Samuel Lunenfeld Research Institute, Mount Sinai Hospital, 600 University Avenue, Toronto, Ontario M5G 1X5, Canada and'Department of Molecular and Medical Genetics, University of Toronto, Canada ondary removal of the selectable marker by intrachromosomal recombination" or by a second targeting event” have been described, but depend on the efficiency of homologous recombination at the locus in question. Use of the Cre re-combinase to remove the selectable marker gene removes this dependency. A targeting vector carrying the mutation of interest and the neogene flanked by loxP sites in a nearby noncoding re-gion is introduced into the host gene (Fig. 1). After identification of the correctly targeted cells, the neogene can be removed by Cre expression. One loxP site remains after excision but is un-likely to affect gene expression. This approach has been successfully applied to mutation of the Switch control element in the IgH locus" and to generation of a point mutation in the leucine zipper region of the N-myc gene (AN et. al., manuscript in preparation).

Tissue-specific knock-out and repair Many genes have multiple roles in different tissues and at different times during development that cannot be revealed by simple “knock-out'of gene function. Restriction of gene disruption to certain lineages or stages would allow a more detailed dissection of gene function. Expression of the Cre recombinase under a tissue-specific promoter has been proposed as a route to solve this problem. The idea is to insert, by homologous recombination, two loxP sites into the gene of interest, such that they flank an essential region of the gene but do not affect wild-type gene function (Fig. 2). If the Cre recombinase is expressed in a lineage-specific manner in mice homozygous for the loxP insertion, then the gene should be inactivated only in those cells. The one published example to date illustrates both the feasibility and the prob-lems of the approach”. Inactivation of the DNA polymerase B gene was