The Prokaryotic β-Recombinase Catalyzes Site-specific Recombination in Mammalian Cells*

The development of new strategies for thein vivo modification of eukaryotic genomes has become an important objective of current research. Site-specific recombination has proven useful, as it allows controlled manipulation of murine, plant, and yeast genomes. Here we provide the first evidence that the prokaryotic site-specific recombinase (β-recombinase), which catalyzes only intramolecular recombination, is active in eukaryotic environments. β-Recombinase, encoded by the β gene of the Gram-positive broad host range plasmid pSM19035, has been functionally expressed in eukaryotic cell lines, demonstrating high avidity for the nuclear compartment and forming a clear speckled pattern when assayed by indirect immunofluorescence. In simian COS-1 cells, transient β-recombinase expression promoted deletion of a DNA fragment lying between two directly oriented specific recognition/crossing over sequences (six sites) located as an extrachromosomal DNA substrate. The same result was obtained in a recombination-dependent lacZ activation system tested in a cell line that stably expresses the β-recombinase protein. In stable NIH/3T3 clones bearing different number of copies of the target sequences integrated at distinct chromosomal locations, transient β-recombinase expression also promoted deletion of the intervening DNA, independently of the insertion position of the target sequences. The utility of this new recombination tool for the manipulation of eukaryotic genomes, used either alone or in combination with the other recombination systems currently in use, is discussed.

Several methods have been developed allowing the manipulation of mammalian genomes in order to elucidate the relevance and function of particular genes of interest. Among them, the development of transgenic mouse strains and gene targeting technologies has been particularly useful (1,2). These techniques have experienced a new advance with the characterization and application of site-specific recombinases (3).
Site-specific recombinases can be clustered into two major families. The Int family comprises those enzymes that catalyze recombination between sites located either in the same DNA molecule (resolution and inversion) or in separate DNA molecules (integration) (4 -7). The latter property has been exploited to allow targeted insertion of specific sequences at precise locations (8,9). The recombinases currently used to manipulate mammalian genomes are mainly the Cre and Flp proteins, both members of the Int family (3). The target sequences for these enzymes, loxP sites for the Cre enzyme and FRT for Flp, consist of a short inverted repeat to which the protein binds. The recombination process is operative through long distances (up to 70 kilobases) in the genome. Using these enzymes, several authors have reported site-and tissue-specific DNA recombination in murine models (10 -13), chromosomal translocations in plants and animals (14 -16), and targeted induction of specific genes (17). For instance, expression of Cre from the lck proximal promoter leads to specific recombination in thymus (10). The gene encoding DNA polymerase ␤ has been tissue-specifically deleted using the same strategy (11). In a different approach, the SV40 tumor antigens have been specifically activated in the lenses of mice, resulting in tumors at that location and not in the rest of the animal (17). The Cre-loxP strategy has also been used in combination with inducible promoters, as in the case of an interferon-responsive promoter that was used to provoke gene ablation in liver with high efficiency and, to a lesser extent, in other tissues (12).
The second family of recombinases includes those enzymes that catalyze recombination only when the sites are located in the same DNA molecule (resolution and/or inversion); they are collectively termed resolvases/invertases (18). ␤-Recombinase, which belongs to this family, catalyzes exclusively intramolecular deletions and inversions of DNA sequences located between two target sites for the recombinase, called six sites (19,20). Each six site comprises 90 bp 1 (see Fig. 1) and is composed of two subsites, termed I and II, to which the recombinase binds (19,21). ␤-Recombinase is encoded by the ␤ gene of the Grampositive broad host range plasmid pSM19035 (22,23).
In this study, we have explored the use of the prokaryotic site-specific ␤-recombinase for the manipulation of mammalian genomes. We describe the cloning and expression in eukaryotic cells of the gene coding for ␤-recombinase and show its ability to catalyze site-specific resolution (deletion) of DNA sequences when the target sequences are either in a plasmid (extrachromosomal target) introduced into the cell by transfection or integrated in the genome as chromatin-associated structures at several locations. The possible applications and potential advantages of this new system, specifically in combination with those already in use, are discussed.

