Mutagenesis of mouse embryonic stem cells with ethylmethanesulfonate.
Unraveling the function of the mammalian genome relies heavily on analyses of the laboratory mouse. Because of its powerful genetics and available technologies to manipulate the genome, plus its developmental and physiological similarities to humans, it has become a goal to generate mutations in all mouse genes and analyze the phenotypic consequences. Gene targeting in embryonic stem (ES) cells is the method of choice for making null mutations in known genes of interest. However, forward genetics approaches, in which mutations are produced randomly throughout the genome, has the advantage of producing alleles of varying severity both within known genes, in non-coding regulatory elements, or in other unannotated functional elements. Such forward genetic mutation screens in mice have typically involved treating male mice with N-ethyl-N-nitrosourea (ENU), followed by three generations of breeding to render potential recessive mutations homozygous, at which time phenotype screens can be performed. An alternative strategy for randomly mutagenizing the mouse genome is by chemical treatment of ES cells. This enables the use of multiple alternative chemicals with different mutational spectra, can reduce breeding to two generations, and impart a higher mutational load. Furthermore, ES cell mutagenesis can be used to create banks of clones that can be screened for point mutations in genes of interest, and to conduct forward genetic screens in vitro to detect potential phenotypes prior to generation of mice. In this chapter, we provide a detailed protocol for mutagenizing ES cells with the point mutagen ethylmethanesulfonate (EMS).
Munroe
Robert
R
College of Veterinary Medicine, Cornell University, Ithaca, NY, USA.
Schimenti
John
J
D000818:Animals; D053595:Embryonic Stem Cells / Q000187:drug effects*; D005020:Ethyl Methanesulfonate / Q000633:toxicity*; D051379:Mice; D016296:Mutagenesis; D009153:Mutagens / Q000633:toxicity*
D009153:Mutagens; D005020:Ethyl Methanesulfonate