Site-specific recombinase technology: Difference between revisions

Revision as of 00:30, 15 February 2017

Revision as of 00:36, 15 February 2017

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===Cre recombinase===

[[Cre recombinase|Cre]] ("causes recombination") is able to recombine specific sequences of DNA without the need for cofactors. The enzyme recognizes 34 base pair DNA sequences called ''lox''P ("locus of crossover in phage P1"). Depending on the orientation of target sites with respect to one another, Cre will integrate/excise or invert DNA sequences. Upon the excision (called "resolution" in case of a circular substrate) of a particular DNA region, normal gene expression is considerably compromised or terminated.<ref>{{cite journal |doi=10.1007/s10616-006-6550-0 |title=Recommended Method for Chromosome Exploitation: RMCE-based Cassette-exchange Systems in Animal Cell Biotechnology |year=2006 |last1=Oumard |first1=André |last2=Qiao |first2=Junhua |last3=Jostock |first3=Thomas |last4=Li |first4=Jiandong |last5=Bode |first5=Juergen |journal=Cytotechnology |volume=50 |pages=93–108 |pmid=19003073 |issue=1–3 |pmc=3476001}}</ref>

Due to the pronounced resolution activity of Cre, one of its initial applications was the excision of ''lox''P-flanked ("floxed") genes leading to cell-specific gene knockout of such a floxed gene after Cre becomes expressed in the tissue of interest. Current technologies incorporate methods, which allow for both the spatial and temporal control of Cre activity. A common method facilitating the spatial control of genetic alteration involves the selection of a tissue-specific [[promotor (biology)|promoter]] to drive Cre expression. If placed under the control of such a promoter, in turn allows for the localized expression of Cre in certain tissues. As an example, Leone et al. have placed the transcription unit under the control of the regulatory sequences of the [[myelin]] proteolipid protein (PLP) gene, leading to induced removal of targeted gene sequences in [[oligodendrocytes]] and [[Schwann cells]].<ref name = "leone">{{cite journal |doi=10.1016/S1044-7431(03)00029-0 |title=Tamoxifen-inducible glia-specific Cre mice for somatic mutagenesis in oligodendrocytes and Schwann cells |year=2003 |last1=Leone |first1=Dino P |last2=Genoud |first2=S.Téphane |last3=Atanasoski |first3=Suzana |last4=Grausenburger |first4=Reinhard |last5=Berger |first5=Philipp |last6=Metzger |first6=Daniel |last7=MacKlin |first7=Wendy B |last8=Chambon |first8=Pierre |last9=Suter |first9=Ueli |journal=Molecular and Cellular Neuroscience |volume=22 |issue=4 |pages=430–40 |pmid=12727441 }}</ref> The specific DNA fragment recognized by Cre remains intact in cells, which do not express the PLP gene; this in turn facilitates empirical observation of the localized effects of genome alterations in the myelin sheath that surround nerve fibers in the [[central nervous system]] (CNS) and the [[peripheral nervous system]] (PNS).<ref name=koenning>{{cite journal |doi=10.1523/JNEUROSCI.1069-12.2012 |title=Myelin Gene Regulatory Factor is Required for Maintenance of Myelin and Mature Oligodendrocyte Identity in the Adult CNS |year=2012 |last1=Koenning |first1=M. |last2=Jackson |first2=S. |last3=Hay |first3=C. M. |last4=Faux |first4=C. |last5=Kilpatrick |first5=T. J. |last6=Willingham |first6=M. |last7=Emery |first7=B. |journal=Journal of Neuroscience |volume=32 |issue=36 |pages=12528–42 |pmid=22956843}}</ref> Selective Cre expression has been achieved in many other cell types and tissues as well.

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[[Image:Fig3 Tag & Exchange.png|thumb|520px|'''A unified "tag-&-exchange strategy.''' Tag-and-exchange strategy relying on homologous recombination (HR; tagging step) followed by RMCE (SSR; exchange step). The figure 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 Recombinaserecombinase===

In its natural host (S. cerevisiae) the [[FLP-FRT recombination|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—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- <ref name="nern" /> 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 refinementtsrefinements 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”"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"knock-out”out"-) reaction the gene was tagged with a selectable marker, typically by insertion of a hygtk ([+/-]) cassette providing G418 resistance. In the following “knock"knock-in”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"negative selection”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 Integraseintegrase===

Without much doubt, Ser integrases[[integrase]]s 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 ''att''P site in that they attract an ''att''B-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 ''att''P (acceptor) and ''att''B 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 "''att''P-pseudosites"). Available information suggests that considerable DNA sequence requirements let the integrase recognize fewer sites than retroviral or even transposase-based integration systems openig 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.<ref name="karow" />

Exploiting the fact of specific (''att''P x ''att''B) recombination routes, [[Recombinase-mediated cassette exchange|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.<ref name="turan" /> Further restrictions are imposed by the fact that irreversibility does not permit standard [[Recombinase-mediated cassette exchange|multiplexing-RMCE]] setups including "serial RMCE" reactions, i.e., repeated cassette exchanges at a given genomic ''locus''.

== Outlook and perspectives ==