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© 1995 Oxford University Press 4608-4613

Footnote

Multiplex Cre/lox recombination permits selective site-specific DNA targeting to both a natural and an engineered site in the yeast genome

Multiplex Cre/<I>lox</I> recombination permits selective site-specific DNA targeting to both a natural and an engineered site in the yeast genome

Brian Sauer*

National Institutes of Health, National Institute of Diabetes, Digestive and Kidney Disease, Bethesda, MD 20892-1800, USA

Received September 18, 1996Revised and Accepted October 18, 1996

ABSTRACT

Variant lox sites having an altered spacer region (heterospecific lox sites) are not proficient for Cre-mediated recombination with the canonical 34 bp loxP site, but can recombine with each other. By placing different heterospecific lox sites at different genomic locations, Cre can catalyze independent DNA recombination events at multiple loci in the same cell without concern that unwanted inter-locus recombination events will be generated. Such heterospecific lox sites also allow Cre to specifically target efficient integration of exogenous DNA to endogenous lox-like sequences that naturally occur in the genome. Specific targeting occurs only with a DNA vector carrying a heterospecific lox site in which the spacer region has been redesigned to match the `spacer' region of the targeted chromosomal element. Moreover, in cells expressing a catalytically active Cre recombinase, naturally occurring lox-like sequences can exhibit almost 20% mitotic recombination. Thus, in the same cell, heterospecific lox sites can be used independently at multiple loci for integration, for deletion and for enhanced mitotic recombination, thereby increasing the repertoire of genomic manipulations catalyzed by the Cre recombinase.

INTRODUCTION

Site-specific DNA recombinases have become important tools for in vivo manipulation of eukaryotic genomes (1-6). Cre recombinase and its DNA recognition site loxP have been used both for activation and for elimination of genes in a variety of transgenic and gene-modified mice (7-9). In these genome manipulation strategies loxP sites are introduced into the genome using either traditional zygote injection techniques or by homologous recombination in pluripotent embryonic stem cells such that they flank the segment of DNA that is to be deleted from the genome. Upon expression of the Cre protein, efficient site-specific conservative recombination occurs at the loxP sites to excise the intervening segment of DNA from the genome. Because recombination occurs only in cells expressing Cre, tissue-specific deletion or activation of a gene can be made to occur by controlling the expression of the cre gene. Prior placement of Cre's DNA recognition site loxP into the genome also permits Cre-mediated integration of a loxP-containing plasmid to that chromosomal site, facilitating construction of isogenic cell lines and mice (10,11). Precise genome manipulation is possible not only because Cre is an efficient recombinase in eukaryotic cells, but also because the loxP size is sufficiently large (34 bp) that it is unlikely to occur naturally in any eukaryotic genome. Rare recombination events have been detected, however, with cryptic lox sites in both the Escherichia coli and the yeast genomes (12,13).

The loxP site consists of two 13 bp inverted repeats to which Cre binds (14,15) and an intervening 8 bp core. Only pairs of sites having identity in the central 6 bp of the core region are proficient for recombination; sites having non-identical core sequences (heterospecific lox sites) do not efficiently recombine with each other (16). Thus, the core region of the lox site determines the specificity of recombination with a lox partner. For example, a variant heterospecific lox site `loxY' would not be able to recombine with the canonical loxP site, but would be proficient for loxY × loxY recombination.

The existence of multiple heterospecific lox sequences suggests several novel recombinational strategies for genome manipulation. First, selective Cre-mediated targeting of different chromosomal lox sites may be achievable by placing heterospecific lox sites into the genome at different loci and then specifying the core sequence on the lox targeting plasmid to direct integration to the corresponding locus. Second, use of different pairs of heterospecific sites would allow Cre-mediated gene excision at different chromosomal locations without generating complicating chromosome inversions, translocations or large scale deletions. Third, because Cre requires as few as 8-10 bp of each 13 bp binding domain (17,18), eukaryotic genomes may naturally contain one or more functional lox-like sequences, but which differ from loxP in the core region. The feasibility of these strategies is demonstrated here using the genome of Saccharomyces cerevisiae.

