Two of the most exciting and versatile genetic tools designed in the last 30 years are the Cre-lox and FLP-FRT technologies. Both allow the location and timing of gene expression to be closely regulated. This article briefly outlines how the two systems work and can be used in mouse models.
Cre-lox technology was introduced in the 1980s (Sauer and Henderson 1988; Sternberg and Hamilton 1981) and patented by DuPont Pharmaceuticals. It has been successfully applied in yeasts, plants, mammalian cell cultures and mice (Araki et al. 1987). It is based on the ability of the P1 bacteriophage cyclization recombination (Cre) recombinase gene (cre) to effect recombination between pairs of loxP sites. Such recombination in a "Cre-lox" mouse (see below) can either activate or inactivate a gene of interest.
To use Cre-lox technology, an investigator has to produce a Cre-lox mouse, typically by breeding a Cre mouse to a loxP mouse. A Cre mouse contains a Cre recombinase transgene under the direction of a tissue-specific promoter; a loxP mouse contains two loxP sites that flank a genomic segment of interest, the "floxed" locus. Typically, Cre and loxP mice are produced by using transgenic technology (Nagy 2000). Depending on the promoters and other regulatory controls used to construct them, Cre mice can be designed to express Cre recombinase only under certain conditions, including the following: in certain tissues, when a mouse's diet is supplemented with substances such as doxycycline, tetracycline, RU486 and tamoxifen (Brocard et al. 1998; Kellendonk et al. 1999; Utomo et al. 1999) and during certain developmental stages.
Depending on the location and orientation of the loxP sites in a Cre-lox mouse, Cre recombinase can initiate deletions, inversions, and translocations of the floxed locus (Nagy 2000) (Fig. 1).
Cre-lox reactions are affected by the orientation and location of loxP sites. Paired loxP sites (triangles) have directionality and may be placed in a cis (same DNA strand) or trans (different DNA strands) arrangement. (A) If the loxP sites flank a DNA segment (rectangle) in a cis arrangement and are oriented in the same direction, Cre recombinase mediates excision or circularization of the segment. (B) If the loxP sites flank the DNA segment in a cis arrangement and are oriented in opposite directions, Cre recombinase mediates the inversion of the segment. (C) If the loxP sites are located on different strands of DNA and are oriented in the same direction, Cre recombinase mediates a translocation of the segment.
Usually, Cre and loxP strains are developed independently and then crossed. Many different Cre strains, each containing a Cre transgene under the direction of a different tissue-specific promoter, may be crossed with a single loxP strain. Depending on which strains are mated, a variety of Cre-mediated model systems can be constructed, including transgenics, knockouts, hypomorphs, repairable hypomorphs, chromosome aberrants and diet-induced mutants. In effect, by mixing and matching Cre and loxP strains, an investigator can study a gene's effects in tissue-specific and developmental stage-specific ways that were previously impossible.
The FLP-FRT system is similar to the Cre-lox system and is becoming more frequently used in mouse-based research. It involves using flippase (FLP) recombinase, derived from the yeast Saccharomyces cerevisiae (Sadowski 1995). FLP recognizes a pair of FLP recombinase target (FRT) sequences that flank a genomic region of interest.
Araki K, Imaizumi T, Okuyama K, Oike Y, Yamamura K. 1997. Efficiency of recombination by Cre transient expression in embryonic stem cells: comparison of various promoters. J Biochem (Tokyo) 122:977-82.
Brocard J, Feil R, Chambon P, Metzger D. 1998 A chimeric Cre recombinase inducible by synthetic, but not by natural ligands of the glucocorticoid receptor. Nucleic Acids Res 26:4086-90.
JAX NOTES. 1999. NIH, Jackson Laboratory and DuPont Pharmaceuticals sign Cre-lox technology use agreements. JAX NOTES 476:4.
Kellendonk C, Tronche F, Reichardt HM, Schutz G. 1999. Mutagenesis of the glucocorticoid receptor in mice. J Steroid Biochem Mol Biol 69:253-9.
Nagy A. 2000. Cre recombinase: the universal reagent for genome tailoring. Genesis 26:99-109.
Sadowski P. 1995. The Flp recombinase of the 2-µm plasmid of Saccharomyces cerevisiae. Prog Nucleic Acid Res Mol Biol 51:53-91.
Sauer B, Henderson N. 1988. Site-specific DNA recombination in mammalian cells by the Cre recombinase of bacteriophage P1. Proc Natl Acad Sci U S A 85:5166-70.
Sternberg N, Hamilton D. 1981. Bacteriophage P1 site-specific recombination. I. Recombination between loxP sites. J Mol Biol 150:467-86.
Utomo AR, Nikitin AY, Lee WH. 1999. Temporal, spatial, and cell type-specific control of Cre-mediated DNA recombination in transgenic mice. Nat Biotechnol 17:1091-6.