12月 14, 2007 at 10:52 am | RNAi
- Posted by qixinming |
Silencing of mammalian genes by tetracycline-inducible shRNA expression
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| 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. |
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