The Cre/loxP recombination system in transgenic mice


This page contains some general aspects of the Cre/loxP recombination system for an analysis of gene function in vivo. For targeting Cre to the mammary gland see our tool page.


Background

Cre is a 38 kDa recombinase protein from bacteriophage P1 which mediates intramolecular (excisive or inversional) and intermolecular (integrative) site specific recombination between loxP sites (see review article by Brian Sauer in Methods of Enzymology; 1993, Vol. 225, 890-900).

A loxP site (locus of X-ing over) consists of two 13 bp inverted repeats separated by an 8 bp asymmetric spacer region (Diagram)

One molecule of Cre binds per inverted repeat or two Cre molecules line up at one loxP site. The recombination occurs in the asymmetric spacer region. Those 8 bases are also responsible for the directionality of the site. Two loxP sequences in opposite orientation to each other invert the intervening piece of DNA, two sites in direct orientation dictate excision of the intervening DNA between the sites leaving one loxP site behind. This precise removal of DNA can be used to:

1. Elimination of genes (conditional gene deletion)

The Cre/loxP system is a tool for tissue-specific (and in connection with the tet system also time-specific) knockout of such genes which cannot be investigated in differentiated tissues because of their early embryonic lethality in mice with conventional knockouts. It can also be used for the removal of a transgene (which was overexpressed in a specific tissue) at a certain time point to study the invert effect of a downregulation of the transgene in a time course experiment.

Strategy:
Two mouse lines are required for conditional gene deletion. First, a conventional transgenic mouse line with Cre targeted to a specific tissue or cell type, and secondly a mouse strain that embodies a target gene (endogenous gene or transgene) flanked by two loxP sites in a direct orientation ("floxed gene"). Recombination (excision and consequently inactivation of the target gene) occurs only in those cells expressing Cre recombinase. Hence, the target gene remains active in all cells and tissues which do not express Cre
(Figure 1.).

Examples:

A. Excision of a reporter transgene
In a first experiment, a group led by Marth in Vancouver, Canada (Orban et al.; 1992 Proc. Natl. Acad. Sci. 89 (15): 6861-5), showed that Cre could be used at a high efficiency to excise a transgene in vivo. Two transgenic mice strains were established and crossed. Double transgenic mice carried a lck-Cre and a floxed lck-LacZ as a reporter gene. The reporter gene was removed exclusively in T cells.

B. Excision of a functional endogenous gene
Rajewsky's group in Cologne, Germany (Gu et al. 1994 Science 265: 103-106), used Marth's Cre expressing mice to inactivate for the first time an endogenous mouse gene. The ubiquitously expressed DNA polymerase beta was partly deleted in T cells. In a later experiment they used a mouse Mx1 promoter to drive Cre (Kuehn et al. 1995 Science 269: 1427-29). Here, the DNA polymerase was deleted in several tissues with different efficiency.


2. Activation of genes

The Cre's DNA excising capability can also be used to turn on a foreign gene by cutting out an intervening stop sequence between the promoter and the coding region of the transgene.

Strategy:
Again, one mouse strain is a conventional transgenic mouse line with Cre targeted to a special tissue or cell type. The second mouse strain has a transgene incorporated whose 5' regulatory elements are separated from the coding region with a floxed stop sequence. The recombination, i.e. excision of the stop signal, occurs only in those cells expressing Cre depending on the specificity of the Cre construct. Therefore, the transgene can be transcribed by the DNA polymerase in this special cell type whereas the transgene remains inactive in all other Cre nonexpressing cells.

Example:
This kind of experiment was first done by Heiner Westphal's group at the NIH in Bethesda (Maryland). They combined the oncogene coding region (large-T antigen or TAg) from simian virus 40 (SV40) and a murine alpha A crystallin promoter (m-alphaA) separated by a 1.3 kb regulatory stop sequence. These mice with a dormant transgene were bred into strains expressing Cre. The recombinase which is under control of either a hCMV or m-alphaA promoter, cuts out the stop signal resulting in a malignant transformation of lens cells
(Figure 2.).

References
Lakso et al. (1992) Proc. Natl. Acad. Sci. 89: 6232-36
Pichel et al. (1993) Oncogene 8: 3333-42


Note

The Cre recombinase has now been targeted to the mammary gland. For further information enter our toolpages (WAP-Cre mice or WAP based mammary expression vectors ).


Submitted by Kay-Uwe Wagner
National Institutes of Health
Bldg. 10, Rm. 9N113
Bethesda
Phone: 301-435-8907
Fax: 301-496-0839
kayuwew@bdg10.niddk.nih.gov

Contact for further information
Kay-Uwe Wagner


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contributed: February 1996
last update: June 1998