利用PCR方法获得shRNA抑制内外源性基因的表达

Suppression of the expression of exo- or endogenous genes by shRNA with the aid of PCR

目的探讨PCR法获得的shRNA对外源性基因和内源性基因表达的抑制效果。方法选择绿色荧光蛋白(GFP)和大鼠肾脏系膜细胞高丰度表达的纤维连接蛋白(FN)分别作为外源性和内源性基因的靶基因,设计特异性引物,利用PCR扩增获得shGFPRNA和shFNRNA。前者与pEGFP质粒共转染至293T细胞,48h后进行激光共聚焦检测细胞表达GFP阳性率;后者转染至大鼠系膜细胞(RMC)中,48h后提取总RNA逆转录成cDNA,经realtimeRTPCR检测FN的mRNA表达水平,提取细胞总蛋白进行Westernblot检测FN蛋白表达水平。结果PCR获得的shGFPRNA产物能有效抑制外源性基因GFP的表达,抑制效率可达70%±3.2%;shRNA转染组内源性基因FN的表达水平明显下降,与对照组相比有显著性差异(P<0.05)。结论PCR方法可快速简单、廉价地获得shRNA,对内源性或外源性基因进行特异性抑制。

Objective To investigate the effects of shRNA produced by PCR on the suppression of the expression of exo- or endogenous genes. Methods The specific primers were designed, with which the shGFP-RNA and ShFN-RNA were produced by PCR. The shGFP-RNA was transfected into 293T cell lines together with pEGFP plasmid, then the cells were detected by laser confocal microscopy 48h later. The shFN-RNA was transfected into rat mesenterie cell lines, then the cells were collected 48h later and the total RNA was extracted, which was reversely transcripted to cDNA. Then the expression level of FN mRNA was examined with real-time PCR, and the expression level of FN protein was examined with Western blot analysis. Results The results of laser confocal microscopy indicated that the EGFP could be successfully suppressed by shGFP-RNA produced by PCR; the results of real-time PCR and Western blot analysis revealed that FN expression level of the cells transfected with shFN-RNA was down regulated, and the level was much lower than those tansfected with independent shRNA （P〈0. 05）. Conclesions The specific shRNA, which could suppress an exo- or endogenous gene, may be obtained by employing PCR quickly, simply and economically.