Genetic modification of mouse embryonic stem (ES) cells is a powerful technology that enabled the generation of a plethora of mutant mouse lines to study gene function and mammalian biology. Here we describe ES cell culture and transfection techniques used to manipulate the ES cell genome to obtain targeted ES cell clones. We include the standard gene targeting approach as well as the application of the CRISPR/Cas9 system that can improve the efficiency of homologous recombination in ES cells by introducing a double-strand DNA break at the target site.
Keywords: CRISPR associated protein (Cas9); Clustered regularly interspaced short palindromic repeats (CRISPR); Electroporation; Embryonic stem (ES) cells; Gene targeting; Mouse.