Сайт-специфическая рекомбинация: инструменты для редактирования генома и изучения регуляторных сетей.

Site specific recombinase technologies are genome engineering tools that depend on recombinase enzymes to replace targeted sections of DNA.

In the late 1980s gene targeting in murine embryonic stem cells (ESCs) enabled the transmission of mutations into the mouse germ line, and emerged as a novel option to study the genetic basis of regulatory networks as they exist in the genome. Still, classical gene targeting proved to be limited in several ways as gene functions became irreversibly destroyed by the marker gene that had to be introduced for selecting recombinant ESCs. These early steps led to animals in which the mutation was present in all cells of the body from the beginning leading to complex phenotypes and/or early lethality. There was a clear need for methods to restrict these mutations to specific points in development and specific cell types. This dream became reality when groups in the USA were able to introduce bacteriophage and yeast derived site specific recombination (SSR ) systems into mammalian cells as well as into the mouse.

The mode integration/resolution and inversion (INT/RES and INV) depend on the orientation of recombinase target sites (RTS), among these pairs of attP and attB. Section C indicates, in a streamlined fashion, the way recombinase mediated cassette exchange (RMCE) can be reached by synchronous double reciprocal crossovers (rather than integration, followed by resolution). Tyr Recombinases are reversible, while the Ser Integrase is unidirectional. Of note is the way reversible Flp (a Tyr recombinase) integration/resolution is modulated by 48 bp (in place of 34 bp minimal) FRT versions: the extra 13 bp arm serves as a Flp "landing path" contributing to the formation of the synaptic complex, both in the context of Flp INT and Flp RMCE functions (see the respective equilibrium situations). While it is barely possible to prevent the (entropy driven) reversion of integration in section A for Cre and hard to achieve for Flp, RMCE can be completed if the donor plasmid is provided at an excess due to the bimolecular character of both the forward and the reverse reaction. Posing both FRT sites in an inverse manner will lead to an equilibrium of both orientations for the insert (green arrow). In contrast to Flp, the Ser integrase PhiC31 (bottom representations) leads to unidirectional integration, at least in the absence of an recombinase directionality (RDF )factor. Relative to Flp RMCE, which requires two different ("heterospecific") FRT spacer mutants, the reaction partner (attB) of the first reacting attP site is hit arbitrarily, such that there is no control over the direction the donor cassette enters the target (cf. the alternative products). Also different from Flp RMCE, several distinct RMCE targets cannot be mounted in parallel, owing to the lack of heterospecific (non crossinteracting) attP/attB combinations.

Cre recombinase (Cre) is able to recombine specific sequences of DNA without the need for cofactors. The enzyme recognizes 34 base pair DNA sequences called loxP ("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. Due to the pronounced resolution activity of Cre, one of its initial applications was the excision of loxP 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 promoter to drive Cre expression. Placement of Cre under control of such a promoter results in localized, tissue specific expression. 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. 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). Selective Cre expression has been achieved in many other cell types and tissues as well. In order to control temporal activity of the excision reaction, forms of Cre which take advantage of various ligand binding domains have been developed. One successful strategy for inducing specific temporal Cre activity involves fusing the enzyme with a mutated ligand binding domain for the human estrogen receptor (ERt). Upon the introduction of tamoxifen (an estrogen receptor antagonist), the Cre ERt construct is able to penetrate the nucleus and induce targeted mutation. ERt binds tamoxifen with greater affinity than endogenous estrogens, which allows Cre ERt to remain cytoplasmic in animals untreated with tamoxifen. The temporal control of SSR activity by tamoxifen permits genetic changes to be induced later in embryogenesis and/or in adult tissues. 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.

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.

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.