Abstract
Gene silencing by RNA interference (RNAi) has become a routine method for extracting function from the mammalian genome. Short hairpin (sh) RNAs expressed from stably integrated vectors mediate RNAi both in cultured cells and mice and present therefore a fast alternative to conventional knockout approaches. We describe three strategies to control gene silencing in mice by shRNA expression that can be applied to any transcript of interest. The strategies include germline and inducible cell type-specific knockdowns, which depending on the molecular switch applied can be either permanent (Cre/loxP) or reversible (tetO/tTA). For reliable expression the shRNAs of interest are knocked into a pre-engineered Rosa26 docking site by recombinase mediated cassette exchange (RMCE). ES cells expressing the shRNA of interest can then be used to generate shRNA transgenic mice. The high efficiency of RMCE in ES cells enables the fast production of knockdown mice for in vivo functional analysis.
Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