MATERIALS AND METHODS

Plasmids

The yeast integrating vector pRB19 contains the LEU2 gene for selection in yeast, but no lox site (10,19). Removal of a single loxP site from the lox2 LEU2 construct pBS30 (20) generated the loxP LEU2 integrating vector pBS222. The mock loxFAS1 vector pBS214 was constructed by inserting the annealed oligos, 5[prime]-AGC TTC GTA TAT ACC TTT CTA TAC GAA GTT GTG-3[prime] and 5[prime]-GAT CCA CAA CTT CGT ATA GAA AGG TAT ATA CGA-3[prime], between the HindIII and BamHI sites of pRB19. The GAL1-cre yeast expression CEN plasmids pBS49 (20) and pBS126 (21) respectively carry the selectable markers URA3 and ILV2-4-10 (sulfometuron methyl resistance) for selection in yeast (22). Plasmid pBS394, GAL1-creY324C, was constructed by replacing the wild-type cre sequence of pBS126 with that of the `creNA2' mutant (23).

Cre reactions

Cre reactions (10 µl) were performed under standard conditions at 30°C for 1 h and contained 2% polyvinyl alcohol (24,25), 20-30 fmol of each lox site and 2 pmol Cre protein or as indicated. Reaction products were visualized after gel electrophoresis by ethidium bromide staining or by autoradiography when using 33P-end-labeled loxFAS1 fragments generated by polymerase chain reaction (PCR) amplification and treatment with T4 kinase. Ethidium bromide stained gels were digitally photographed using a Foto/Eclipse camera system (Fotodyne, Inc.) and quantitated using NIH Image software. Radiolabeled gels were quantitated using the Fuji BAS 1500 phosphorimager.

Yeast strains and genetic manipulation

Strains used in this work are listed in Table 1. Culture conditions (S medium: minimal defined medium; YEPD: yeast extract, peptone, dextrose) and selection procedures have been described (20). BSY659 is a ura1::LEU2 insertion/deletion derivative of DBY745 made by homologous recombination (26). In the ura1::LEU2 insertion the LEU2 gene replaces a 328 bp segment between the EcoRV and NcoI sites of URA1 (a 986 bp fragment amplified from genomic DNA with the oligos 5[prime]-CAG GTC GAC TCT AGA GGA CCA AAC ATG ACA GCC AG-3[prime] and 5[prime]-GTA ACC CGG GAT CCA AGC TTA AAT GCT GTT CAA CTT CC-3[prime]). Plasmids pBS126 and pBS394 were introduced into and maintained in BSY695 by selection for resistance to sulfometuron methyl (a gift of S. C. Falco, DuPont Co.). DNA transformation was with the Frozen-EZ Yeast Transformation Kit (Zymo Research).

Table 1 Strain genotypes
Strain Genotype Reference
2132 Mata trp1 leu2 cdc16 mak11-1 (40)
DBY745 Mat[agr] ade1-100 leu2-3, 112 ura3-52 (17)
DBY931 Mata his4 leu2-3, 112 ura3 met8-1 can1-101 (17)
BSY3 Mat[agr] ade1-100 leu2-3, 112 ura3 (pBS49) (18)
BSY103 Mat[agr] ade1-100 leu2-3, 112 ura3 Ch VII::loxP (pBS49) (10)
BSY659 Mat[agr] ade1-100 leu2-3, 112 ura3-52 ura1::LEU2 This work
BSY671 Mat[agr]ade1 1eu2 ura3 +    +    +    (pBS49)
Mata +   leu2 ura3 met8 his4 can1		 BSY3 × DBY931
BSY695 Mat[agr] leu2 ura3 ura1::LEU2 +    +    +    ade1
Mata leu2 +    +    trp1 cdc16 mak11 + 		BSY659 × 2132

For determination of the efficiency of Cre-mediated targeting, yeast strains (± a resident chromosomal loxP target and containing the cre gene under GAL1 control) were induced for cre expression by pregrowth in S + galactose medium, transformed with the indicated amount of the appropriate LEU2 targeting vector, and selected for leucine prototrophy.

