Stable production of mutant mice from double gene converted ES cells with puromycin and neomycin.

The antibiotic puromycin is an effective inhibitor of protein synthesis and puromycin N-acetyl transferase gene could be used as a dominant selection marker. We report the effective production of mutant mice from double gene-converted ES cells by selection with G418 and puromycin. We confirmed that (i) puromycin efficiently inhibited the growth of ES cells at a low-dose (0.1 microgram/ml) and for a short time (2 days), independent of G418 selection; (ii) when these selected ES cells were injected into eight-cell stage embryos, the cells produced chimeras with high levels of chimerism; (iii) these chimeric males were fertile and exclusively yielded ES cell-derived offspring; and (iv) each offspring contained both neomycin transferase and puromycin N-acetyl transferase genes.