Several key components of the RNA interference (RNAi) pathway were identified in genetic screens performed in non-mammalian 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 deficient (Blm-deficient). We constructed a reporter cell line in Blm-deficient ES cells to isolate RNAi mutants using 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 Argonaute 2, a vital component of the RNAi pathway.
Keywords: Argonaute 2; Bloom-deficient mouse embryonic stem cells; RNA interference; Recessive genetic screen; Retroviral gene-trap mutagenesis; Splinkerette PCR.