For analysis of mitotic recombination, overnight cultures of the heterozygote BSY695 (Fig. 3) carrying the indicated cre expression construct were grown in SD (S + dextrose), washed, and diluted (final concentration: 1 × 106 cells/ml) into S medium supplemented with adenine, leucine, uracil and either galactose or dextrose. Cells were grown for the indicated time and samples plated for individual colonies on YEPD plates at 30°C. Phenotypes were determined by replica plating to SD plates with the appropriate nutritional supplements.

Polymerase chain reaction

Analysis of the loxFAS1 region and preparation of loxFAS1 fragments for recombination in vitro (above) was with the following primers: a, 5[prime]-GGT CCA GAA GCA AGT ATG TCT ATG G-3[prime]; b, 5[prime]-CGC TGT TGC GTA ATT ATG CTT GGC-5[prime]; 97, 5[prime]-ATC AAG ACC AGG AAC AAT ACC-3[prime]; 98, 5[prime]-GCA CCT CGT GTA TCG TGA TGC-3[prime]; L2b, 5[prime]-GAC GAT TGC TAA CCA CCT ATT GG-3[prime]; U, 5[prime]-CAG GGT TAT TGT CTC ATG AGC GG-3[prime]. Amplification was by 30 cycles: 30 s at 94°C, 30 s at 65°C, 60 s at 71°C, in a Perkin Elmer 9600 thermocycler with Taq polymerase (Perkin Elmer), using [sim]50 ng yeast genomic DNA in a 50 µl reaction volume.

RESULTS

Recombination at candidate lox-like sequences

A minimum requirement for a functional lox site is two 13 bp inverted repeat elements (for Cre binding) separated by 8 nt. Although several cryptic lox sites in the yeast genome have been identified previously, none contain two `good' 13 bp repeat elements (13). Do there exist in eukaryotic genomes lox-like sequences having functional Cre-binding inverted repeats and a correctly sized heterologous spacer? A search of the GenBank genome database (Release 89.0) for lox-like DNA sequences with strong homology to the two loxP 13 bp inverted repeats found no eukaryotic sequences with identity in the inverted repeat regions of SYMBOL 179 \f "Symbol" \s 129 bp and separated by a non-specified 8 nt core region. However, a potential recombination site with 9 bp of identity adjacent to the loxP core on one side, and a single mismatch in the 10 bp adjacent to the core on the other, was found in S.cerevisiae on chromosome XI, just upsteam of the FAS1 gene (Fig. 1A) which encodes the [bgr] subunit of fatty acid synthetase (27,28). The core region of this loxFAS1 site bears almost no homology to the loxP core. To determine the recombinational competence of this site, a mock loxFAS1 site was synthesized having two functional inverted repeats flanking a core region identical to that of the loxFAS1 site. The synthetic site was cloned into a LEU2 integrative yeast vector for comparison with a similar loxP vector (Fig. 1B).


Figure 1. The lox recombination sites and vectors. (A) Alignment of loxP with the yeast loxFAS1 site (accession no. X03977; nucleotides 505-538) and the synthetic mock loxFAS1 site. Positions identical to those in loxP (41) are boxed in grey. The 13 bp inverted repeats are indicated by the arrows. Identity in the core region is emphasized by vertical lines. (B) lox integrating vectors. Both carry the LEU2 gene for selection in yeast. The no lox control pRB19 is identical to pBS214 but carries no lox site. B, BamHI; H, HindIII; RI, EcoRI; S, SalI. (C) In vitro recombination assay. Linear lox recombination substrates were generated by restriction digest of either pBS222 (loxP) or pBS214 (mock loxFAS1). Production of new longer or shorter DNA fragments is indicative of Cre-mediated recombination, as sketched here for loxP site recombination using pBS222. Similar strategies were used to provide distinctly-sized loxP and mock loxFAS1 substrates.

