The ability to generate mutations is a prerequisite to functional genetic 
analysis. Despite a long history of using mice as a model system for genetic 
analysis, the scientific community has not generated a comprehensive collection 
of multiple alleles for most mouse genes. The chemical mutagen of choice for 
mouse has been N-ethyl-N-nitrosourea (ENU), an alkylating agent that mainly 
causes base substitutions in DNA, and therefore allows for recovery of complete 
and partial loss-, as well as gain-, of-function alleles . Specific locus tests 
designed to detect recessive mutations showed that ENU is the most efficient 
mutagen in mouse with an approximate mutation rate of 1 in 1,000 gametes. In 
fact, several genome-wide and region-specific screens based on phenotypes have 
been carried out. The anticipation of the completion of the human and mouse 
genome projects, however, now emphasizes genotype-driven genetics--from sequence 
to mutants. To take advantage of the mutagenicity of ENU and its ability to 
create allelic series of mutations, we have developed a complementary approach 
to generating mutations using mouse embryonic stem (ES) cells. We show that a 
high mutation frequency can be achieved and that modulating DNA-repair 
activities can enhance this frequency. The treated cells retain germline 
competency, thereby rendering this approach applicable for efficient generation 
of an allelic series of mutations pivotal to a fine-tuned dissection of 
biological pathways.