New methods for gene targeting in mouse ESC's.

27 November 2017

Hendrik Marks and Henk Stunnenberg, theme Cancer development and immune defense, together with researchers at the Sanger Institute (Hinxton, UK), recently published in Nucleic Acid Research (NAR) a new methods for gene targeting in mouse ESCs.

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. For confirmation, we exploited the flexibility of our system, enabling tamoxifen inducible conditional gene ablation while controlling for genetic background and tamoxifen effects. Our strategy to generate null mutant mouse ES cells is applicable to thousands of genes and repurposes existing IKMC Intermediate Vectors.

Publication link.

 
Hendrik Marks                                                 Henk Stunnenberg