Proc. Nail. Acad. Sci. USA Vol. 85, pp. 5166-5170, July 1988 Genetics Site-specific DNA recombination in mammalian cells by the Cre recombinase of bacteriophage P1 (genome rearrangement/herpesviruses/Cre-lox/metalothionein) BRIAN SAUER AND NANCY HENDERSON E. I. du Pont de Nemours and Company, Inc., Central Research and Development Department, Experimental Station, Wilmington, DE 19898 Communicated by Robert A. Weinberg, March 28, 1988 ABSTRACT The Cre protein encoded by the coliphage P1 give inversion of the intervening DNA segment. Both intra- is a 38-kDa protein that efficiently promotes both intra- and and intermolecular recombination is catalyzed by Cre with intermolecular synapsis and recombination of DNA both in either supercoiled or linear DNA. Recently we have shown Escherichia coli and in vitro. Recombination occurs at a specific that the Cre-lox system can function efficiently in yeast to site, called lox, and does not require any other protein factors. cause recombination on chromosomes present in the nucleus The Cre protein is shown here also to be able to cause synapsis (5). We now demonstrate that this site-specific recombination of DNA and site-specific recombination in a mammalian cell system also functions in a mammalian cell. line. A stable mouse cell line was established that expresses the Cre protein under the control of the Cd2+-inducible metallo- MATERIALS AND METHODS thionein I gene promoter. DNA recombination was monitored with DNA substrates containing two directly repeated lox sites. Plasmids. The Cre expression plasmid pBS31 and its One such substrate is a circular plasmid with two directly parent, pBMTx (obtained from G. Pavlakis, Frederick Can- repeated lox sites (lox2) flanking a marker gene and was cer Research Facility), have been described (5, 6), as have introduced into cells by Ca3(P04)2 transformation. As a second pRH43 (7), pBS64, and pBS109 (8). M13mpll neoAS10, substrate we used a pseudorabies virus (a herpesvirus) con- which contains an internal fragment of the kanamycin- taining a lox2 insertion designed to provide a sensitive detection resistance gene (kan') from transposon 5 (TnS), was used for system for recombination. In both cases, site-specific recom- DNA hybridization analysis and was thE gift of N. Steinberg bination in vivo is dependent on the presence of the Cre protein (DuPont). Plasmids and the replicative form of M13 were and occurs specifically at the 34-base-pair lox sites. These prepared by standard techniques (9). results demonstrate the controlled site-specific synapsis ofDNA Mammalian Cells and Viruses. The swine kidney cell line and recombination by a prokaryotic protein in mammalian PK15, the Becker strain of pseudorabies virus (PRV-Be), cells and suggest that Cre-mediated site-specific recombination pseudorabies virus 42 (PRV42), and PRV42::pBS64 have may be a useful tool for and been described (8), as has the procedure for the black-plaque understanding modulating genome assay (10). The mouse cell line C-127 (11) was transformed rearrangements in eukaryotes. with the bovine papilloma virus-based vectors pBS31 and pBMTx [both containing the metallothionein I (MT-I) gene The processes governing DNA recombination in mitotic promoter] to establish cell lines 55 and 3C-5, respectively. mammalian cells have been the subject of intense investiga- The cell lines were obtained after colony purification of tion in recent years. Recombinational events have been transformed cells from foci generated after DNA transfor- shown to be important in a variety of both normal and mation (12). Southern blot analysis indicated that each cell abnormal processes in mammalian cells. Mitotic recombina- line contained at least 60 extrachromosomal copies of the tion plays a central role in the development and function of indicated plasmid, as anticipated (13). the immune system. DNA recombination is also likely to be DNA Transformation. Ca3(PO4)2-mediated DNA transfor- involved in both gene amplification events leading to drug mation of mammalian cells was exactly as described by Lin resistance and the generation of the chromosomal transloca- et al. (14). Transient DNA transformation was performed in tions associated with some lymphomas and leukemias-for the absence of carrier DNA with 0.5 pug of pRH43 per 10-cm example, those in Burkitt lymphoma (1). The mechanism(s) diameter dish containing 5 x 105 cells of the indicated cell by which recombination occurs in mammalian cells is poorly line. understood. Therefore, the introduction of a well-character- Analysis of DNA and RNA. Either total cellular DNA (15) ized site-specific recombination system into mammalian cells or Hirt supernatant DNA (16) was prepared from cells and may provide some insight into certain aspects of recombina- then analyzed by Southern blots (5, 17). DNA was electro- tion. The constraints imposed on DNA synapsis ofchromatin phoresed in 0.8% agarose gels containing Tris borate buffer structure, for example, could be one such concern. A and no ethidium bromide (9). PRV DNA was prepared from site-specific recombination system would also be useful in nucleocapsids (18). Polyadenylylated RNA was prepared (19, directed manipulation of the eukaryotic genome. 