Intermolecular recombination in vitro with purified Cre recombinase (using the strategy shown in Fig. 1C) showed that the synthetic mock loxFAS1 site was as proficient for Cre-mediated site-specific recombination as an authentic loxP site (Fig. 2A: compare lanes 1 and 2 with 3 and 4). Thus, this altered spacer itself does not markedly affect self by self recombination. As expected, recombination between heterospecific sites (loxP × mock loxFAS1) was not detectable and Cre did not generate a novel band diagnostic of loxP × mock loxFAS1 recombination (lanes 5 and 6). The natural loxFAS1 site was also proficient in DNA recombination with the synthetic mock loxFAS1 site (lanes 7-10); the 3-fold reduced efficiency probably reflects a non-optimal interaction of Cre with the natural loxFAS1 site that results in reduced recombinational proficiency. Consistent with this idea, a 3-fold molar increase of the loxFAS1 site enhanced recombination whereas a 3-fold increase in the mock loxFAS1 substrate had little effect. Intermolecular recombination between substrates carrying the natural loxFAS1 sites is barely detectable in vitro (Fig. 2B), and is far less efficient than loxFAS1 × mock loxFAS1 recombination. These results suggested that the natural loxFAS1 site would be targetable in vivo using the mock loxFAS1 site, i.e. Cre may be able to target integration of an exogenous DNA carrying the synthetic lox site specifically to the endogenous yeast sequence upstream of the FAS1 gene.


Figure 2. Recombination in vitro. (A) Linear lox containing substrates (Fig. 1) were mixed and treated with Cre as described in Materials and Methods. Reaction products were visualized by ethidium bromide staining. Arrows mark the position of predicted recombination products in `+ Cre' lanes. Lanes 1 and 2: pBS222 × EcoRI + pBS222 × SalI; lanes 3 and 4: pBS214 × EcoRI + pBS214 × BamHI; lanes 5 and 6: pBS214 × SalI + pBS222 × EcoRI; lanes 7-10: pBS214 × EcoRI + 528 bp loxFAS1 PCR fragment (primers 97 + 98; Fig. 3). Lanes 8 and 9 contain a 3-fold molar excess of the loxFAS1 fragment; lane 10 contains a 3-fold molar excess of the mock lox plasmid substrate. M: size markers, a HindIII digest of phage [lgr]. (B) A 33P-end-labeled 440 bp loxFAS1 PCR fragment (primers a + b; Fig. 3) was tested for recombination as in (A) with either a 2-fold molar excess (lanes 1 and 2) or equal molar (lane 3) unlabeled 528 bp loxFAS1 fragment and with an equal molar amount of the mock lox plasmid pBS214 linearized with EcoRI (lane 4).


Figure 3. Analysis of pBS214 integrants. (A) Map of the wild-type and targeted loxFAS1 region. PCR primers diagnostic for the undisrupted genomic locus and for the predicted junction fragments after pBS214 integation at the loxFAS1 site are shown. The lox sites are represented by stubby black arrows. (B) PCR analysis. For each of the three strains targeted with pBS214 in Table 2 genomic DNA was prepared from four independent Leu+ transformants and from the Leu- parent (P) and then amplified with one of the three sets of primers diagnostic for the endogenous loxFAS1 locus, for the left junction or for the right junction, as predicted after site-specific integration. Analysis of an additional 26 Leu+ transformants showed identical specificity in loxFAS1 targeting by pBS214.

