title: A recessive genetic screen for components of the RNA interference pathway in mouse embryonic stem cells.
abstract: 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.
authors.affiliation: Department of Biochemistry, Northwestern University, Evanston, IL, USA.
authors.forename: Melanie I
authors.initials: MI
authors.lastname: Trombly
authors.forename: Xiaozhong
authors.initials: X
authors.lastname: Wang
mesh_terms: D000818:Animals; D002460:Cell Line; D053595:Embryonic Stem Cells / Q000378:metabolism*; D051379:Mice; D008957:Models, Genetic; D016133:Polymerase Chain Reaction; D034622:RNA Interference / Q000502:physiology*; D012190:Retroviridae / Q000235:genetics; D015398:Signal Transduction / Q000235:genetics / Q000502:physiology