Site-specific cassette exchange and germline transmission with mouse ES cells expressing phiC31 integrase.
Currently two site-specific recombinases are available for engineering the mouse genome: Cre from P1 phage and Flp from yeast. 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. Furthermore, strategies have been designed to achieve site-specific insertion or cassette exchange. 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. None of the systems, however, allowed efficient selection-free identification of insertion or cassette exchange. Recently, an integrase from Streptomyces phage phiC31 has been shown to function in Schizosaccharomyces pombe and mammalian 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 integrase. Therefore, the phiC31 integrase is ideal to facilitate site-specific insertions into the mammalian genome.
Belteki
Gusztav
G
Samuel Lunenfeld Research Institute, Mount Sinai Hospital, 600 University Avenue, Toronto, Ontario M5G 1X5, Canada.
Gertsenstein
Marina
M
Ow
David W
DW
Nagy
Andras
A
D000818:Animals; D002460:Cell Line; D029721:Drosophila Proteins / Q000235:genetics / Q000378:metabolism; D018095:Germ-Line Mutation / Q000235:genetics; D019426:Integrases / Q000235:genetics* / Q000378:metabolism*; D051379:Mice; D016254:Mutagenesis, Insertional / Q000379:methods*; D016297:Mutagenesis, Site-Directed; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D013234:Stem Cells / Q000378:metabolism*
D029721:Drosophila Proteins; D011994:Recombinant Proteins; C100924:att-ORFB protein, Drosophila; D019426:Integrases