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* [[Genetic recombination]]
* [[Homologous recombination]]
illustrates analogous double-reciprocal crossover principles for HR and RMCE, the major difference being the dramatically different requirements for homologous sequences, which are in the kb-range for HR but as short as ~50 bp for SSRs
Flp recombinase
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). 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 loxP 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. 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 FRTs. These facts have enabled, since 1994, the development and continuous refinements of recombinase-mediated cassette exchange (RMCE-)strategies permitting the clean exchange of a target cassette for an incoming donor cassette.
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, 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. 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 could be streamlined after the advent of RMCE, which could take over and add efficiency to the knock-in step.
PhiC31 integrase
Without much doubt, Ser integrases are the current tools of choice for integrating transgenes into a restricted number of well-understood genomic acceptor sites that mostly (but not always) mimic the phage attP site in that they attract an attB-containing donor vector. At this time the most prominent member is PhiC31-INT with proven potential in the context of human and mouse genomes.
Contrary to the above Tyr recombinases, PhiC31-INT as such acts in a unidirectional manner, firmly locking in the donor vector at a genomically anchored target. An obvious advantage of this system is that it can rely on unmodified, native attP (acceptor) and attB donor sites. Additional benefits (together with certain complications) may arise from the fact that mouse and human genomes per se contain a limited number of endogenous targets (so called "attP-pseudosites"). Available information suggests that considerable DNA sequence requirements let the integrase recognize fewer sites than retroviral or even transposase-based integration systems opening its career as a superior carrier vehicle for the transport and insertion at a number of well established genomic sites, some of which with so called "safe-harbor" properties.
Exploiting the fact of specific (attP x attB) recombination routes, RMCE becomes possible without requirements for synthetic, heterospecific att-sites. This obvious advantage, however comes at the expense of certain shortcomings, such as lack of control about the kind or directionality of the entering (donor-) cassette. Further restrictions are imposed by the fact that irreversibility does not permit standard multiplexing-RMCE setups including "serial RMCE" reactions, i.e., repeated cassette exchanges at a given genomic locus.
Outlook and perspectives
Annotation of the human and mouse genomes has led to the identification of >20 000 protein-coding genes and >3 000 noncoding RNA genes, which guide the development of the organism from fertilization through embryogenesis to adult life. Although dramatic progress is noted, the relevance of rare gene variants has remained a central topic of research.
As one of the most important platforms for dealing with vertebrate gene functions on a large scale, genome-wide genetic resources of mutant murine ES cells have been established. To this end four international programs aimed at saturation mutagenesis of the mouse genome have been founded in Europe and North America (EUCOMM, KOMP, NorCOMM, and TIGM). Coordinated by the International Knockout Mouse Consortium (IKSC) these ES-cell repositories are available for exchange between international research units. Present resources comprise mutations in 11 539 unique genes, 4 414 of these conditional.
The relevant technologies have now reached a level permitting their extension to other mammalian species and to human stem cells, most prominently those with an iPS (induced pluripotent) status.
See also
Site-specific recombination
Recombinase-mediated cassette exchange
Cre recombinase
Cre-Lox recombination
FLP-FRT recombination
Genetic recombination
Homologous recombination
== References ==
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