20) and then analyzed by Northern blots (9). Restriction The Cre-lox site-specific recombination system of coli- enzymes and topoisomerase I were obtained from Bethesda phage P1 is particularly simple and well characterized (2-4). Research Laboratories and used as recommended by the A single 38-kDa protein, Cre, is both necessary and sufficient supplier. Cre protein was the gift of Ken Abremski (DuPont) to catalyze recombination between two lox sites, each of and was used as described (8, 21). which is 34 bp in length. Recombination can occur between Antibody and Immunoblot Analysis. Antibody to Cre and directly repeated sites on the same molecule to excise the immunoblot analysis have been described (5). Additional intervening DNA segment or between inverted lox sites to antibody to the Cre protein was produced at Hazelton Bio- technologies (Denver, PA) by injection of a New Zealand The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" Abbreviations: PRV, pseudorabies virus; kanr, gene encoding ka- in accordance with 18 U.S.C. §1734 solely to indicate this fact. namycin resistance; MT-I, metallothionein I. 5166 Downloaded by guest on September 25, 2021 Genetics: Sauer and Henderson Proc. Natl. Acad. Sci. USA 85 (1988) 5167 White rabbit with 1 mg ofpurified Cre. Protein extracts ofcells A B were prepared as described for yeast (5) with the omission of 1 2 3 4 5 6 7 1 2 3 4 3 6 7 Zymolyase treatment. Protein extract (50 Ag per lane) was electrophoresed on an 8% polyacrylamide gel containing sodium dodecyl sulfate and electrophoretically transferred to nitrocellulose for detection ofthe 38-kDa Cre protein by using NR- __m Cre-specific antibody and '25I-labeled protein A (5). The amount ofCre in cell extracts was determined by densitometry ofthe 38-kDa Cre protein and a comparison to a titration series of a known amount ofpurified Cre electrophoresed in parallel R -. R _* on the same gel. RESULTS The phage P1 cre gene was placed under the control of the Cd2 + -inducible MT-I promoter on a plasmid vector contain- ing the bovine papilloma virus replicon and transforming region (5, 6). The resulting plasmid, pBS31 (Fig. lA), was introduced into the C-127 mouse cell line, and transformants FIG. 2. Detection by Southern blot analysis of the excised kanr were identified by their ability to form foci. Cell line 55 is a fragment from pRH43. (A) Cells were transformed with pRH43 DNA subclone of that by Ca3(PO4)2 coprecipitation. Fresh medium was added, and 5 hr one such focus contains =60 extra- later cells were induced for 4 hr with 8 ,uM CdCl2 (protocol I) or 24 chromosomal copies of pBS31. hr later cells were induced for 4 hr with 20 ,uM CdCl2 (protocol II). RNA blot analysis revealed that cre-specific RNA was After induction, cells were washed with phosphate-buffered saline, induced -50-fold in cell line 55 after treatment of cells with and fresh medium was added (22). Total DNA (15) was prepared 30 8 AM CdCl2 for 4 hr (data not shown). Immunoblot analysis hr after removal of the Ca3(PO4)2 coprecipitate for all cultures, ofcell line 55 also showed CdCl2 induction ofthe Cre protein, digested with Bgl II, and analyzed by Southern blotting with a kanr as described in more detail below. To determine whether the probe. Lanes: 1, 1.3 ng of pRH43 DNA treated with Cre protein in Cre recombinase expressed in cell line 55 was functional, we vitro and digested with Bgl II; 2, uninduced C-127; 3, protocol used a recombination assay with I-treated C-127; 4, protocol II-treated C-127; 5, uninduced cell line plasmid pRH43 (Fig. 1B). 55; 6, protocol I-treated 55; 7, protocol lI-treated 55. (B) A four-fold Plasmid pRH43 contains two directly repeated loxP sites overexposure of the blot shown in A. NR, nonrecombinant (4.3-kb, flanking the kanr gene from TnS (7). Recombination by Cre linear fragment); R, recombinant (1.9-kb, linear fragment). at the loxP sites results in excision of the kanr gene from pRH43 as a 1.9-kilobase (kb) circular DNA molecule con- as the 1.9-kb fragment diagnostic of Cre-mediated recombi- taining a single Bgl II site. Calcium phosphate-mediated DNA nation (R in Fig. 2). It was not present in uninduced cells (lane transformation was used to introduce pRH43 into 55. Two 5), nor was it present in protocol II-induced cells (lane 7), different induction protocols were used to elicit Cre expres- which were allowed only a short expression time.