Review Article Conditional RNAi in mice Aljoscha Kleinhammer a,1 , Jan Deussing c , Wolfgang Wurst a,b,c,2 , Ralf Kühn a,b,⇑ a Institute for Developmental Genetics, Helmholtz Center Munich – German Research Center for Environmental Health, Ingolstädter Landstr. 1, 85764 Neuherberg/Munich, Germany b Technical University Munich, Munich, Germany c Max-Planck-Institute of Psychiatry, Molecular Neurogenetics, Kraepelinstrasse 2-10, 80804 Munich, Germany article info Article history: Available online 10 August 2010 Keywords: RNAi Transgenic mice Rosa26 Cre/loxP Doxycycline RMCE shRNA abstract RNA interference (RNAi)-mediated gene knockdown has developed into a routine method to assess gene function in cultured mammalian cells in a fast and easy manner. For the use of RNAi in mice, short hairpin (sh) RNAs expressed stably from the genome are a fast alternative to conventional knockout approaches. We developed a strategy for complete or conditional gene knockdown in mice, where the Cre/loxP system is used to activate RNAi in a time and tissue dependent manner. Alternatively doxycycline controlled shRNA expression vectors can be used for conditional gene silencing. Single copy RNAi constructs are placed into the Rosa26 locus of ES cells by recombinase mediated cassette exchange and transmitted through the germline of chimeric mice. The shRNA transgenic offspring can be either directly used for phenotypic analysis or are further crossed to a Cre transgenic strain to activate conditional shRNA vec- tors. The site specific insertion of single copy shRNA vectors allows the expedite and reproducible pro- duction of knockdown mice and provides an easy and fast approach to assess gene function in vivo. Ó 2010 Elsevier Inc. All rights reserved. 1. Introduction Silencing of gene expression by RNA interference (RNAi) has become a widely used tool for functional genomics in mammalian cells. RNAi is a sequence specific gene silencing process that occurs at the messenger RNA (mRNA) level. In invertebrate cells, long double stranded RNAs (dsRNA), which are processed into short interfering RNAs (siRNA) by the ribonuclease Dicer, induce efficient and specific gene silencing. In this process the siRNA antisense strand serves as a template for the RNA-induced silencing complex (RISC). RISC recognises and cleaves the complementary mRNA, which is then rapidly degraded [1]. In mammalian cells, long dsR- NAs (>30 bp) elicit an interferon response resulting in the global inhibition of protein synthesis and non-specific mRNA degrada- tion. However, short synthetic dsRNAs trigger the specific knock- down of mRNAs in mammalian cells without interferon activation, if their length is below 30 bp [2]. Such synthetic siRNAs can be introduced into cultured cells and induce a transient knock- down that enables the study of mammalian gene function within a short time frame. Due to advances in the delivery and design of siRNAs, gene silencing developed into a routine method for in vitro use. After the establishment of siRNA mediated transient gene silencing, DNA based expression vectors were developed that allow the endogenous production of small dsRNAs in mammalian cells [3,4]. These vector derived transcripts are designed to contain a sense and an antisense region that is complementary to a selected mRNA segment. These transcripts can fold back into a stem-loop structure and form short hairpin RNAs (shRNAs) that are processed by Dicer in a similar way as siRNAs. Since shRNA expression vectors can be stably integrated into the genome, they allow permanent, long lasting gene silencing in cell lines and organisms. Upon the establishment of RNAi in cultured cells it was obvious to explore the utility of gene silencing also in mice. Reports on the generation of shRNA vector transgenic mice include the pronuclear injection of vectors, the infection of zygotes or ES cells with lentiv- iral vectors, random integration into ES cells, homologous recombi- nation and targeted transgenesis (knock-in) in ES cells [5–7]. The efficiency of gene silencing in transgenic mice can achieve levels of 90% knockdown or more. The phenotypes of knockdown and the corresponding knockout mice were compared in several instances and found to be identical or very similar. Vector based transgenic RNAi provides a tool to achieve effi- cient gene silencing during embryonic development as well as in organs of adult mice. As compared to the delivery of siRNA or viral vectors to somatic tissues, for which the uptake or infection rate is a critical issue, transgenic animals harbour the shRNA expression vector in all cells and provide a well defined experimental setup. 1046-2023/$ - see front matter Ó 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.ymeth.2010.08.003 ⇑ Corresponding author at: Institute for Developmental Genetics, Helmholtz Center Munich – German Research Center for Environmental Health, Ingolstädter Landstr. 1, 85764 Neuherberg/Munich, Germany. Fax: +49 (0)89 3187 3099. E-mail addresses: aljoscha.kleinhammer@helmholtz-muenchen.de (A. Klein- hammer), deussing@mpipsykl.mpg.de (J. Deussing), wurst@helmholtz-muenchen. de (W. Wurst), ralf.kuehn@helmholtz-muenchen.de (R. Kühn). 1 Fax: +49 (0)89 3187 3099. 2 Fax: +49 (0)89 3187 3099. Methods 53 (2011) 142–150 Contents lists available at ScienceDirect Methods journal homepage: www.elsevier.com/locate/ymeth