Mouse splice mutant generation from ENU-treated ES cells--a gene-driven approach.
Mutant mice are important for elucidating mammalian gene functions and for modeling human disease phenotypes. In recent years, chemical mutagenesis has become an increasingly popular method to disrupt gene functions due to its high efficiency of inducing mutations throughout the genome. Mutagenesis of embryonic stem (ES) cells offers the possibility of gene-driven approaches, which, however, require efficient mutation detection procedures to screen archives of mutated samples for lesions in particular genes. We have developed an approach that focuses on the detection of splice mutations in highly pooled cDNA samples using exon-skipping PCR primers. As a proof of concept, splice mutants for the Kit gene were isolated from a library comprising approximately 40,000 ES cell clones treated with N-ethyl-N-nitrosourea followed by transmission through the mouse germ-line. The approach will be useful for the production of mouse models for human disease-related splice mutations and as a general gene disruption strategy.
Greber
Boris
B
Department of Vertebrate Genomics, Max-Planck-Institute of Molecular Genetics, Ihnestrasse 73, 14195 Berlin, Germany.
Lehrach
Hans
H
Himmelbauer
Heinz
H
D017398:Alternative Splicing / Q000235:genetics*; D000818:Animals; D004252:DNA Mutational Analysis / Q000379:methods*; D017931:DNA Primers; D018076:DNA, Complementary / Q000235:genetics; D004622:Embryo, Mammalian / Q000166:cytology*; D005038:Ethylnitrosourea; D015723:Gene Library; D051379:Mice / Q000235:genetics*; D009154:Mutation / Q000235:genetics*; D019009:Proto-Oncogene Proteins c-kit / Q000235:genetics; D013234:Stem Cells
D017931:DNA Primers; D018076:DNA, Complementary; D019009:Proto-Oncogene Proteins c-kit; D005038:Ethylnitrosourea