Genotype-based screen for ENU-induced mutations in mouse embryonic stem cells

The ability to generate mutations is a prerequisite to functional genetic a nalysis. Despite a long history of using mice as a model system for genetic analysis, the scientific community has not generated a comprehensive colle ction of multiple alleles for most mouse genes. The chemical mutagen of cho ice for mouse has been N-ethyl-N-nitrosourea (ENU). an alkylating agent tha t mainly causes base substitutions in DNA, and therefore allows for recover y of complete and partial loss-, as well as gain-, of-function alleles', Sp ecific locus tests designed to detect recessive mutations showed that ENU i s the most efficient mutagen in mouse with an approximate mutation rate of 1 in 1,000 gametes(2,3). In fact, several genome-wide(4-7) and region-speci fic(8-10) screens based on phenotypes have been carried out. The anticipati on of the completion of the human and mouse genome projects, however, now e mphasizes genotype-driven genetics-from sequence to mutants. To take advant age of the mutagenicity of ENU and its ability to create allelic series of mutations, we have developed a complementary approach to generating mutatio ns using mouse embryonic stem (ES) cells. We show that a high mutation freq uency can be achieved and that modulating DNA-repair activities can enhance this frequency. The treated cells retain germline competency, thereby rend ering this approach applicable for efficient generation of an allelic serie s of mutations pivotal to a fine-tuned dissection of biological pathways.