A recessive genetic screen for components of the RNA interference pathway performed in mouse embryonic stem cells
Methods in Molecular Biology, ISSN: 1064-3745, Vol: 1622, Page: 111-129
2017
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Book Chapter Description
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.
Bibliographic Details
http://www.scopus.com/inward/record.url?partnerID=HzOxMe3b&scp=85021761473&origin=inward; http://dx.doi.org/10.1007/978-1-4939-7108-4_9; http://www.ncbi.nlm.nih.gov/pubmed/28674805; http://link.springer.com/10.1007/978-1-4939-7108-4_9; https://dx.doi.org/10.1007/978-1-4939-7108-4_9; https://link.springer.com/protocol/10.1007/978-1-4939-7108-4_9
Springer Science and Business Media LLC
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