Chapter title |
A Recessive Genetic Screen for Components of the RNA Interference Pathway Performed in Mouse Embryonic Stem Cells
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Chapter number | 9 |
Book title |
RNAi and Small Regulatory RNAs in Stem Cells
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Published in |
Methods in molecular biology, July 2017
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DOI | 10.1007/978-1-4939-7108-4_9 |
Pubmed ID | |
Book ISBNs |
978-1-4939-7106-0, 978-1-4939-7108-4
|
Authors |
Melanie I. Trombly, Xiaozhong Wang |
Editors |
Baohong Zhang |
Abstract |
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. |
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