Selection-marker-free modification of the murine ss-casein gene using a lox2722 site

Gene targeting and site-specific recombination strategies allow the precise modification of the eukaryotic genome. Many of the recombination strategie s currently used, however, will introduce a selection marker gene at the mo dified site. DNA sequences of prokaryotic origin like vector sequences, sel ection marker, and reporter genes have been shown to markedly influence the regulation of the modified genomic loci. In order to avoid the insertion o f excess sequences, a biphasic recombination strategy involving homologous recombination and Cre-recombinase-mediated cassette exchange (RMCE) was dev ised and used to insert a foreign gene into the beta -casein gene in murine embryonic stem cells. The incompatibility of the heterospecific Lox sites used for the recombinase-mediated cassette exchange was found to be critica l for the success of the strategy. The frequently used mutant site lox511, which differs from the natural loxP site by a single point mutation, proved unsuitable for this approach. A mutant lox site carrying two point mutatio ns, however, was highly effective and 90% of the selected cell clones carri ed the desired modification. This biphasic recombination strategy allows fo r the efficient and precise modification of gene loci without the concomita nt introduction of a selectable marker gene. (C) 2001 Academic Press.