通过四环素诱导的短发夹RNA表达使哺乳动物基因沉默。

Silencing of mammalian genes by tetracycline-inducible shRNA expression.

作者信息

Kappel Sven, Matthess Yves, Kaufmann Manfred, Strebhardt Klaus

机构信息

Department of Gynaecology and Obstetrics, School of Medicine, J.W. Goethe-University, Theodor-Stern-Kai 7, D-60590 Frankfurt/Main, Germany.

出版信息

Nat Protoc. 2007;2(12):3257-69. doi: 10.1038/nprot.2007.458.

DOI:10.1038/nprot.2007.458

PMID:18079726

Abstract

Conditional gene silencing in mammalian cells, via the controlled expression of short hairpin RNAs (shRNAs), is an effective method for studying gene function, particularly if the gene is essential for cell survival or development. Here we describe a simple and rapid protocol for the generation of tetracycline (Tet)-inducible vectors that express shRNAs in a time- and dosage-dependent manner. Tet-operator (TetO) sequences responsive to occupation by the Tet-repressor (TetR) were inserted at alternative positions within the wild-type H1 promoter and cloned into a eukaryotic expression vector. Additional cloning sites downstream of the promoter enable the insertion of shRNA sequences. This Tet-inducible shRNA expression system can be used for both transient and stable RNA interference (RNAi) approaches to control gene function in a spatiotemporal fashion. The entire protocol (preparation of constructs, generation of stable cell lines and functional analysis) can be completed in 3 months.

摘要

通过控制短发夹RNA（shRNA）的表达来实现哺乳动物细胞中的条件性基因沉默，是研究基因功能的一种有效方法，特别是当该基因对细胞存活或发育至关重要时。在此，我们描述了一种简单快速的方案，用于生成以时间和剂量依赖性方式表达shRNA的四环素（Tet）诱导型载体。将对Tet阻遏物（TetR）占据有反应的Tet操纵子（TetO）序列插入野生型H1启动子内的替代位置，并克隆到真核表达载体中。启动子下游的额外克隆位点使得能够插入shRNA序列。这种Tet诱导型shRNA表达系统可用于瞬时和稳定的RNA干扰（RNAi）方法，以时空方式控制基因功能。整个方案（构建体的制备、稳定细胞系的生成和功能分析）可在3个月内完成。