Introduction to Cre-lox technology

The Cre-lox system is a sophisticated tool for general knockouts, conditional knockouts, and reporter strains.

The Cre-lox mechanism was discovered in P1 bacteriophage as part of this organism's normal viral live cycle (Sauer and Henderson 1988; Sternberg and Hamilton 1981). The bacteriophage uses Cre-lox recombination to circularize and facilitate replication of its genomic DNA when reproducing. Since being discovered, the bacteriophage's recombination strategy has been developed as a technology for genome manipulation and successfully applied in mammalian cell cultures, yeasts, plants, mice, and other organisms (Araki et al. 1987). Much of the success of Cre-lox is due to its simplicity. It requires only two components:

  1. Cre recombinase: an enzyme that catalyzes recombination between two loxP sites
  2. LoxP sites: specific 34-base pair (bp) sequences consisting of an 8-bp core sequence, where recombination takes place, and two flanking 13-bp inverted repeats (Figure 1)

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Figure 1. The 34-base loxP sequence. The core sequence (shaded) is flanked by two inverted repeats.

Because the cre gene and loxP sites are not native to the mouse genome, they must be introduced by transgenic technology (Nagy 2000). The orientation and location of the loxP sites determine whether Cre recombination induces a deletion, inversion, or chromosomal translocation (Nagy 2000) (Figure 2). The vast majority of Cre-lox strains being developed are designed to produce deletions.

Figure 2 showing inversion, translocation, and deletion

Figure 2. The outcome of a Cre-lox recombination is determined by the orientation and location of flanking loxP sites. (A) If the loxP sites are oriented in opposite directions, Cre recombinase mediates the inversion of the floxed segment. (B) If the loxP sites are located on different chromosomes (trans arrangement), Cre recombinase mediates a chromosomal translocation. (C) If the loxP sites are oriented in the same direction on a chromosome segment (cis arrangement), Cre recombinase mediates a deletion of the floxed segment.

Cre and loxP mouse strains

Typically, cre and loxP strains are developed separately and crossed to produce a Cre-lox strain (Nagy 2000). The majority of cre and loxP strains being developed fall into one of the following categories:

  • Cre expressing strains: contain a transgene that expresses cre under the control of a widespread (general) or tissue-specific (conditional) promoter. They are used to produce general or conditional knockouts respectively.
  • Inducible Cre strains: contain a transgene that expresses a modified form of Cre recombinase that is non-functional until an inducing agent (such as doxycycline, tetracycline, RU486, or tamoxifen) is administered at a desired time point in embryonic development or adult life
  • LoxP-flanked (floxed) strains: contain loxP sites flanking (on each side of) a critical portion of a target gene or genomic region of interest
  • Cre reporter strains: contain loxP sites in combination with visible (fluorescent or lacZ) marker proteins used to trace Cre recombination success and/or alterations in gene expression.

References

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. (PMID: 9443813)

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. (PMID: 9705523)

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. (PMID: 10418999)

Nagy A. 2000. Cre recombinase: the universal reagent for genome tailoring. Genesis 26:99-109. (PMID: 10686599)

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. (PMID: 2839833)

Sternberg N, Hamilton D. 1981. Bacteriophage P1 site-specific recombination. I. Recombination between loxP sites. J Mol Biol 150:467-86. (PMID:6276558)

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. (PMID: 10545915)