EXPERIMENTAL PROCEDURES
Plasmids and Cloning-Plasmids pBT338 and pCB8, carrying either one or two directly oriented six sites (19), and pLXSN, which carries the resistance marker for neomycin (G418) (24), have been previously described. A eukaryotic expression vector with the SV40 early promoter, pSV2 (25), was kindly provided by Dr. J. Ortín (Centro Nacional de Biotecnología). The expression plasmid pSV␤2 was constructed by PCR amplification of the coding sequence for the ␤ gene from plasmid pBT233 (22). The primers used for PCR were as follows: betaUP, 5Ј-GAGAGAAAGCTTGGTTGGTTGAAAATGGCT-3Ј; and betaDO, 5Ј-GAGAGATGATCAGTACTCATTAACTATCCC-3Ј. These oligonucleotides contain restriction sites for HindIII and BclI, respectively, which were used to clone the amplified gene in the pSV2 vector following standard methods (26). Since BclI is sensitive to methylation, the pSV2 plasmid was isolated from the BZ101 (dam Ϫ ) bacterial strain. The relevant structures are depicted in Fig. 1.
Culture and Cell Lines-Transient expression assays were performed in the simian COS-1 cell line, kindly provided by Dr. J. Ortín. Stable clones with the DNA substrate for ␤-recombinase integrated at different chromatin sites were established in the murine cell line NIH/ 3T3. Both cell lines were cultured in Dulbecco's modified Eagle's medium (Life Technologies, Inc.) supplemented with 10% fetal calf serum (Cultek, Madrid, Spain), 2 mM L-glutamine (Merck, Darmstadt, Germany), and the antibiotics streptomycin (0.1 mg/ml; Sigma) and penicillin (100 units/ml; Sigma).
Transfection Conditions and Plasmid DNA Extraction-The transient expression experiments were performed in COS-1 cells by DEAEdextran transfection as described (26). Cells were harvested 48 h after transfection, and the extrachromosomal DNA was extracted using the method described by Hirt (27). In brief, cell pellets were lysed with SDS (Merck) and treated with proteinase K (Boehringer, Mannheim, Germany) at 37°C. The genomic DNA was precipitated with 1 M NaCl (Merck). Upon centrifugation, the supernatant was phenol-extracted, and plasmid DNA was precipitated with ethanol and resuspended in water for further experiments.
Stable cell clones with pCB8 DNA randomly inserted at different genome sites were obtained by electroporation, in a Bio-Rad Gene Pulser, of 2 ϫ 10 6 NIH/3T3 cells at 220 V and 960 microfarads with 20 g of pCB8 DNA and pLXSN DNA at a 10:1 ratio. Transfected cells were selected with 1 mg/ml G418 (Sigma) for ϳ2 weeks. The stable clones obtained were analyzed in Southern experiments (26) or by immunofluorescence as described below.
Immunoblotting and Immunofluorescence-Rabbit polyclonal antibodies against the purified ␤-recombinase were obtained by conventional techniques (26). ␤-Recombinase was detected by indirect immunofluorescence or by SDS-polyacrylamide gel electrophoresis followed by immunoblotting.
Transfected cells were cultured on coverslips. After 48 h, cells were fixed in methanol/acetone (1:1) at Ϫ20°C for 5 min, air-dried, and rehydrated with phosphate-buffered saline. Cells were then incubated with rabbit polyclonal anti-␤-recombinase antibodies (1:5000 dilution) at room temperature for 30 min, washed three times for 5 min with phosphate-buffered saline, and reincubated with a fluorescein-conjugated anti-rabbit IgM antibody (Dako, Glostrup, Denmark) for 1 h at 37°C in phosphate-buffered saline. The cells were mounted on microscope slides and photographed in a fluorescence microscope.