Genomic targeting

Both the efficiency and independence of genomic targeting at the loxFAS1 site in vivo were examined using the loxP and mock loxFAS1 yeast integration vectors carrying the LEU2 marker (Fig. 1B). Such circular DNA vectors integrate at low efficiency by homologous recombination into the yeast genome at the chromosomal LEU2 locus (29). However, Cre directs integration of the loxP vector into a chromosomal loxP site at high efficiency (10). The mock loxFAS1 plasmid pBS214 was used to test for targeting in both haploid (BSY3 and BSY103) and diploid (BSY671) leu2 auxotrophs carrying a GAL1-cre expression plasmid. Test strains were induced for recombinase expression by growth on galactose and then transformed with either the loxP or the mock lox plasmid. Leu+ transformants were selected on plates containing glucose (to repress expression of cre), thereby trapping any LEU2 plasmids integrated into the genome by Cre-mediated recombination (Table 2). As shown previously, in strain BSY103 (which has been enginneered to have a loxP site near PDR1 on chromosome VII), Cre-dependent targeting integrates the pBS222 loxP plasmid 50-fold more efficiently than a plasmid having no lox site (pRB19). In contrast, integration occurs at the same frequency with both pBS222 and pRB19 in strain BSY3, which lacks a chromosomal loxP site. Cre-mediated targeting of the mock loxFAS1 plasmid pBS214 results in a 25-fold stimulation of Leu+ transformants in all three strains compared with the no lox control pRB19, regardless of the presence or absence of a chromosomal loxP site. This result suggests (i) that Cre is directing integration of the mock loxFAS1 site into an endogenous yeast locus, presumably the loxFAS1 site, and (ii) that integration does not disrupt an essential yeast gene.

Table 2 Selective targeting of chromosomal lox sites by Cre
Strain µg DNA Leu+ transformants
pBS222
(loxP)		 pBS214
(mock lox)		 pRB19
(no lox site)
BSY103 1 65 24 0
(loxP target) 4 119 47 2
BSY3 1 3 84 3
(no loxP target) 4 6 324 13
BSY671 1 - - -
(no loxP target) 4 1 149 -

Inspection of the Cre-mediated Leu+ transformants obtained with the mock loxFAS1 vector showed that integration was specific to the genomic loxFAS1 site. Importantly, the presence of the loxP site in strain BSY103 did not misdirect integration of the mock loxFAS1 plasmid. PCR analysis on four pBS214 Leu+ yeast transformants from each of the three strains targeted in Table 2 confirmed that all had integrated a copy of the mock loxFAS1 integration plasmid at the loxFAS1 chromosomal target (Fig. 3). As expected, diploid strains retained, in addition, one intact copy of the loxFAS1 locus on the untargeted chromosome homologue.

Stimulation of mitotic crossover events

The in vitro results indicated that the natural loxFAS1 site is proficient for a low level of self × self recombination. Such Cre catalyzed site-specific recombination in vivo should be manifest as a hotspot for mitotic recombination, but only under conditions of Cre induction. Figure 4 shows a test of this prediction schematically using leu2/leu2 diploid cells heterozygous at the URA1 and CDC16 loci. Crossover events centromere proximal to the URA1/ura1::LEU2 heteroalleles predict the production of Ura- and Leu- segregants to a maximum of 50% if crossovers occur in every cell. To test Cre's ability to generate such mitotic recombinants an appropriately marked diploid carrying the cre gene under GAL1 control was grown in either glucose- (non-inducing) or galactose-containing media and the generation of auxotrophs was monitored. After 18-24 h of Cre induction (+ galactose) there is a remarkable rise in the incidence of mitotic recombination to a level of almost 20% with equal numbers of Ura- and Leu- segregants being produced (Table 3). These auxotrophs are not generated by a chromosome loss event: such Leu- segregants would necessarily be temperature-sensitive since the URA1 chromosome carries the centromere proximal cdc16 marker, yet none of the auxotrophs obtained was temperature-sensitive. The lack of temperature-sensitive auxotrophs also indicates that the crossover events must have taken place centromere distal to cdc16, consistent with recombination having occurred at the loxFAS1 site. Note that although BSY695 is heterozygous at the ADE1 locus, no Ade- auxotrophs were induced by Cre, indicating that Cre does not result in a general stimulation of mitotic crossing-over. These results clearly show that Cre provokes a dramatic elevation in the incidence of mitotic crossover events on chromosome XI, as expected for site-specific recombination at the loxFAS1 site.


