An efficient method for generation of bi-allelic null mutant mouse embryonic stem cells and its application for investigating epigenetic modifiers.
Cynthia L Fisher, Hendrik Marks, Lily Ting-Yin Cho, Robert Andrews, Sam Wormald, Thomas Carroll, Vivek Iyer, Peri Tate, Barry Rosen, Hendrik G Stunnenberg, Amanda G Fisher, William C Skarnes
Nucleic Acids Res | Published : 2017
Mouse embryonic stem (ES) cells are a popular model system to study biological processes, though uncovering recessive phenotypes requires inactivating both alleles. Building upon resources from the International Knockout Mouse Consortium (IKMC), we developed a targeting vector for second allele inactivation in conditional-ready IKMC 'knockout-first' ES cell lines. We applied our technology to several epigenetic regulators, recovering bi-allelic targeted clones with a high efficiency of 60% and used Flp recombinase to restore expression in two null cell lines to demonstrate how our system confirms causality through mutant phenotype reversion. We designed our strategy to select against re-targ..View full abstract
Awarded by Medical Research Council