For immunoblotting analysis, transiently transfected cells were harvested 48 h after transfection and lysed in radioimmune precipitation assay buffer (137 mM NaCl, 20 mM Tris-HCl (pH 8), 1 mM MgCl 2 , 1 mM CaCl 2 , 10% glycerol, 1% Nonidet P-40, 0.5% sodium deoxycholate, and 0.1% SDS; Merck). The lysed fraction was separated on SDS-polyacrylamide gel; blotted onto nitrocellulose membrane (Bio-Rad); and incubated with polyclonal anti-␤-recombinase antibodies, previously blocked with COS-1 total cell lysate (1:500 dilution). Peroxidase-conjugated anti-IgM antibody (Dako) was used as secondary antibody. Membranes were processed using the ECL chemiluminescence detection kit (Amersham Pharmacia Biotech, Aylesbury, United Kingdom). Subcellular fractionation was performed by detergent lysis of transiently transfected cells essentially as follows. 48 h after transfection, cells were trypsinized, washed twice with phosphate-buffered saline, and harvested by centrifugation. Each cell pellet was resuspended in TM-2 buffer (10 mM Tris-HCl (pH 7.4), 2 mM MgCl 2 , and 0.5 mM phenylmethylsulfonyl fluoride) and incubated on ice for 5 min. Then, Triton X-100 was added to each pellet to a final concentration of 0.5% and incubated on ice for 5 min. Cells were sheared by gentle pipetting, monitoring the appearance of free nuclei in a phase-contrast microscope. When essentially all nuclei were free of cytoplasmic tags, they were collected by centrifugation. The proteins of the cytoplasmic fraction were stored frozen for further Western analysis. The nuclei were washed twice with TM-2 buffer, and the proteins were extracted as described before. ␤-Recombinase detection was performed on Western blots as described. A monoclonal anti-␤-actin antibody (Sigma) was used as a marker for cytoplasmic fraction detection. Alternatively, the presence of nuclear fraction proteins was monitored with a monoclonal anti-histone antibody (Chemicon International, Inc., Temecula, CA). Peroxidase-conjugated anti-mouse IgM antibody (Dako) was used as secondary antibody for both purposes.
Analysis of Recombination Products-PCR was performed with the GeneAmp PCR System 2400 from Perkin-Elmer equipped with a heating cover. Each reaction was carried out using 0.5 g of genomic DNA or one-tenth of the Hirt preparation according to the supplier's instructions. Taq polymerase (2.5 units; Perkin-Elmer) was added with Perfect Match PCR Enhancer (Stratagene, La Jolla, CA) after an initial denaturation (94°C, 10 min). The procedure (Touch-Down) was thereafter performed as follows: 80°C for 2 min, five cycles of denaturation (94°C, 1 min) and annealing/extension (72°C, 2 min), and five cycles of 1 min at 94°C and 2 min at 70°C. This was coupled to 25 cycles of denaturation (94°C, 1 min), annealing (68°C, 30 s), and extension (72°C, 2 min) and one additional extension step at 72°C for 5 min. For the PCR analysis of the Hirt preparations, we used the 16-mer reverse sequencing primer (No. 1201) and the 17-mer universal sequencing primer (No. 1211) from New England Biolabs Inc. (Beverly, MA). These primers are hereafter referred to as a and b, respectively.
The primers used for PCR amplification of the Hirt preparations were unsuitable for the analysis of genomic DNA preparations (low T m ). A new pair of primers was thus designed: pBT338UP158, 5Ј-CCG-GCTCGTATGTTGTGTGGAAT-3Ј; and pBT338DO802, 5Ј-TGGCGAA-AGGGGGATGTGCTG-3Ј. These primers are hereafter referred to as aЈ and bЈ, respectively.
Southern analysis of the PCR products was performed by blotting the DNA separated on agarose gels onto nylon membranes (Amersham Pharmacia Biotech). Filters were hybridized at 42°C in 250 mM phosphate buffer (pH 7.2), 50% formamide, 250 mM NaCl, 1 mM EDTA, and 7% SDS and washed in 1ϫ SSC and 0.1% SDS at room temperature for 30 min, at least twice. The washing temperature was increased when needed. The radioactive labeling of probes was performed with the Prime-It random primer labeling kit (Stratagene). Nucleotide sequences from the PCR bands of interest were determined by automated fluorescent sequencing and were analyzed using Seq-Ed 1.0.3 software (Applied Biosystems Inc.).
Recombination-activated Gene Expression-To obtain further evidence of recombination due to ␤-recombinase, a recombination-dependent gene expression system was constructed, as depicted in Fig. 5, for the reporter gene lacZ. For analysis of ␤-galactosidase expression, plasmids were transiently transfected in a cell line constitutively expressing ␤-recombinase activity, 2 and 48 h after transfection, the proteins were extracted according to the protocol from Luminescent ␤-galactosidase detection kit II (CLONTECH). lacZ gene expression was measured in a scintillation counter for each condition.

