Site-specific recombinase technology: Difference between revisions

Revision as of 14:53, 21 November 2007

Revision as of 11:17, 10 April 2008

Line 1:

{{Unreferenced|date=April 2008}}

'''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, [[Cre-Lox Recombination|CreloxP]] (i.e. locus of [[chromosomal crossover]] (x) in the [[bacteriophage]] P1), facilitates the recombination of specific sequences of DNA with high fidelity.

'''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, [[Cre-Lox Recombination|CreloxPCre-loxP]] (i.e. locus of [[chromosomal crossover]] (x) in the [[bacteriophage]] P1), facilitates the recombination of specific sequences of DNA with high fidelity.

==Mechanism==

Line 5 ⟶ 7:

[[Image:creexcision.png|thumb|right|250px]]

[[Cre recombinase|Cre]] belongs to a family of [[enzymes]] called [[recombinases]]. Cre ('''c'''yclic '''re'''combinase) is able to recombine specific sequences of DNA without the need for [[Cofactor (biochemistry)|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''loxP'' 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 CreloxPCre-''loxP'' system, normal gene expression is considerably compromised or eliminated.

==Regulating Cre expression==

SSR technology involving the CreloxPCre-''loxP'' 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]].[[Fact|date=April 2008}} 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.{{Fact|date=April 2008}}

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.

Line 15 ⟶ 17:

==Current challenges==

In addition to the two CreloxPCre-''loxP''-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==