Figure 4. Generation of auxotrophs by Cre-mediated DNA crossover and mitotic segregation. In a diploid leu2/leu2 strain heterozygous for a LEU2 insertion at URA1 (URA1/ura1::LEU2), mitotic recombination at the centromere proximal loxFAS1 site followed by mitotic segregation predicts the induction of Ura- and Leu- segregants. Cre-mediated recombination at the loxFAS1 site is shown occurring just after chromosome XI replication but before centromere (CEN) segregation in a diploid heterozygote. After mitotic crossover, 50% of the centromere segregation events result in equal numbers of Leu- and Ura- singly auxotrophic segregants (as shown) and half regenerate Leu+ Ura+ heterozygous progeny (not shown). This gives the frequency of mitotic recombination as twice the incidence of auxotrophy. Inclusion of the recessive temperature-sensitive cdc16 marker distinguishes mitotic crossover events from chromosome loss events.

Table 3 Cre-mediated mitotic crossover on chromosome XIa
Cre h post-shift Carbon source No. colonies Auxotrophs Leu- Ura+ Leu+ Ura- Leu- Ura- ts auxotrophs % Mitotic crossover
WT 0 Dex 298 0 - - - - 0
18 Dex 344 1 0 1 0 0 0.58
24 Dex 419 0 - - - - 0
18 Gal 377 31 14 17 0 0 16
24 Gal 316 27 12 15 0 0 17
Y324C 0 Dex 485 0 - - - - 0
18 Dex 408 0 - - - - 0
24 Dex 409 0 - - - - 0
18 Gal 425 2 2 0 0 0 0.94
24 Gal 815 0 - - - - 0
aStrain BSY695 contained either pBS126 (wild-type Cre) or pBS394 (CreY324C) as indicated.

The crossover events detected after Cre induction are most likely catalyzed directly by Cre recombinase although the frequency observed is surprisingly high given the low frequency of recombination between loxFAS1 sites in vitro. To rule out the possibility that Cre binding and ensuing synapsis alone are sufficient for increased crossing over, the assay was repeated using a mutant Cre protein (Y324C) specifically lacking Cre catalytic activity due to mutation of the catalytic tyrosine (23). As shown in Table 3, expression of the mutant cre gene did not stimulate mitotic recombination. Thus, Cre catalysis is required for these crossover events at the endogenous loxFAS1 site.

DISCUSSION

I have shown here that a naturally occurring sequence near the FAS1 gene of yeast is proficient for Cre-mediated DNA recombination. By respecifying the core region of a synthetic lox site to match that of the yeast genomic sequence, Cre specifically targets exogenous DNA bearing the synthetic site to that chromosomal locus, and does so efficiently: targeting of the endogenous FAS1 locus with the mock lox vector is only 2-fold less efficient than loxP vector targeting of an authentic loxP site previously engineered into the yeast genome.

How many heterospecific lox sites are proficient for recombination? If the central six positions of the core could accept any base then there would be 46 = 4096 possible sites. However, not all of these sites are proficient for self × self recombination. Hoess et al. (16) showed that mutation of the central TpA dinucleotide in the loxP core to TpG abolishes recombination and proposed that this region is required for unwinding of the lox site during strand exchange. The necessity for a central TpA dinucleotide in loxP suggests that proficient sites require a region within the core that facillitates DNA unwinding. Since the central dinucleotide of the loxFAS1 and the synthetic mock loxFAS1 sites is CpT, these sites must bypass an absolute TpA requirement by a compensating feature in these sites' core region. The trinucleotide TpTpT may play this role in the loxFAS1 site.

