在哺乳动物细胞中构建和转染表达小干扰RNA（siRNA）或短发夹RNA（shRNA）的PCR产物。

Construction and transfection of PCR products expressing siRNAs or shRNAs in mammalian cells.

作者信息

Castanotto Daniela, Rossi John J

机构信息

Departmetn of Biology and Molecular Biology, Beckman Research Institute of the City of Hope, Duarte, CA, USA.

出版信息

Methods Mol Biol. 2004;252:509-14. doi: 10.1385/1-59259-746-7:509.

DOI:10.1385/1-59259-746-7:509

PMID:15017076

Abstract

In mammalian cells, the RNA interference (RNAi) effect has been observed through expression of 21-23 base transcripts capable of forming duplexes, or via expression of short hairpin RNAs. Here, we describe a facile polymerase chain reaction (PCR)-based strategy for rapid synthesis and evaluation of small interfering RNAs (siRNA) expression units in mammalian cells. The siRNA expression constructs are constructed by PCR, and the PCR products are directly transfected into mammalian cells for functional testing. This method is fast and inexpensive, allowing several different siRNA gene candidates to be rapidly screened for efficacy.

摘要

在哺乳动物细胞中，通过表达能够形成双链体的21 - 23个碱基的转录本，或通过短发夹RNA的表达，已观察到RNA干扰（RNAi）效应。在此，我们描述了一种基于聚合酶链反应（PCR）的简便策略，用于在哺乳动物细胞中快速合成和评估小干扰RNA（siRNA）表达单元。siRNA表达构建体通过PCR构建，PCR产物直接转染到哺乳动物细胞中进行功能测试。该方法快速且廉价，能够快速筛选几种不同的siRNA基因候选物的有效性。