Expression of the Prokaryotic ␤-Recombinase in Mammalian
Cells-The coding sequence for ␤-recombinase was cloned in the pSV2 vector under the control of the SV40 early promoter. A control plasmid that does not contain the ␤-gene was also generated during this process (pSVc). The resulting constructs, pSV␤2 (Fig. 1B) and pSVc, respectively, were transiently transfected in COS-1 cells, which express SV40 T-antigen. Under these conditions, the expression from plasmids that contain the SV40 early promoter (included in pSV␤2) is amplified.
Transiently transfected cells were stained with rabbit polyclonal anti-␤-recombinase antibodies. Fluorescence microscopy of the pSV␤2-transfected cells showed a strong speckled signal located specifically in the cell nucleus (Fig. 2, D and E). However, very faint staining was detected in the mock and control transfections (Fig. 2, A and B, respectively) as well as in pSV␤2-transfected cells incubated first with preimmune rabbit serum instead of the polyclonal anti-␤-recombinase antibodies ( Fig. 2C). Similar results were obtained when expression was tested by immunoblotting (Fig. 2F). A specific 25-kDa band, with a mobility corresponding to that of purified ␤-protein (Fig.  2F, c lane), was developed by the anti-␤-recombinase antibodies when COS-1 cells were transfected with the pSV␤2 plasmid (ϩ lane), but not in the mock-transfected cells (Ϫ lane). Definitive evidence for the preferential nuclear location of ␤-recombinase was provided by subcellular fractionation experiments. As shown in Fig. 3, the specific band corresponding to ␤-recombinase appeared only on the nuclear enriched fraction of the pSV␤2-transfected cells.
These results indicate that ␤-recombinase can be expressed in eukaryotic environments, showing strong avidity for the nuclear compartment. Additional experiments with stable ␤-recombinase-expressing clones showed the same cellular distribution, without affecting cellular viability. 3 ␤-Recombinase Catalyzes Site-specific Recombination in Transiently Transfected Mammalian Cells-Unlike integrases with simple recombination sites, such as Cre and Flp, which catalyze inter-and intramolecular recombination and do not require additional protein factors (4, 5, 7), ␤-recombinase catalyzes intramolecular recombination and has a strict requirement for a chromatin-associated protein to mediate DNA recombination (19,20). ␤-Recombinase binds to the six sites and, with the help of a chromatin-associated protein, promotes strand exchange (Fig. 1A). The accessory factor is a chromatinassociated protein such as prokaryotic HU or eukaryotic HMG1 protein (20,28,29).
To determine whether eukaryotic cells could provide this host factor, recombination activity due to ␤-recombinase was first tested by transient cotransfections in COS-1 cells with plasmids pSV␤2 (bearing the ␤-recombinase gene) and pCB8 (the substrate DNA containing two target sites for ␤-recombinase in direct orientation flanking the xylE gene; see Fig. 1B). Upon recombination, two derivatives of pCB8, with a single six site each, should be obtained. The presence of one of these recombination products can be easily monitored by PCR amplification of Hirt extracts using primers complementary to the sequences located upstream of one of the six sites (primer a in Fig. 1B and under "Experimental Procedures") and downstream of the second six site (primer b in Fig. 1B and under "Experimental Procedures"). In pCB8, these two primers hybridize to sequences located Ͼ2.7 kilobases apart. Under our PCR conditions, this fragment was not efficiently amplified; nevertheless, a 555-bp DNA segment should be amplified from the recombination product. A band of similar length should be obtained when using the same primers and plasmid pBT338 as template, which contains a single six site and was used as positive control (Fig. 1B). After transfection of the COS-1 cells (48 h), the extrachromosomal fraction (Hirt extraction) of the cells was therefore purified, and the presence of recombination products was analyzed by PCR. An amplified band of the expected length (555 bp) was observed only when both pCB8 and pSV␤2 plasmids were cotransfected (Fig. 4); this band was absent when the two DNAs were transfected separately or when pCB8 was cotransfected with pSVc, the negative control plasmid. The specificity of the amplified band was further confirmed by Southern hybridization (Fig. 4, lower panel) with a probe specific for the six site (see Fig. 1A). A positive signal of the correct size was detected only in the positive control lane (Fig. 4, pBT338 (ϩ))and in the pCB8/pSV␤2 cotransfection sample. In lanes corresponding to transfections containing the pSV␤2 plasmid, the additional band of smaller size detected on the agarose gel was demonstrated to be nonspecific, as it did not hybridize to the probe containing the six site sequence. These results indicate that ␤-recombinase is active in a eukaryotic environment, using the machinery/factors provided by the host cell.