One other striking feature of the loxP site is the unusual alternating pattern of purine and pyrimidine bases throughout the entire spacer region. In contrast, both the mock loxFAS1 and the natural loxFAS1 sites contain a 4 nt homopyrimidine stretch in the core region. Although at times functional significance has been ascribed to unusual sequence patterns, the work here clearly indicates that the alternating purine-pyrimidine character of the loxP spacer is not critical for efficient Cre-mediated recombination either in vitro or in vivo.

Use of heterospecific lox sites allows multiplexing of Cre-mediated recombination and thus has important practical implications for the use of Cre in genetically manipulating the genomes of all eukaryotes. Placement of multiple independently acting lox sites into the genome at defined locations by homologous recombination allows subsequent high efficiency Cre-mediated targeting of a transgene construct to different chromosomal locations in the same cell by simply specifying the corresponding lox site on the targeting vector. Such a strategy will be of particular utility in mammalian systems which show considerable variability of transgene expression depending on the site of transgene integration.

Genetic manipulation of endogenous genes in eukaryotes by homologous recombination with an incoming exogenous DNA (gene targeting) necessarily involves the use of a selectable marker gene as only a small percentage of cells incorporate DNA after transfection. In some cases, however, it would be highly desirable to remove that selectable marker. For example there is only a small number of selectable markers available for genetic manipulation and often it would be advantageous to re-use the same marker gene in subsequent rounds of gene targeting. An additional consideration supporting removal of the selectable marker is that the promoter and enhancer elements used to drive expression of the marker gene have the potential to interfere with correct expression of neighboring endogenous genes (30,31). Cre recombinase has become a useful tool for the removal of loxP-flanked selectable marker genes and other unwanted DNA both in yeast (20,21) and in higher eukaryotes (25,32-35), allowing selectable markers to be recycled for subsequent re-use. Multiple rounds of selectable marker removal will, however, leave multiple loxP sites in the genome. Recombination between these chromosomal loxP sites by Cre recombinase will generate complicating and unwanted chromosomal translocations, deletions and/or inversions. A second situation in which this problem can potentially occur is when Cre is used to make a tissue-specific or conditional knockout of two or more genes in the same cell. Certainly in yeast interchromosomal recombination is efficiently catayzed by Cre both between homologues, as shown here, and between heterologous chromosomes (unpublished data), similar to results obtained with the R recombinase of pSR1 (36). Indeed, in Drosophila melanogaster interhomologue FLP-mediated recombination occurs at sufficiently high efficiency that it has been used to routinely generate genetic mosaics (37,38). Although Cre-mediated interchromosomal recombination has not always occurred at high frequency in mouse embryonic stem cells (35,39,40), at least when the sites are very far apart, it is unlikely that lox-tagged adjacent genes would likewise be refractory to efficient Cre-mediated inter-locus recombination. Simply employing non-interacting heterospecific lox sites such as those described here at each chromosomal locus at which a Cre-mediated recombination event is desired would circumvent such potential complications.

Lastly it is likely that, as in yeast, potentially functional lox sequences exist in the much more complex mammalian genome. Such mammalian sites, especially if they occur at a nonessential chromosomal locus, present a unique strategy for Cre-mediated gene targeting of an endogenous chromosomal site using a targeting vector carrying an appropriately designed mock lox sequence. If sufficiently efficient, Cre-mediated targeting of such natural sites in the genome would facillitate the generation of transgenic animals by eliminating position and copy number effects on transgene expression. Efficient Cre-mediated targeting of an endogenous lox-like site in the human genome could lead to alternative strategies in gene therapy that eliminate problems due to random DNA integration by permitting precise stable integration of exogenous DNA at a defined genomic locus.

ACKNOWLEDGEMENTS

I thank R. Wickner for the gift of yeast strain 2132, R. Hoess for providing the mutant creNA2 and M. Brennan for helpful comments on the manuscript.

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