Cre Recombinase Activity

In natural Cre molecules, helices B and D are in contact with the LoxP site, but ring A / B interacts with helix E of the same or adjacent Cre protein (39), and these interactions are separated N. - terminating fragment Furthermore, considering the interaction of helices A and B with helices C and D, the isolated N-terminal fragment of Cre can have a structure similar to that used in intact Cre molecules, Also contributes to its ability to be stable and complex.

(A) Schematic of the target genomic modification of mice used to generate a conditional knockout allele carrying the Hsp60 (Flox allele). Hsp60 knockout is specifically produced in intestinal epithelial cells (IEC) or intestinal stem cells (ISC) by expressing Cre recombinase and inducing Cre activity by tamoxifen. (B) Schedule of oral tamoxifen administration to induce IEC's HSP60 deficiency in adult mice. Agarose gels showed the presence of knockout alleles, especially in IEC isolates. (C) Representative H & E and HSP 60 IHC staining of Hsp60flox / flox and Hsp60Δ / ΔIEC mice along the intestinal tract. Higher magnification HSP 60 IHC images show HSP 60 positive crypt region from HSP 60 deficient villi to jejunum. HSP60 positive crypt nodules in Hsp60Δ / ΔIEC mice were counted along the intestine using HSP60 IHC staining. The figure shows the quantification of Hsp60Δ / ΔIEC mice (N = 6) with> 100 crypts per animal number.

If an important gene is knocked out, it may be fatal to the organism. Site-specific recombinase (SSR) was used to study the function of these genes. The two most common types are the Cre-LoxP and Flp-FRT systems. Cre recombinase is an enzyme that removes DNA by homologous recombination between binding sequences called Lox-P site. The Flip-FRT system works in the same way, and the Flip recombinase recognizes the FRT sequence. By hybridizing an organism containing a recombinase site adjacent to the gene of interest to an organism expressing the SSR under the control of a tissue specific promoter, the gene can be knocked out or turned on only in certain cells. These techniques are also used to remove marker genes from transgenic animals. Further modifications to these systems allowed researchers to induce recombination only under certain conditions and allow the genes to be knocked out or expressed at the time or stage of development.