Cre Recombinase Activity Essay

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The major conclusion of this study is that our novel split-Cre complementation system introduces both temporal and special control of site specific recombination using Cre recombinase enzyme. This system solved many drawbacks have emerged during the extensive use of Cre recombinase in molecular biology. The complemented protein is almost as efficient as the Full CRE in the recombination activity (~95%). Moreover, each fragment lacks the recombinase activity. This system allows precise genetic manipulation. It has a special importance in neuroscience lacking selective promoter region for conditional knocking our specific gene at specific brain tissue. Many websites, such as GenePaint; a digital atlas for gene expression pattern in mouse…show more content…
They depended on the linker used by Jullien et al (19). We depended on the designing criteria of the linker for efficiently separating domains in bifunctional proteins (40). They reported to use rapamycin-dependent dimerization or ɑ-helix interactions for enhancing posttranslational association between the two fragments. Some trials used artificially designed antiparallel Leucine Zipper to assist protein fragment reconstitution (41). In our study, we used yeast GCN4 coil/coil Leucine Zipper domain which previously used by some split-Cre systems (19). This Leucine Zipper does not interfere with normal cellular physiology (30). Each protein fragment behaves as an independent protein. It has different kinetics than the other protein fragment. We tested the recombinase activity on synthetic construct and on mammalian genome. We have optimized the system to choose the best spatial and temporal control of the Cre recombinase. Meanwhile, the reconstituted CRE recombination was not present in all cells. We had to sort cells expressed both nCre and cCre fragments. There are two possibilities for this finding. First, the reconstituted CRE is too low to induce complementation. Second, there is difference in the expression pattern between the two promoters in each cell due to epigenetic factors. Tyrosine family site-specific recombinases and type IB topoisomerases are characterized by a
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