U6和H1双启动子载体用于RNAi的实验研究  

RNA Interference Mediated by U6 and H1 Dual Promoter Vector

作　　者：蹇锐[1] 程小星[1] 彭涛[1] 邓少丽[1] 蒋静[1] 

机构地区：[1]重庆市第三军医大学微生物学教研室,重庆400038

出　　处：《中国生物工程杂志》2004年第11期26-29,31,共5页China Biotechnology

基　　金：国家自然科学基金资助项目 ( 3 0 3 70 60 3)

摘　　要：为建立一条简便、有效的构建哺乳动物细胞随机RNAi文库技术路线 ,首先通过PCR将 1 9～ 2 1ntsiRNA编码序列及终止信号等元件引入U6启动子下游 ,再将该扩增产物定向克隆至H1启动子的下游。构建的双启动子载体中 ,U6和H1相对排列 ,均以其间插入的靶序列为模板 ,分别转录出其正义和反义链 ,形成功能性siRNA。以EGFP和bcl 2为靶基因的实验表明 ,该方法构建的载体可有效地干扰相应基因的表达。To facilitate the construction of randomised RNAi expression library of mammalian cells,a dual promoter vector composed of two different opposing polymerase Ⅲ promoters was produced. The 19～21nt siRNA coding sequence and terminate signal sequence were inserted between the mouse U6 and human H1 promoters by PCR. Upon transfection into mammalian cells, the sense and antisense strands of the duplex were transcribed by these two convergent promoters from the same template to form functional siRNA. Experiments performed to inhibit the expression of both the EGFP and bcl-2 genes demonstrated that this vector design could be used to mediate potent gene-specific silencing via RNAi.;

关 键 词：RNA 载体 反义 启动子 实验研究 bcl-2 靶基因 哺乳动物细胞 定向克隆 转录 

分 类 号：Q78[生物学—分子生物学] Q785