Site-specific recombinase technology

Site-specific recombinase (SSR) technology allows for the manipulation of genetic material in order to explore gene function. The success of the Human Genome Project has made recombinant DNA technology an inevitable next step in molecular biology and genetics. As a mechanism of DNA recombination, site-specific recombinase (SSR) technology is transforming mouse genetics. One specific SSR system, CreloxP (i.e. locus of chromosomal crossover (x) in the bacteriophage P1), facilitates the recombination of specific sequences of DNA with high fidelity.

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Mechanism

Cre belongs to a family of enzymes called recombinases. Cre (cyclic recombinase) is able to recombine specific sequences of DNA without the need for cofactors. Cre recombinase recognizes a 34 base pair DNA sequence called loxP. Upon encountering two separate loxP sites flanking a target nucleotide sequence along a linear DNA fragment, Cre deletes this intervening sequence. Tissue-specific gene knockout is achieved by the excision of a lox-P flanked (floxed) critical region of a gene after Cre is expressed in the tissue of interest. Depending on the orientation of target sites with respect to one another, Cre will excise, exchange, integrate, or invert DNA sequences. The excision reaction is effectively irreversible, and has been most successfully carried out in the mouse. Upon the excision of a particular region of DNA by the CreloxP system, normal gene expression is considerably compromised or eliminated.

Regulating Cre expression

SSR technology involving the CreloxP system incorporates methods which allow for both the spatial and temporal control of SSR activity. A common method facilitating the spatial control of genetic alteration involves the selection of a tissue-specific promoter that drives Cre activity. Cre expression is placed under the control of a specific promoter sequence, which in turn allows for the localized expression of Cre in certain tissues. For example, Cre has been placed 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 targeted by Cre will remain 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 surrounding the central nervous system (CNS) and the peripheral nervous system (PNS). Selective Cre expression has been achieved in many other cells and tissue regions as well.

In order to control temporal activity of the Cre excision reaction, forms of Cre which take advantage of various ligand binding domains have been developed. One successful strategy for inducing temporally specific Cre activity involves fusing the enzyme with a mutated ligand-binding domain of the human estrogen receptor (ERt). Upon the introduction of the drug 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. This allows researchers to bypass embryonic lethality while still investigating the function of targeted genes.

Current challenges

In addition to the two CreloxP-mediated recombinant systems discussed above, there are even more powerful systems which induce Cre expression in a spatially as well as temporally controlled manner. These systems give researchers greater empirical accuracy than ever before, allowing scientists to investigate genetic contributions with remarkable specificity. However, there are a number of different challenges facing SSR technology. Many issues revolve around the ability to choose promoters which isolate Cre activity sufficiently in order to investigate spatially controlled genetic alterations. In the absence of a sufficiently localized promoter, Cre expression becomes too widespread, and this compromises experimental control. Also, when investigating temporally activated Cre systems it is necessary to monitor Cre activity at certain time points in order to verify that Cre was not active previously during development. In order to address this issue, scientists have come up with a number of reporter lines which facilitate the supervising of Cre expression.

Scientific implications

Nearly every human gene has a counterpart in the mouse. Because of this homology between the two species, the mouse is uniquely suited to the task of elucidating the ways in which our genetic material encodes information. SSR technology provides researchers with a powerful new way to manipulate the mouse genome in pursuit of the elucidation of human gene function. For the scientist, witnessing the effect of an altered or mutated gene on the function of an organism at the level of development and behavior helps greatly to illuminate the unique role this gene plays.

Because many genes serve an essential function, eliminating or compromising gene activity throughout the entire animal often causes either embryonic death, which prevents the analysis of genetic function altogether, or causes other genes to compensate or take over the function of the compromised or eliminated gene. This in turn prevents researchers from identifying the unique role this gene plays in disease and development. Site-specific recombinase (SSR) technology gives scientists the ability to overcome these difficulties because it allows for the introduction of controlled genetic mutations in mice. These mutations can be isolated to a particular organ or biological area, or they can be activated at a certain stage in development. Because of this control, researchers are able to bypass a number of problems which seemed absolutely insurmountable only a few years ago, and which prevented much research into gene function from progressing. In short, a new and revolutionary biology is made possible through the application of this technology.