在细胞内表达siRNA引发基因沉默[选购宝典]

生物通技术专刊：RNAi技术专辑之四

在细胞内表达siRNA引发基因沉默     RNAi在哺乳动物细胞中通常用于阻断特定基因的表达从而研究基因的功能，常见的做法是将靶向特定基因的大约21碱基长短的双链siRNAs(small interfering RNAs)，或者是45—50-mer的发夹结构RNA（small hairpin RNA, shRNA）转染到细胞。shRNA在细胞内会自动被加工成为siRNA，从而引发基因沉默或者表达抑制。现在有多个报道说通过质粒表达siRNAs同样可以抑制特定基因的表达。尽管细节各有不同，但载体大都是用PolIII启动子启动编码shRNA(small hairpin RNA)的序列。选用PolIII启动子的原因在于这个启动子总是在离启动子一个固定距离的位置开始转录合成RNA，遇到4—5个连续的U即终止，非常精确。当这种带有PolIII 启动子和shRNA编码序列的质粒转染哺乳动物细胞时，这种能表达siRNA的质粒确实能够下调特定基因的表达，抑制范围包括外源基因和内源基因。采用这种方法的优点在于，通过siRNA表达质粒的选择标记，siRNA载体能够更长时间地抑制目的基因表达。当然还有一点，那就是由于质粒可以复制扩增，相比起化学合成来说，这就能够显著降低制备siRNA的成本。因为质粒的价钱大约是3000多人民币，还不到化学合成1条21nt的RNA价格的一半，但是就可以自行制备siRNA而不受数量的限制。生物通在这里为大家介绍几种不同的siRNA合成质粒，但是大家要注意的是这里介绍的siRNA质粒是不同于体外转录合成RNA的质粒（生物通在后文会继续为大家介绍），因为这里制备的是用于哺乳动物细胞实验的siRNA，而不是长链的RNA（长链的dsRNA不适宜用在哺乳动物细胞，因为会引发非特异抑制，参考RNAi——回顾）     pSilencer  siRNA 表达载体系列是用于在培养的哺乳动物细胞中表达siRNA的一系列载体。在载体上包含有RNA聚和酶III （Pol III）启动子，氨苄抗性基因和大肠杆菌复制子，只要将编码一小段对应目的基因特异序列的、其RNA能形成发夹结构（就是回文结构）的DNA序列插入载体中Pol III 启动子的下游，转入哺乳动物细胞，载体就能表达出发夹结构的RNA，这种RNA很快在细胞中加工成为siRNA。     这些表达载体已经成功的在Hela细胞中抑制了包括Cyclophilin、GAPDH、p53、c-myc在内的多个基因的表达。实验表明：能够被化学合成或者体外合成的siRNAs抑制的基因同样可以被表达相同序列的载体生成的siRNAs抑制。 pSilence  1.0-U6     载体pSilence  1.0-U6是采用小鼠U6 Pol III启动子。这个由哈佛大学开发的载体已经成功的在Hela、H1299, U-2 OS和C-33A细胞中敲除了cdk-2和laminA/C的表达。这个载体经过Apa I和EcoR I酶切线性化和纯化，你只需要合成一对55-mer的寡核苷酸，带有4个碱基的突出（当然是对应Apa I和EcoR I酶切位点咯），一起退火、快速连接就可以转化了。在购买线性化的质粒同时，厂家还提供带有GAPDH siRNA的环形质粒对照（万一线性质粒不够用还可以自己扩增再切嘛！） pSilencer 2.0-U6 and pSilencer 3.0-H1     这两个载体带有不同的RNA Pol III 启动子，pSilencer 2.0-U6带的是人的U6启动子，pSilencer 3.0-H1则是用H1 RNA启动子（RNase P的一个组成部分）。这两个载体都是用BamH I和Hind III线性化并且经过纯化的，方便定向克隆。这两个载体都是以线性化的质粒供应，同时还提供GAPDH对照和一个空白对照质粒，以及DNA退火的Buffer。 pSilencer™ siRNA Expression Vectors; Maps and siRNA Design For each target gene two complementary 55-60 mer oligonucleotides must be prepared. The oligonucleotides should encode the 19 mer hairpin sequences specific to the mRNA target, a loop sequence separating the two complementary domains, and a polythymidine tract to terminate transcription by RNA Pol III. The insert design shown above is specific for the pSilencer 2.0-U6 and 3.0-H1 Expression Vectors and contains the overhanging 5’ ends to facilitate efficient and directional cloning into these plasmids. The insert for pSilencer 1.0-U6 would contain the appropriate end sequences for cloning into the Apa I and EcoR I sites. Early indications suggest that a great deal of latitude is available in the design of the loop; here we provide our default loop sequence that we find works well. 　 　 　 　 　 pSilencer-Induced Reductions in Target Gene Expression pSilencer 2.0-U6 and 3.0-H1 vectors encoding hairpin siRNAs specific to GAPDH, cyclophilin, or a non-genomic sequence were transfected into HeLa cells. Forty-eight hours post-transfection, target RNA and protein levels were assessed. (A) 1 ug of total RNA isolated from various cell samples was assessed by Northern analysis using the NorthernMax™ procedure (Ambion). RNA probes specific to GAPDH, cyclophilin, and 28S rRNA were used to probe the Northerns. The specific activity of the 28S rRNA probe was approximately 100,000-fold lower than the mRNA-specific probes. (B) GAPDH protein levels in cells transfected with pSilencer 2.0-U6-GAPDH were analyzed by immunofluorescence using a GAPDH-specific antibody (Ambion). Green: anti-GAPDH antibody detected with fluorescein labeled secondary antibody; Blue: DAPI stained nuclei. 　 Figure 1. Plasmid Vector Expression of siRNA Cause Gene Silencing. HeLa cells were plated at 60,000 cells per well in a 24 well tissue culture plate containing glass cover slips and transfected 24 hours later using 0.8 mg of pSilencer™ 1.0-U6 GAPDH plasmid. The cells were subsequently split and replated onto glass cover slips to obtain appropriate density. A. Immunofluorescence for GAPDH protein, 96 hours post transfection. B. Fluorescent signal, standard deviation, and cell number of the transfected and non-transfected samples, quantitated using MetaMorph software (Nikon). The fluorescent signal was normalized to cell number and the percent of non-transfected levels was calculated using Microsoft Excel. C. The graph shows an 85% reduction of GAPDH protein levels in cells transfected with the pSilencer 1.0-U6 GAPDH plasmid when compared to non-transfected levels. 相关文章： RNAi——回顾 RNAi研究相关的产品介绍之一 利用试剂盒，自己制备siRNAs或者dsRNA