title: Target validation in mice by constitutive and conditional RNAi.
abstract: Gene silencing by RNA interference (RNAi) has become a standard method for the characterization of gene function in mammalian cells. Short hairpin (sh) RNAs expressed from stably integrated vectors mediate gene knockdown both in cultured cells and in mice, presenting a fast alternative to gene knockout approaches. We describe three strategies to control gene silencing in mice that can be applied to any transcript of interest. This shRNA based approach enables either i) constitutive body-wide knockdown, ii) cell type-specific knockdown controlled by Cre recombinase, or iii) inducible body-wide knockdown controlled by doxycycline. For reliable expression the shRNA vector of interest is inserted into a Rosa26 docking site of ES cells by a site-specific recombinase. These ES cells can then be used to generate shRNA transgenic mice. This technology enables the production of adult knockdown mice within 11 months for an expedite in vivo validation of drug targets.
authors: Institute for Developmental Genetics, Helmholtz Zentrum München, Munich, Germany.
authors: Aljoscha
authors: A
authors: Kleinhammer
authors: Wolfgang
authors: W
authors: Wurst
authors: Ralf
authors: R
authors: Kühn
mesh_terms: D000818:Animals; D002478:Cells, Cultured; D004254:DNA Nucleotidyltransferases; D004318:Doxycycline / Q000494:pharmacology; D015195:Drug Design; D053595:Embryonic Stem Cells; D005818:Genetic Engineering / Q000379:methods*; D019426:Integrases; D051379:Mice; D008822:Mice, Transgenic; D058990:Molecular Targeted Therapy; D011401:Promoter Regions, Genetic; D011506:Proteins / Q000235:genetics; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000235:genetics*; D022661:RNA, Untranslated
chemical_list: C110853:Gt(ROSA)26Sor non-coding RNA, mouse; D011506:Proteins; D034741:RNA, Small Interfering; D022661:RNA, Untranslated; C045073:Cre recombinase; D004254:DNA Nucleotidyltransferases; D019426:Integrases; C077570:Site-specific recombinase; D004318:Doxycycline