting this third mode of dsRNA introduction, RNAi by feeding, which allows rapid and effective analysis of gene function in C. elegans. Furthermore, we have constructed a library of bacterial strains corresponding to roughly 86% of the estimated 19,000 predicted genes in C. elegans, and we have used it to perform genome-wide analyses of gene function. This library is publicly available, reusable resource allowing for rapid large-scale RNAi experiments. We have used this library to perform genome-wide analyses of gene function in C. elegans. Here, we describe the protocols used for bacterial library construction and for high-throughput screening in C. elegans using RNAi by feeding.     

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。     

Strategies for generation of an siRNA expression library directed against the human genome

RNA interference (RNAi) is a phenomenon whereby expression of an individual gene is specifically silenced by the introduction of a double-stranded RNA (dsRNA) whose sequence is homologous to that of the gene in question. The generation of a small interfering RNA (siRNA) expression library directed against the entire human genome is a project that requires solutions to many difficult technical problems. We present here some strategies for solving some of these problems, including the development of genetically stable and highly active siRNA expression vectors, a procedure for selection of favorable target sites, and an efficient and inexpensive procedure for constructing an siRNA expression library.     

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.     

Genome-wide RNAi as a route to gene function in Drosophila.

With the sequencing of the human genome and the genomes of most major model organisms completed, the systematic characterisation of gene functions remains a key challenge. During the past few years, RNA interference (RNAi) has become a powerful tool to silence the expression of genes and analyse their loss-of-function phenotype when mutant alleles are not available. Genome-wide RNAi screens against all predicted genes have been successfully used to dissect a variety of biological processes in Caenorhabditis elegans. Recently, a genome-wide library of double-stranded RNAs, that target every gene in the Drosophila genome and that is suitable for high throughput cell-based assays, was published. In this paper, recent advances will be summarised. Screening strategies and applications as a route to comprehensively characterising gene function will be discussed.     

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.