A system for rapid generation of coat color-tagged knockouts and defined chromosomal rearrangements in mice

Gene targeting in mouse embryonic stem (ES) cells can be used to generate s ingle gene mutations or defined multi-megabase chromosomal rearrangements w hen applied with the Cre-loxP recombination system. While single knockouts are essential for uncovering functions of cloned genes, chromosomal rearran gements are great genetic tools for mapping, mutagenesis screens and functi onal genomics. The conventional approach to generate mice with targeted alt erations of the genome requires extensive molecular cloning to build target ing vectors and DNA-based genotyping for stock maintenance. Here we describ e the design and construction of a two-library system to facilitate high th roughput gene targeting and chromosomal engineering. The unique feature of these libraries is that once a clone is isolated, it is essentially ready t o be used for insertional targeting in ES cells. The two libraries each bea r a complementary set of genetic markers tailored so that the vector can be used for Cre-loxP-based chromosome engineering as well as single knockouts . By incorporating mouse coat color markers into the vectors, we illustrate a widely applicable method for stock maintenance of ES cell-derived mice w ith single gene knockouts or more extensive chromosomal rearrangements.