Recombinases

Since the development of the original Cre-ER system, mutagenesis and screening strategies have identified modified ER ligand-binding domains that have reduced responsiveness to E2 but can mediate tamoxifen-dependent recombination [48]. It is important to make the distinction that these modified ligand-receptor pairs do not necessarily form transcriptionally active complexes. Since the first report of the Cre-ER system, several new systems 

1 Chemical Biology of NHRs and the Potential of Engineered Nuclear Receptors 

---

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. 

Classical global knockouts may have a developmental or lethal phenotype and thus preclude the analysis of the phenotypic consequences of the lack of a gene in specific tissues in adult animals. With the development of the cre/loxP and flp/FRT systems, it has become possible to excise defined DNA fragments from the genome of specified cells. Cre and Flp are bacterial and yeast recombinases, respectively, which recognize loxP and FRT sequences, respectively. The most common... 

Sanchez-Contreras et al. 2002), and is mediated by the site-specific recombinases Sss and XerD (Martmez-Granero et al. 2005, 2006). An analogous phenomenon occurred in Pseudomonas sp. strain PCL1171 in which mutations in the genes gacA and gacS were shown to be responsible (van den Broek et al. 2005). 

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... 

Marker excision by site-specific recombination Very clean excision, small footprint Complex cloning procedure. Requires additional transgene encoding Cre recombinase 39... 

P = constitutive promoter, tetR = TETrepressor gene,TETR = TET repressor protein, tetO = TET operator sequence, = tetracycline, ere/CRE = Cre recombinase gene/protein, PLEA = late embryogenesis abundant promoter, RIP = gene for ribosome inactivating protein, shaded blocks are loxP sites in orientation shown by solid triangle. After [48]... 

Yang, W. and Steitz, T. A. (1995). Crystal structure of the site-specific recombinase yS resolvase complexed with a 34 bp cleavage site. Cell 82,193-207. 

A widely used inducible system in the mouse is the bacteriophage Pl-derived Cre-LoxP system (25). The site-specific Cre recombinase recognizes specifically 34 bp loxP sites. Generating mice that harbor a DNA sequence flanked by two loxP sites and... 

Sauer B, Henderson N (1988) Site-specific DNA recombination in mammalian cells by the Cre recombinase of bacteriophage PI. Proc Natl Acad Sci USA 85 5166-5170... 

Zhu XD, Sadowski PD (1995) Cleavage-dependent ligation by the FLP recombinase. Characterization of a mutant FLP protein with an alteration in a catalytic amino acid. J Biol Chem 270 23044-23054... 

Lee CL, Moding EJ, Huang X et al (2012) Generation of primary tumors with Flp recombinase in FRT-flanked p53 mice. Dis Model Mech. doi 10.1242/dmm.009084... 

Key words RNAi, Conditional, ShRNA, Cre recombinase, TetR/O, Transgenic mice, Rosa26,... 

Delete the lox-STOP-lox cassette in vitro by incubating 1 pg conditional shRNA plasmid DNA with 4 units of Cre recom-binase (New England Biolabs) in a total volume of 50 pi lx Cre buffer at 37°C for 30 min. Inactivate the recombinase for 10 min at 70°C. Eliminate nonrecombined plasmids by EcoRI digestion (see Note 8). 

---