Cre-lox system

Cre-Lox system, including temporal and spatial conditional mice... 

Sauer B (1998) Inducible gene targeting in mice using the Cre/lox system. Methods 14 381-392... 

The Cre/lox system requires the generation of two separate lines of transgenic mice that are crossed to produce a conditional knockout in the offspring as follows (Fig. 9-20). One parental strain of transgenic mice is generated expressing... 

Fig. 9-20 The Cre/lox system for generating tissue-specific deletion of transgenes.

In the Cre/lox system, why is the gene of interest not simply expressed from a tissue-specific promoter SOLUTION... 

Heitzer, M. and Zschoernig, B. (2007) Construction of modular tandem expression vectors for the green alga Chlamydomonas reinhardtii using the Cre/lox-system. Biotechniques, 43 (3), 324, 326, 328 passim. 

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... 

The most efficient strategy may be to use the topoisomerase cloning method in conjunction with the Crs-lox or X recombination systems. In this way, PCR products can be efficiently inserted into the Univector or Entry Clone with minimal additional sequences added to PCR primers. Once the genes are inserted into the starting vectors they can rapidly be converted to functional vectors using either Cre-lox or X recombination. 

Corneille, S. et al. (2001). Efficient elimination of selectable marker genes from the plastid genome by the CRE-lox site-specific recombination system. Plant. . 27 171-178. 

Forlino, A., Porter, F. D., Lee, E. J., West-phal, FL and Marini, J. C. (1999) Use of the Cre/lox recombination system to develop a non-lethal knock-in murine model for osteogenesis imperfecta with an alphal(I) G349C substitution. Variability in phenotype in BrtllV mice. J Biol Chem 274, 37923-37931. 

Forlino A, Porter FD, Lee EJ, et al. Use of the Cre/lox recombination system to develop a non-lethal knock-in murine model for osteogen-... 

One remaining problem with tissue-specific null mutants that exploit the Cre/ lox-P system is that early excision at the time of birth of sensory neurons may allow developmental compensatory mechanisms to occur, again complicating phenotypic analysis, particularly in terms of behaviour. It would thus be ideal to be able to delete genes in adult animals, whose behaviour under control conditions has already been assessed. Recent advances in gene dehvery suggest that this approach may be feasible. 

Hoess, R. H. snd Abremski, K. (1985) Mechanism of strand cleavage and exchange in the Cre-lox site-specific recombination system. J. Mol. Biol. 181,351-362. 

Osborne BI, Wulz U, Baker B (1995) A system for insertional mutagenesis and chromosomal rearrangement using the Ds ttansposon and Cre-lox. Plant 7 687-701. 

Lambert, J.M., Bongers, R.S., and Kleerebezem, M. (2007). Cre-lox-based system for multiple gene deletions and selectable-marker removal in Lactobacillus plantarum. Appl Environ Microbiol 73, 1126-1135. 

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