Site-specific DNA recombination in mammalian cells by the Cre recombinase of bacteriophage P1.

Abstract

The Cre protein encoded by the coliphage P1 is a 38-kDa protein that efficiently promotes both intra-and intermolecular synapsis and recombination of DNA both in Escherichia coli and in vitro. Recombination occurs at a specific site, called lox, and does not require any other protein factors. The Cre protein is shown here also to be able to cause synapsis of DNA and site-specific recombination in a mammalian cell line. A stable mouse cell line was established that expresses the Cre protein under the control of the Cd2+-inducible metallothionein I gene promoter. DNA recombination was monitored with DNA substrates containing two directly repeated lox sites. One such substrate is a circular plasmid with two directly repeated lox sites (lox2) flanking a marker gene and was introduced into cells by Ca3(P04)2 transformation. As a second substrate we used a pseudorabies virus (a herpesvirus) containing a lox2 insertion designed to provide a sensitive detection system for recombination. In both cases, site-specific recombination in vivo is dependent on the presence of the Cre protein and occurs specifically at the 34-base-pair lox sites. These results demonstrate the controlled site-specific synapsis ofDNA and recombination by a prokaryotic protein in mammalian cells and suggest that Cre-mediated site-specific recombination may be a useful tool for understanding and modulating genome rearrangements in eukaryotes.

Modeling human lung cancer in mice: similarities and shortcomings

“…Indeed, a useful model for melanoma was created in this fashion (Chin et al, 1997). Alternatively, Cre/LoxP technology could be used to create conditionally targeted alleles of genes known to be mutated in human lung cancer, such as Kras, p53, p16 INK4a and Rb (Sauer and Henderson, 1988). Mice with single or multiple conditional alleles can then be analysed following the cre-induced somatic cell gene activation of K-ras and inactivation of tumor suppressor genes.…”

Ablation of de novo DNA methyltransferase Dnmt3a in the nervous system leads to neuromuscular defects and shortened lifespan

“…In order to control the ablation of Dnmt3a, we designed a conditional knockout version of the Dnmt3a gene that could be used in conjunction with the Cre-loxP recombination system (Sauer and Henderson, 1988). In the targeting construct, exons encoding the catalytic domains were flanked by loxP sites (Fig.…”

Flp recombinase regulated lacZ expression at the ROSA26 locus

“…The recombinase system most commonly used for conditional alleles in mouse is Cre/loxP derived from bacteriophage P1 (Sauer and Henderson, 1988). While less utilized, FLP/FRT derived from S. cerevisiae (O'Gorman et al, 1991) has been elegantly used by Martin and colleagues in combination with Cre to create a series of alleles for Fgf8 of varying strength (Meyers et al, 1998).…”