Efficient biallelic mutagenesis with Cre/loxP-mediated inter-chromosomal recombination.
The isolation of mutant cells with phenotypes caused by random mutagenesis has been hampered in mammalian cells because there are two alleles per gene and the disruption of both alleles is extremely rare. We describe a method for the efficient biallelic mutagenesis in embryonic stem cells. loxP sites were introduced near the centromeric regions of a pair of chromosome 1s. A mutant neo gene was inserted at the distal part of one of the loxP sites so that biallelic mutants would be selected by high-dose G418. Expression of Cre induced the recombination between homologous chromosomes and led to an elevation in the number of biallelic mutants. This system will facilitate phenotype-driven gene function study in the mammalian system.
Koike
Hiroko
H
Department of Social and Environmental Medicine, Graduate School of Medicine, Osaka University, Suita, Osaka 565-0871, Japan.
Horie
Kyoji
K
Fukuyama
Hidehiro
H
Kondoh
Gen
G
Nagata
Shigekazu
S
Takeda
Junji
J
D000818:Animals; D002875:Chromosomes; D005839:Gentamicins / Q000378:metabolism; D006720:Homozygote; D019426:Integrases / Q000378:metabolism*; D019868:Kanamycin Kinase / Q000378:metabolism; D051379:Mice; D016296:Mutagenesis; D017473:Receptors, Opioid, kappa / Q000235:genetics / Q000378:metabolism; D011995:Recombination, Genetic; D013234:Stem Cells; D014764:Viral Proteins / Q000378:metabolism*
D005839:Gentamicins; D017473:Receptors, Opioid, kappa; D014764:Viral Proteins; C010680:antibiotic G 418; D019868:Kanamycin Kinase; C045073:Cre recombinase; D019426:Integrases