三种启动子shRNA表达框的制备及其在筛选有效SiRNA中的应用

为能快速经济地获取小干扰核糖核酸 (small interfering RNA siRNA)，设计采用特异性延伸引
物和上游、下游两条通用引物，通过重叠延伸一步聚合酶链反应(PCR)法一次性快速、简捷地制备包含U6
+1、H1或tRNAVal在内的三种人小RNA多聚酶III启动子 小发夹状RNA（shRNA）表达框.用该方法制备的增
强型绿色荧光蛋白（EGFP）特异性三种启动子 shRNA表达框在转染HepG2细胞后有效地抑制了EGFP转基因
表达，其中以tRNAVal shRNA表达框抑制效果最显著，且未检测到干扰素应答基因2'5'OAS mRNA的表达，
表明该表达框可被有效转染并启动产生特异基因的RNA干扰效应，进而用于快速筛选最佳siRNA位点及其最
适搭配启动子.

Generation of three kinds of promoter-shRNA expression cassettes and their application in screening of efficient siRNA

For the purpose of rapid screening of efficient siRNA, one-step overlapping extension polymerase chain reaction(PCR)strategy with specific extension primer, upstream (universal) primer and downstream (universal) primer, was developed to promptly and economically prepare small hairpin RNA (shRNA) expression cassettes (SECs) containing three kinds of RNA polymerase III (RNA Pol III) promoter, U6 1, H1 or tRNA~(val).Flow cytometric analysis showed that transfection of PCR-based enhanced green fluorescent protein (EGFP)-specific SECs into HepG2 cells resulted in inhibition of transient expression of EGFP, and that tRNA~(Val)-SEC demonstrated much more inhibition effect compared with U6 1-and H1-SEC in HepG2 cells. Furthermore, no interferon-induced 2'5'OAS mRNA expression was detected in any of the SEC-transfected cells. The results indicated that the SECs could be transfected into cells effectively to induce RNA interference (RNAi), and the PCR-based SEC strategy can be applied in identification of optimal siRNA and its matching promoters.