A simple procedure for the efficient derivation of mouse ES cells

Embryonic stem (ES) cells were first derived from inner cell mass (ICM) explants of preimplantation stage mouse blastocysts some 30 years ago. ES cells are of primary interest as they are used to genetically modify the genome of mice via gene targeting. Although many founder ES lines have been established, there is still a need to obtain new ES lines or their derivatives, often from new mutant mouse lines, to study the function of a mutated gene in different cell types. Existing methods for isolating ES cell lines are inefficient. Here, we describe a reproducible, efficient, and economical method to derive ES cells from different mouse strains using a defined serum-free, serum replacement (KO-SR) media, with 50-85% efficiency. We have derived over 100 ES lines, which when karyotyped>70% were euploid. Two of these lines, when tested, produced germ-line chimeras. We also present procedures for the routine maintenance and karyotyping of the ES cells.