Nature Biotechnology
21, 321 - 324 (2003)
Published online: 3 February 2003; | doi:10.1038/nbt787
Site-specific cassette exchange and germline transmission with mouse ES cells expressing  C31 integraseGusztav Belteki1, 2, Marina Gertsenstein1, David W. Ow3
& Andras Nagy1, 41
Samuel Lunenfeld Research Institute, Mount Sinai Hospital, 600 University Avenue, Toronto, Ontario M5G 1X5, Canada. 2
Semmelweis University, Budapest, Hungary. 3
Plant Gene Expression Center, US Department of Agriculture−Agricultural Research Service, Albany, CA 94710, and Plant and Microbial Biology, University of California, Berkeley, CA 94720, USA. 4
Department of Molecular and Medical Genetics, University of Toronto, Toronto, Ontario M5S 1A8, Canada.
Correspondence should be addressed to Andras Nagy nagy@mshri.on.caCurrently two site-specific recombinases are available for engineering the mouse genome: Cre from P1 phage1,
2 and Flp from yeast3,
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 sites5,
6. Furthermore, strategies have been designed to achieve site-specific insertion or cassette exchange7,
8,
9,
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 identified8,
9,
10,
11,
12. None of the systems, however, allowed efficient selection-free identification of insertion or cassette exchange. Recently, an integrase from Streptomyces phage  C31 has been shown to function in Schizosaccharomyces pombe
13 and mammalian14,
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 C31 integrase is ideal to facilitate site-specific insertions into the mammalian genome.
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C31 integrase
