Gene silencing in mammalian cells by PCR-based short hairpin RNA
Gene silencing in mammalian cells by PCR-based short hairpin RNA
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DOI：
10.1016/s0014-5793(03)00630-6
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发表时间：
2003-07-31
期刊：
影响因子：
3.5
通讯作者：
Liu, L
中科院分区：
文献类型：
--
作者：
Gou, DM;Jin, NL;Liu, L
RNA干扰（RNAi）是一种在多种系统中以序列特异性方式沉默基因的有力工具。然而，并非所有序列都能有效抑制RNA干扰介导的基因沉默。在这项研究中，我们开发了一种基于聚合酶链反应（PCR）的RNAi策略，用于快速筛选小干扰RNA（siRNA）的效率。该方法利用两步PCR产生含有U6启动子或巨细胞病毒启动子和短发夹DNA的嵌合DNA模板。我们证明了将PCR产物转染到哺乳动物细胞中导致外源性（荧光素酶、绿色荧光蛋白和β-半乳糖苷酶）和内源性（膜联蛋白II）基因表达的特异性抑制。这种PCR策略提供了一种快速、简便和廉价的方法来检测候选siRNA序列，是亚克隆的一种有吸引力的替代方法。(C)2003年由Elsevier Science B. V.代表欧洲生物化学学会联合会出版。
RNA interference (RNAi) provides a powerful tool to silence genes in a sequence-specific manner in a variety of systems. However, not all sequences are effective in the RNAi-mediated gene silencing. In this study, we developed a polymerase chain reaction (PCR)-based RNAi strategy for a quick screening of small interfering RNA (siRNA) efficiency. This method utilized a two-step PCR to generate a chimeric DNA template containing the U6 promoter or cytomegalovirus promoter and short hairpin DNA. We demonstrated that the transfection of the PCR products into mammalian cells resulted in specific depressions of exogenous (luciferase, green fluorescent protein and beta-galactosidase) and endogenous (annexin II) gene expressions. This PCR strategy provides a rapid, easy and cheap approach for testing candidates siRNA sequences and is an attractive alternative to subcloning. (C) 2003 Published by Elsevier Science B.V. on behalf of the Federation of European Biochemical Societies.