Genotype-based screen for ENU-induced mutations in mouse embryonic stem cells.
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.
Chen
Y
Y
Department of Genetics, Case Western Reserve University, Cleveland, Ohio, USA.
Yee
D
D
Dains
K
K
Chatterjee
A
A
Cavalcoli
J
J
Schneider
E
E
Om
J
J
Woychik
R P
RP
Magnuson
T
T
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*
D003062:Codon; D009153:Mutagens; D019853:O(6)-Methylguanine-DNA Methyltransferase; D007041:Hypoxanthine Phosphoribosyltransferase; D005038:Ethylnitrosourea