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

TLDR

The 38‑kDa Cre protein from bacteriophage P1 mediates efficient intra‑ and intermolecular synapsis and recombination at lox sites in E. coli and in vitro without additional factors. A Cd2⁺‑inducible mouse cell line expressing Cre was used to monitor recombination with substrates bearing two directly repeated lox sites, including a plasmid flanking a marker gene and a pseudorabies virus carrying a lox2 insertion.

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(PO4)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 of DNA 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.