RNAi在哺乳動物細胞中通常用于阻斷特定基因的表達從而研究基因的功能,，常見的做法是將靶向特定基因的大約21堿基長短的雙鏈siRNAs(small interfering RNAs)，或者是45—50-mer的發(fā)夾結構RNA（small hairpin RNA, shRNA）轉染到細胞。shRNA在細胞內會自動被加工成為siRNA,，從而引發(fā)基因沉默或者表達抑制。現(xiàn)在有多個報道說通過質粒表達siRNAs同樣可以抑制特定基因的表達,。盡管細節(jié)各有不同,，但載體大都是用PolIII啟動子啟動編碼shRNA(small hairpin RNA)的序列。選用PolIII啟動子的原因在于這個啟動子總是在離啟動子一個固定距離的位置開始轉錄合成RNA,，遇到4—5個連續(xù)的U即終止,，非常精確。當這種帶有PolIII 啟動子和shRNA編碼序列的質粒轉染哺乳動物細胞時,，這種能表達siRNA的質粒確實能夠下調特定基因的表達,，抑制范圍包括外源基因和內源基因,。采用這種方法的優(yōu)點在于，通過siRNA表達質粒的選擇標記,，siRNA載體能夠更長時間地抑制目的基因表達,。當然還有一點，那就是由于質?？梢詮椭茢U增,，相比起化學合成來說，這就能夠顯著降低制備siRNA的成本,。因為質粒的價錢大約是3000多人民幣,，還不到化學合成1條21nt的RNA價格的一半，但是就可以自行制備siRNA而不受數(shù)量的限制,。生物通在這里為大家介紹幾種不同的siRNA合成質粒,，但是大家要注意的是這里介紹的siRNA質粒是不同于體外轉錄合成RNA的質粒（生物通在后文會繼續(xù)為大家介紹），因為這里制備的是用于哺乳動物細胞實驗的siRNA,，而不是長鏈的RNA（長鏈的dsRNA不適宜用在哺乳動物細胞,，因為會引發(fā)非特異抑制）
pSilencer siRNA 表達載體系列是用于在培養(yǎng)的哺乳動物細胞中表達siRNA的一系列載體。在載體上包含有RNA聚和酶III （Pol III）啟動子,，氨芐抗性基因和大腸桿菌復制子,，只要將編碼一小段對應目的基因特異序列的、其RNA能形成發(fā)夾結構（就是回文結構）的DNA序列插入載體中Pol III 啟動子的下游,，轉入哺乳動物細胞,，載體就能表達出發(fā)夾結構的RNA，這種RNA很快在細胞中加工成為siRNA,。
這些表達載體已經(jīng)成功的在Hela細胞中抑制了包括Cyclophilin,、GAPDH、p53,、c-myc在內的多個基因的表達,。實驗表明：能夠被化學合成或者體外合成的siRNAs抑制的基因同樣可以被表達相同序列的載體生成的siRNAs抑制。
pSilence 1.0-U6
載體pSilence 1.0-U6是采用小鼠U6 Pol III啟動子,。這個由哈佛大學開發(fā)的載體已經(jīng)成功的在Hela,、H1299, U-2 OS和C-33A細胞中敲除了cdk-2和laminA/C的表達。這個載體經(jīng)過Apa I和EcoR I酶切線性化和純化,，你只需要合成一對55-mer的寡核苷酸,，帶有4個堿基的突出（當然是對應Apa I和EcoR I酶切位點咯），一起退火,、快速連接就可以轉化了,。在購買線性化的質粒同時，廠家還提供帶有GAPDH siRNA的環(huán)形質粒對照（萬一線性質粒不夠用還可以自己擴增再切嘛?。?/p>
pSilencer 2.0-U6 and pSilencer 3.0-H1
這兩個載體帶有不同的RNA Pol III 啟動子,，pSilencer 2.0-U6帶的是人的U6啟動子,，pSilencer 3.0-H1則是用H1 RNA啟動子（RNase P的一個組成部分）,。這兩個載體都是用BamH I和Hind III線性化并且經(jīng)過純化的,，方便定向克隆。這兩個載體都是以線性化的質粒供應,，同時還提供GAPDH對照和一個空白對照質粒,，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.
