Site-specific cassette exchange and germline transmission with mouse ES cells expressing φC31 integrase

Currently two site-specific recombinases are available for engineering the mouse genome: Cre from P1 phage^1,2 and Flp from yeast^3,4. Both enzymes catalyze recombination between two 34-base pair recognition sites, lox and FRT, respectively, resulting in excision, inversion, or translocation of DNA sequences depending upon the location and the orientation of the recognition sites^5,6. Furthermore, strategies have been designed to achieve site-specific insertion or cassette exchange^7-10. The problem with both recombinase systems is that when they insert a circular DNA into the genome (trans event), two cis-positioned recognition sites are created, which are immediate substrates for excision. To stabilize the trans event, functional mutant recognition sites had to be identified^8-12. None of the systems, however, allowed efficient selection-free identification of insertion or cassette exchange. Recently, an integrase from Streptomyces phage φC31 has been shown to function in Schizosaccharomyces pombe¹³ and mammalian^4,15 cells. This enzyme recombines between two heterotypic sites: attB and attP. The product sites of the recombination event (attL and attR) are not substrates for the integrase¹⁶. Therefore, the φC31 integrase is ideal to facilitate site-specific insertions into the mammalian genome.