Mouse mutants from chemically mutagenized embryonic stem cells

The drive to characterize functions of human genes on a global scale has st imulated interest in large-scale generation of mouse mutants. Conventional germ-cell mutagenesis with N-ethyl-N-nitrosourea (ENU) is compromised by an inability to monitor mutation efficiency, strain(1) and interlocus(2) vari ation in mutation induction, and extensive husbandry requirements. To overc ome these obstacles and develop new methods for generating mouse mutants, w e devised protocols to generate germline chimaeric mice from embryonic stem (ES) cells heavily mutagenized with ethylmethanesulphonate (EMS). Germline chimaeras were derived from cultures that underwent a mutation rate of up to 1 in 1,200 at the Hprt locus (encoding hypoxanthine guanine phosphoribos yl transferase). The spectrum of mutations induced by EMS and the frameshif t mutagen ICR191 was consistent with that observed in other mammalian cells . Chimaeras derived from ES cells treated with EMS transmitted mutations af fecting several processes, including limb development, hair growth, hearing and gametogenesis. This technology affords several advantages over traditi onal mutagenesis, including the ability to conduct shortened breeding schem es and to screen for mutant phenotypes directly in ES cells or their differ entiated derivatives.