ENU-induced mutant mice for a next-generation gene-targeting system

By the N-ethyl-N-nitrosourea (ENU)-based gene-driven mutagenesis, it is now possible to obtain allelic series of mutant mouse strains, each of which carries a different base substitution in any target gene. This new reverse genetic tool has become available based on the ENU mutant mouse library. The ENU mutant mouse library consists of dual archives of frozen sperm and corresponding genomic DNA derived from Generation-1 (G1) male mice, each of which carries thousands of ENU-induced base substitutions. Firstly, ENU-induced mutations in the target gene are screened from the genomic DNA archive by using one of the high-throughput mutation discovery systems. The identified mutations are then revived as live mice by the in vitro fertilization (IVF) and embryo transfer (ET) technology. Just like the knockout (KO) mouse system, the revived mutant strains are finally subjected to the three-generation scheme to reveal the gene function(s) of the target gene. This new reverse genetics or "next-generation gene-targeting system" allows us to elucidate the biological roles of the mouse genome in terms of single base-pair effects not only for the protein-coding sequences but also for any genomic sequences.