RUSH and CRUSH: A rapid and conditional RNA interference method in mice

Summary RNA interference (RNAi) is a powerful approach to phenocopy mutations in many organisms. Gold standard conventional knock‐out mouse technology is labor‐ and time‐intensive; however, off‐target effects may confound transgenic RNAi approaches. Here, we describe a rapid method for conditional and reversible gene silencing in RNAi transgenic mouse models and embryonic stem (ES) cells. RUSH and CRUSH RNAi vectors were designed for reversible or conditional knockdown, respectively, demonstrated using targeted replacement in an engineered ROSA26lacZ ES cell line and wildtype V6.5 ES cells. RUSH was validated by reversible knockdown of Dnmt1 in vitro. Conditional mouse model production using CRUSH was expedited by deriving ES cell lines from Cre transgenic mouse strains (nestin, cTnnT, and Isl1) and generating all‐ES G0 transgenic founders by tetraploid complementation. A control CRUSHGFP RNAi mouse strain showed quantitative knockdown of GFP fluorescence as observed in compound CRUSHGFP, Ds‐Red Cre‐reporter transgenic mice, and confirmed by Western blotting. The capability to turn RUSH and CRUSH alleles off or on using Cre recombinase enables this method to rapidly address questions of tissue‐specificity and cell autonomy of gene function in development. genesis 52:39–48, 2014. © 2013 Wiley Periodicals, Inc.