Traditionally, manipulation of gene expression in the mouse has relied on either the overexpression of cDNAs or modification (deletion/knock-in) of endogenous genes using homologous recombination ( Capecchi, 2005). Genetically engineered mice are invaluable tools for understanding biology and disease. This system provides a cost-effective and scalable platform for the production of RNAi transgenic mice targeting any mammalian gene. In addition, through regulated gene silencing we validate APC/Wnt and p19 ARF as potential therapeutic targets in T cell acute lymphoblastic leukemia/lymphoma and lung adenocarcinoma, respectively. Further, using an shRNA targeting APC, we illustrate how this approach can identify predicted phenotypes and also unknown functions for a well-studied gene. Using this system, we generated eight tet-regulated shRNA transgenic lines targeting Firefly and Renilla luciferases, Oct4 and tumor suppressors p53, p16 INK4a, p19 ARF and APC and demonstrate potent gene silencing and GFP-tracked knockdown in a broad range of tissues in vivo. By combining optimized fluorescence-coupled miR30-based shRNAs with high efficiency ES cell targeting, we developed a fast, scalable pipeline for the production of shRNA transgenic mice. RNA interference is a powerful tool for studying gene function, however, the reproducible generation of RNAi transgenic mice remains a significant limitation.
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