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

Gusztav Belteki, Marina Gertsenstein, David W. Ow, Andras Nagy

Research output: Contribution to journalArticleResearchpeer-review

161 Citations (Scopus)

Abstract

Currently 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 exchange7-10. 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 identified8-12. 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 mammalian4,15 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.

Original languageEnglish
Pages (from-to)321-324
Number of pages4
JournalNature Biotechnology
Volume21
Issue number3
DOIs
Publication statusPublished - 1 Mar 2003
Externally publishedYes

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