Figure 5. Site-specific intermolecular reactions.

Cre can catalyze the recombination between an integrated genomic loxP site and a second site present on an incoming molecule transfected in ES cells. Selection for the desired recombination events requires reconstitution of a bipartite selectable marker, the non-functional components of which are present at the integrated loxP site (M 3′) as well as on the incoming DNA construct (M 5′). Cre-mediated intermolecular recombination reconstitutes a functional cassette (M 5′-loxP-M 3′) which provides the phenotypic selection for the recovery of stable recombinants in transient Cre expression experiments. (A) In site-specific integration reactions, the extra-chromosomal loxP site is present on a circular molecule. Cre can catalyze the integration of this entire construct and yield recombinants in which the integrated DNA is flanked by loxP sites. (B) In recombinase-mediated chromosome truncation, the incoming construct is linear and carries a terminal array of telomere repeats. In this case, the Cre-mediated intermolecular reaction catalyzes the exchange between this incoming artificial telomere and the distal end of the chromosome carrying the docking site. The outcome is the generation of a chromosomal truncation and integration of the incoming construct at the new telomeric end.