A recessive genetic screen for components of the RNA interference pathway in mouse embryonic stem cells

Several key components of the RNA interference (RNAi) pathway were identified in genetic screens performed in nonmammalian model organisms. To identify components of the mammalian RNAi pathway, we developed a recessive genetic screen in mouse embryonic stem (ES) cells. Recessive genetic screens are feasible in ES cells that are Bloom-syndrome protein (Blm-) deficient. Therefore, we constructed a reporter cell line in Blm-deficient ES cells to isolate RNAi mutants through a simple drug-selection scheme. This chapter describes how we used retroviral gene traps to mutagenize the reporter cell line and select for RNAi mutants. Putative RNAi mutants were confirmed using a separate functional assay. The location of the gene trap was then identified using molecular techniques such as Splinkerette PCR. Our screening strategy successfully isolated several mutant clones of Argonaute2, a vital component of the RNAi pathway.