LoxP recombination site

Figure 4.3. Univector plasmid fusion system. Cre- loxP mediated site-specific recombination fuses the pUNI and pHOST plasmids at the loxP site. As a result, the gene of interest is placed under the control of the pHOST promoter and fused to any Tag sequences present in the pHOST plasmid.

This technique employs site-specific DNA recombination sites (called loxP sites) and the enzyme Cre that catalyzes recombination between them. The loxP-Cre recombination system is derived from bacteriophage P1, but this site-specific recombination system also functions when placed in mouse cells. An essential feature of this technique is that expression of Cre is controlled by a cell-type-speclflc promoter. In loxP-Cre mice generated by the procedure depicted in Figure 9-40, inactivation of the gene of Interest (X) occurs only in cells in which the promoter controlling the cre gene is active. 

SPGPTQSHY ASTSSGGGGSITSYSIHYTK 29 VH/VL monomers, but contains an internal protease site. loxP wild-type recombination site, flanked... 

Smith, A. J. H., Sousa, M. A. D., Kwabi-Addo, B., Heppell-Parton, A., Impey, H., and Rabbitts, P. (1995) A site-directed chromosomal translocation induced in embryonic stem cells by Cre-loxP recombination. Nature Genetics 9,376-385. 

Cre is a bacterial recombinase (cre=causes recombination), which recognizes loxP sites of bacteriophage P. If two loxP (loxP= locus of x-ing over of bacteriophage P) sites have a parallel orientation, the DNA segment between these sites will be deleted by the action of the Cre recombinase. 

An alternative to repeated cloning of PCR products is a recombination-based approach developed by Liu et al. (1998) to permit the cloning of a PCR product into a plasmid and the rapid conversion of the plasmid to a number of different expression systems without the necessity of cloning the PCR product multiple, independent times. The method, termed the univector plasmid-fusion system (UPS), involves the insertion of the PCR product into a particular type of plasmid, called the univector, which can then be placed under the control of a variety of promoters or fused in-frame to various tag sequences. The system is based upon plasmid fusion using the Cre-lox site-specific recombination system of bacteriophage PI (Sternberg et al., 1981). The Cre enzyme is a site-specific recombinase that catalyzes recombination between two 34 base pair (bp) loxP sequences and is involved in the resolution of dimers formed during replication of the... 

Gopaul, D. N., Guo, F. and Van Duyne, G. D. (1998). Structure of the Holliday junction intermediate in Cre-loxP site-specific recombination. EMBO J. 17, 4175-4187. 

Analogously to the Cre-LoxP system, the 34 bp Flippase recognition target (FRT) sites are recognized by the Flippase recombination enzyme (FLP) derived from Saccharomyccs cerevisiae (28). The FLP-FRT recombination system is also utilized for the inducible genetic manipulation of the mouse genome (29, 30). 

Van Duyne, G.D. (2001) A structural view of Cre-loxP site-specific recombination. Annu. Rev. Biophys. Biomol Struct. 30, 87-104. 

Other research groups are combining stress inducible promoters with the Cre-LoxP site specific recombination system of PI bacteriophage. Scott et al. (2000) have combined the ionizing radiation inducible EGR-1 promoter with this recombination system to express herpes simplex vims thymidine kinase (HSV-tk). With this combination system, radiation doses relevant for cancer treatment can be used to... 

Sternberg, N. and Hamilton, D. (1981) Bacteriophage PI site-specific recombination. I. Recombination between loxP sites../. Mol. Biol. 150,467-486. 

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