Site-specific recombinase technology: Difference between revisions

Revision as of 09:17, 25 September 2012

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[[Image:Classification_of_site-specific_recombinases_according_to_mechanism.png|''Figure 1: Classification of site-specific recombinases'']]

'''''Figure 1: Tyr- and Ser-SSRs from prokaryotes''' (phages; grey) '''and eukaryotes''' (yeasts; brown); a comprehensive overview (including references) can be found in'' <ref name = "turan"> Turan, S. , Bode, J., 2011. Review: Site-specific recombinases: from tag-and-target- to tag-and-exchange-based genomic modifications. ''FASEB J.'' '''25''', 4088-4107, http://dx.doi.org/10.1096/fj.11-186940 ; pmid=http://www.ncbi.nlm.nih.gov/pubmed/21891781.</ref>.

Our table extends the selection of the conventional SSR systems and groups these according to their performance. All enzymes listed in Fig. 1 recombine two target sites, which are either identical (subfamily A1) or distinct (phage-derived enzymes in A2, B1 and B2).<ref name="turan"/> Whereas for A1 these sites have individual designations ("''FRT''" in case of Flp-recombinase, "''lox''P" for Cre-recombinase), the terms "''att''P" and "''att''B" (attachment sites on the phage and bacterial part, respectively) are valid in the other cases. In case of subfamily A1 we have to deal with short (usually 34 bp-) sites consisting of two (near-)identical 13 bp arms (arrows) flanking an 8 bp spacer (the crossover region, indicated by red line doublets) <ref>{{cite journal|author= T. Schlake, M. Iber, D. Schübeler, J. Seibler, E. Snezhkov, L. Nikolaev, & Bode J.|year=2000 |title=The transgeneticist's toolbox - Novel methods for the targeted modification of eukaryotic genomes| journal=Biol. Chem.| volume=381|pages= 801–813|doi=10.1515/BC.2000.103|pmid=11076013|issue=9–10}}</ref>. Note that for Flp there is an alternative, 48 bp site available with three arms, each accommodating a Flp unit (a so called "protomer"). ''att''P- and ''att''B-sites follow similar architectural rules, but here the arms show only partial identity (indicated by the broken lines) and differ in both cases. These features account for relevant differences:

* recombination of two identical educt sites leads to product sites with the same composition, although they contain arms from both substrates; these conversions are reversible;

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In its natural host (S. cerevisiae) the Flp/''FRT'' system enables replication of a "2μ plasmid" by the inversion of a segment that is flanked by two identical, but oppositely oriented ''FRT'' sites ("flippase" activity according to Fig. 2, section B). This inversion changes the relative orientation of replication forks within the plasmid enabling „rolling circle"―amplification of the circular 2μ entity before the multimeric intermediates are resolved to release multiple monomeric products. Whereas 34 bp minimal ''FRT'' sites favor excision/resolution to a similar extent as the analogue ''lox''P sites for Cre, the natural, more extended 48 bp FRT variants enable a higher degree of integration, while overcoming certain promiscuous interactions as described for phage enzymes like Cre and PhiC31- <ref name="turan"/> An additional advantage is the fact, that simple rules can be applied to generate heterospecific ''FRT'' sites which undergo crossovers with equal partners but nor with wild type ''FRT''s. These facts have enabled, since 1994, the development and continuous refinementts of [[recombinase-mediated cassette exchange]] (RMCE-)strategies permitting the clean exchange of a target cassette for an incoming donor cassette.<ref name="turan"/>

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>{{cite journal|last=Turan|first=S.|coauthors= Galla, M., Ernst, E., Qiao, J., Voelkel, C., Schiedlmeier, B., Zehe C., Bode, J.|year=2011 |title=Review: Site-specific recombinases: from tag-and-target- to tag-and-exchange-based genomic modifications| journal=FASEB J. | volume=25|pages= 4088-4107 |doi=10.1096/fj.11-186940|pmid=21891781 http://www.ncbi.nlm.nih.gov/pubmed/22968824}}</ref>, involving the excision and inversion activities illustrated in Fig. 2, sections A/B. 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 (http://www.knockoutmouse.org/)<ref name = "bradley"> Bradley, A., Anastassiadis, K., Ayadi, A., Battey, J. F., Bell, C. et al.(2012) "The mammalian gene function resource: the international knockout mouse consortium". Mammalian Genome; http://dx.doi.org/10.1007/s00335-012-9422-2 ; PMID: 22968824 </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|last=Askew|first=G.R.|coauthors= T. Doetschman, J.B. Lingrel |year=1993 |title=Site-Directed Point Mutations in Embryonic Stem Cells: a Gene-Targeting Tag-and-Exchange Strategy| journal=Mol. Cell. Biol.| volume=13|pages= 4115-4124}}</ref> could be streamlined after the advent of RMCE, which could take over and add efficiency to the knock-in step (Fig. 3).