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'''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.
===MECHANISM===
Cre belongs to a family of [[enzymes]] called [[recombinases]]. Cre ('''c'''auses '''re'''combination) 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
[[Image:creexcision.png]]
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.
Due to the fact that 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.
===PHILOSOPHICAL IMPLICATIONS===
However, this way of understanding the contributions made by our genetic material to our development and behavior is seemingly taken for granted by biologists. Scientists assume that there is a direct causal link between our genetic material and how we are as complex organisms; moreover, the current scientific consensus seems to be that the technology described above will be able to uncover this link. Philosophers of science have debated the extent to which complex biological systems can be explained in terms of contributions made by the genome. Many have argued that some behaviors are too complex ever to be explained in terms of the contribution of one gene (or many). There is even evidence for the fact that certain diseases (such as Huntington’s) cannot be explained adequately in terms of genetic contributions. More radically, some influential philosophers of biology argue that our genetic material alone cannot even be said to contain information independent of the complex and dynamic cellular environment upon which the expression, modification and biological ‘realization’ of our genes relies. If this were indeed the case, then it seems that scientists and philosophers alike would be obliged to reorient themselves in relation to certain foundational assumptions which have motivated the investigation of the human genome. SSR technology must be examined from a philosophical perspective because it seems to hold a unique possibility for exploring the limits of the scientific investigation into genetic function. How far can we go in attributing our genetic material as the cause or source of complex human traits, such as behavior? If indeed SSR technology holds as much promise as scientists maintain, it might be the case that the development and behavior of complex organisms can be explained more thoroughly in terms of genetic causation than current scientific and philosophical opinion holds possible.
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