Protocol abstract
Nature Protocols 2, - 3257 - 3269 (2007)
Published online: 13 December 2007 | doi:10.1038/nprot.2007.458
Subject Categories: Cell and tissue culture | Genetic analysis | Genetic modification
Silencing of mammalian genes by tetracycline-inducible shRNA expression
Sven Kappel1, Yves Matthess1, Manfred Kaufmann1 & Klaus Strebhardt1
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.
- Department of Gynaecology and Obstetrics, School of Medicine, J.W. Goethe-University, Theodor-Stern-Kai 7, D-60590 Frankfurt/Main, Germany.
Correspondence to: Klaus Strebhardt1 e-mail: Strebhardt@em.uni-frankfurt.de
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- 36.5% Formaldehyde solution(Sigma)
- [
-32P]dCTP(GE Healthcare) - Agarose(Roth)
- Amersham hyperfilm MP(GE Healthcare)
- Blasticidin S HCl(Invitrogen)
- BSA(Sigma)
