Site-Specific Recombination in Eukaryotes and Constructs Useful Therefor

Inventors: Geoff Wahl and Stephen O'Gorman
Potential Uses: Gene Expression
A fusion of the protamine 1 gene promoter with the Cre recombinase gene for efficient manipulation of chromosomal sequences in mammalian systems

The invention relates to the expression of site-specific recombinases in combination with germ-line promoters and embryonic stem cells to markedly facilitate the production of altered chromosomal alleles in intact organisms. For example, by this method it is possible to create alleles in mice that have been homologously recombined and then recombined by a site-specific recombinase in no more time, and with the expenditure of no more resources, than it would typically take to create a simple, homologously recombined allele. In one application of this method the mouse protamine-1 gene promoter was fused to the Cre recombinase gene and inbred strain 129 transgenic mice were prepared. Males that contained the transgenes and a Cre recombination target efficiently recombined the target in their germ cells, but not in other tissues. More than 80% of the progeny of such males inherited the Cre-recombined target. Mouse embryonic stem cells (129 strain) have been derived from one protamine-Cre transgenic line. A variety of target genes have been homologously recombined in these cells using targeting vectors that contain loxP-flanked selectable markers. When targeted embryonic stem cells have been used to prepare chimeras, good germline transmission has been routinely obtained and the selectable marker has been efficiently excised in the germline of the chimeras; most progeny inherit the Cre-recombined allele.