Exchangeable gene trap using the Cre/mutated lox system

The gene trap technique is a powerful approach for characterizing and mutat ing genes involved in mouse development. However, one shortcoming of gene t rapping is the relative inability to induce subtle mutations. This problem can be overcome by introducing a knock-in system into the gene trap strateg y. Here, we have constructed a new gene trap vector, pU-Hachi, employing th e Cre-mutated lox system (Araki et al., 1997), in which a pair of mutant le x, lox71 and lox66, was used to promote targeted integrative reaction by Cr e recombinase. The pU-Hachi carries splicing acceptor (SA)-lox71-internal r ibosomal entry site (IRES)- beta-geo-pA-loxP-pA-pUC. By using this vector, we can carry out random insertional mutagenesis as the first step, and then we can replace the beta-geo gene with any gene of interest through Cre-med iated integration, We have isolated 109 trap clones electroporated with pU- Hachi, and analyzed their integration patterns by Southern blotting to sele ct those carrying a single copy of the trap vector. By use of some of these clones, we have succeeded in exchanging the reporter gene at high efficien cy, ranging between 20-80%, This integration system is also quite useful fo r plasmid rescue to recover flanking genomic sequences, because a plasmid v ector sequence can be introduced even when the pUC sequence of the trap vec tor is lost through integration into the genome. Thus, this method, termed exchangeable gene trapping, has many advantages as the trapped clones can b e utilized to express genes with any type of mutation.