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

Melanie I. Trombly*, Xiaozhong Wang

*Corresponding author for this work

Research output: Chapter in Book/Report/Conference proceedingChapter

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.

Original languageEnglish (US)
Title of host publicationRNAi and MicroRNA-Mediated Gene Regulation in Stem Cells
Subtitle of host publicationMethods, Protocols, and Applications
EditorsBaohong Zhang, Edmund Stellwag
Pages45-63
Number of pages19
DOIs
StatePublished - Dec 1 2010

Publication series

NameMethods in Molecular Biology
Volume650
ISSN (Print)1064-3745

Keywords

  • Bloom-deficient mouse embryonic stem cells
  • RNA interference
  • recessive genetic screen
  • retroviral gene-trap mutagenesis
  • splinkerette PCR

ASJC Scopus subject areas

  • Molecular Biology
  • Genetics
  • Medicine(all)

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  • Cite this

    Trombly, M. I., & Wang, X. (2010). A recessive genetic screen for components of the RNA interference pathway in mouse embryonic stem cells. In B. Zhang, & E. Stellwag (Eds.), RNAi and MicroRNA-Mediated Gene Regulation in Stem Cells: Methods, Protocols, and Applications (pp. 45-63). (Methods in Molecular Biology; Vol. 650). https://doi.org/10.1007/978-1-60761-769-3_4