Site-specific recombinase technology: Difference between revisions

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Based on the RMCE technology, a particular resource of pre-characterized ES-strains that lends itself to further elaboration has evolved in the framework of the EUCOMM (European Conditional Mouse Mutagenesis) program, based on the now established Cre- and/or Flp-based "FlExing" (Flp-mediated excision/inversion) setups,<ref name="turan" /> involving the excision and inversion activities. Initiated in 2005, this project focused first on saturation mutagenesis to enable complete functional annotation of the mouse genome (coordinated by the International Knockout-Mouse Consortium, IKMC) with the ultimate goal to have all protein genes mutated via gene trapping and -targeting in murine ES cells.<ref name = "bradley">{{cite journal |doi=10.1007/s00335-012-9422-2 |title=The mammalian gene function resource: The international knockout mouse consortium |year=2012 |last1=Bradley |first1=Allan |last2=Anastassiadis |first2=Konstantinos |last3=Ayadi |first3=Abdelkader |last4=Battey |first4=James F. |last5=Bell |first5=Cindy |last6=Birling |first6=Marie-Christine |last7=Bottomley |first7=Joanna |last8=Brown |first8=Steve D. |last9=Bürger |first9=Antje |last10=Bult |first10=Carol J. |last11=Bushell |first11=Wendy |last12=Collins |first12=Francis S. |last13=Desaintes |first13=Christian |last14=Doe |first14=Brendan |last15=Economides |first15=Aris |last16=Eppig |first16=Janan T. |last17=Finnell |first17=Richard H. |last18=Fletcher |first18=Colin |last19=Fray |first19=Martin |last20=Frendewey |first20=David |last21=Friedel |first21=Roland H. |last22=Grosveld |first22=Frank G. |last23=Hansen |first23=Jens |last24=Hérault |first24=Yann |last25=Hicks |first25=Geoffrey |last26=Hörlein |first26=Andreas |last27=Houghton |first27=Richard |last28=Hrabé De Angelis |first28=Martin |last29=Huylebroeck |first29=Danny |last30=Iyer |first30=Vivek |journal=Mammalian Genome |volume=23 |issue=9–10 |pages=580–6 |pmid=22968824 |pmc=3463800|display-authors=8 }}</ref> These efforts mark the top of various "tag-and-exchange" strategies, which are dedicated to tagging a distinct genomic site such that the "tag" can serve as an address to introduce novel (or alter existing) genetic information. The tagging step ''per se'' may address certain classes of integration sites by exploiting integration preferences of retroviruses or even site specific integrases like PhiC31, both of which act in an essentially unidirectional fashion.

The traditional, laborious "tag-and-exchange" procedures relied on two successive homologous recombination (HR-)steps, the first one ("HR1") to introduce a tag consisting of a selection marker gene. "HR2" was then used to replace the marker by the "GOI. In the first ("knock-out"-) reaction the gene was tagged with a selectable marker, typically by insertion of a hygtk ([+/-]) cassette providing G418 resistance. In the following "knock-in" step, the tagged genomic sequence was replaced by homologous genomic sequences with certain mutations. Cell clones could then be isolated by their resistance to ganciclovir due to loss of the HSV-tk gene, i.e. ("negative selection"). This conventional two-step tag-and-exchange procedure <ref>{{cite journal |first1=G. Roger |last1=Askew |first2=Thomas |last2=Doetschman |first3=Jerry B. |last3=Lingrel |pmid=8391633 |doi=10.1128/MCB.13.7.4115 |url=http://mcb.asm.org/cgi/pmidlookup?view=long&pmid=8391633 |year=1993 |title=Site-directed point mutations in embryonic stem cells: A gene-targeting tag-and-exchange strategy |volume=13 |issue=7 |pages=4115–24 |pmc=359961 |journal=Molecular and Cellular Biology}}</ref> could be streamlined after the advent of RMCE, which could take over and add efficiency to the knock-in step.

===PhiC31 integrase===