title: Genotype-based screen for ENU-induced mutations in mouse embryonic stem cells.
abstract: 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.
authors.affiliation: Department of Genetics, Case Western Reserve University, Cleveland, Ohio, USA.
authors.forename: Y
authors.initials: Y
authors.lastname: Chen
authors.forename: D
authors.initials: D
authors.lastname: Yee
authors.forename: K
authors.initials: K
authors.lastname: Dains
authors.forename: A
authors.initials: A
authors.lastname: Chatterjee
authors.forename: J
authors.initials: J
authors.lastname: Cavalcoli
authors.forename: E
authors.initials: E
authors.lastname: Schneider
authors.forename: J
authors.initials: J
authors.lastname: Om
authors.forename: R P
authors.initials: RP
authors.lastname: Woychik
authors.forename: T
authors.initials: T
authors.lastname: Magnuson
mesh_terms: D019943:Amino Acid Substitution; D000818:Animals; D002678:Chimera / Q000235:genetics; D003062:Codon / Q000235:genetics; D004249:DNA Damage; D004260:DNA Repair; D005038:Ethylnitrosourea / Q000633:toxicity*; D005838:Genotype; D007041:Hypoxanthine Phosphoribosyltransferase / Q000235:genetics; D051379:Mice; D008810:Mice, Inbred C57BL; D016296:Mutagenesis; D009153:Mutagens / Q000633:toxicity*; D009154:Mutation; D019853:O(6)-Methylguanine-DNA Methyltransferase / Q000037:antagonists & inhibitors / Q000502:physiology; D017354:Point Mutation; D012326:RNA Splicing / Q000235:genetics; D013234:Stem Cells / Q000737:chemistry / Q000187:drug effects*
chemical_list: D003062:Codon; D009153:Mutagens; D019853:O(6)-Methylguanine-DNA Methyltransferase; D007041:Hypoxanthine Phosphoribosyltransferase; D005038:Ethylnitrosourea