GENOMIC TARGETING OF A BICISTRONIC DNA FRAGMENT BY CRE-MEDIATED SITE-SPECIFIC RECOMBINATION

The Cre-recombinase of bacteriophage P1 catalyses site-specific recomb ination between DNA fragments containing loxP sites. Targeting of pred efined genomic loci can be achieved by Cre-mediated linkage of a promo terless resistance marker gene to a flexed promoter pre-existing in th e genome. In order to avoid the introduction of plasmid sequences into the host genome, we have constructed a series of plasmids in which th e DNA segment to be integrated is flanked by two loxP sites. We show h ere that this flexed targeting fragment is reliably and effectively se parated from the vector backbone and integrated into genomic loxP site s by Cre-mediated site-specific recombination in mammalian cells. We a lso demonstrate that by using this approach two convergent, promoterle ss coding regions can simultaneously be linked to two independent prom oter elements at a pre-existing genomic loxP site. This methodology wi ll be particularly useful for genomic targeting experiments in transge nic animals. (C) 1997 Elsevier Science B.V.