Site-specific recombinase technology: Difference between revisions

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Revision as of 15:20, 3 January 2015

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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 (Fig. 2, Section A) or invert DNA sequences (Fig.2, Section B). 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 (Fig.2, section "A") 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|display-authors=9 }}</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.

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.<ref name = "leone" /> This allows researchers to bypass embryonic lethality while still investigating the function of targeted genes.