To provide further experimental evidence of the ␤-recombinase-mediated process in eukaryotic cells, a new set of vectors was constructed for recombination-activated gene expression (Fig. 5A). The assay vector consisted of the lacZ gene separated from the SV40 early promoter by the pac gene (which confers resistance to puromycin in bacteria and eukaryotic cells) flanked by two six sites in direct orientation. Upon recombination, the pac gene should be excised from the plasmid, leaving the lacZ gene under the control of the SV40 promoter, thus rendering expression of ␤-galactosidase activity. This reporter gene can easily be monitored and quantified in cell extracts. The negative control (plasmid pPursixgal) lacks the first six site and is not a suitable substrate for recombination. A positive control (plasmid pgal) was obtained by in vitro recombination (19) of the Recombiner plasmid using purified ␤-recombinase and further isolation and characterization.
Upon transfection of these plasmids in a stable ␤-recombinase-expressing cell line, the whole protein fraction was extracted from each condition and assayed for ␤-galactosidase activity. As shown in Fig. 5B, transfection of the Recombiner construct promoted ␤-galactosidase expression several orders of magnitude higher than the mock and pPursixgal transfec- tions, indicating that recombination had occurred on that substrate. Equivalent transfection experiments on the parental cell line not expressing ␤-recombinase rendered no detectable ␤-galactosidase activity, demonstrating ␤-recombinase dependence of the measured activity.
However, the ␤-galactosidase activity induced by transfection of the Recombiner construct was not in the same range as the one obtained with the positive control (pgal transfection). One plausible reason for this result could be that recombination occurs in a time period close to that used in the experimental conditions. Since pgal is already recombined, expression of ␤-galactosidase from this plasmid occurs early after transfection. This is not the case of Recombiner, which has to become recombined prior to lacZ gene expression. As a result, the number of cells with recombined plasmid is less in Recombiner transfection 48 h later than in pgal transfection, and therefore, ␤-galactosidase accumulation is reduced.
␤-Recombinase Promotes Recombination in Structured Chromatin-The need of supercoiled DNA has been described as a critical condition for ␤-recombinase-mediated deletions between two directly oriented six sites (20). To explore whether ␤-recombinase can promote DNA rearrangement when two six sites form part of the chromatin structure, we established NIH/3T3 cell clones in which the pCB8 construct was integrated at different locations within the mammalian genome. Several stable clones were analyzed by Southern hybridization.
Five of them, each carrying a different copy number of the substrate plasmid (5-75) (data not shown), were chosen for transient transfection with the ␤-recombinase expression plasmid pSV␤2. The presence of recombination products was determined by PCR of genomic DNA preparations using two primers (pBT338UP158 and pBT338LO802 (see "Experimental Procedures"), termed primers aЈ and bЈ, respectively, in Fig.  1B), which should generate a 668-bp amplified DNA fragment. Amplified DNA fragments in high copy number clones could be seen directly on agarose gels (data not shown). In Southern blot assays performed with a probe specific for the six site, however, a band (ϳ660 bp) was detected in all cases in the pSV␤2transfected samples (Fig. 6, ϩ lanes). This DNA fragment did not appear when plasmid pSV␤2 was not included in the transfection (mock transfection; Ϫ lanes). Signal strength appeared to correlate with the copy number of the target construction integrated in the chromosome, suggesting that recombination had occurred at many of the integrated target sequences and regardless of the integration site. Control PCR experiments in mock-transfected NIH/3T3 cells or NIH/3T3 cells transfected with the pSV␤2 plasmid were carried out routinely, and no amplified band of 660 bp was detected (Fig. 6, lanes c and d).
The fidelity of the recombination mechanism was also confirmed by DNA sequencing of the amplified bands in the case of clones 1 and 2 (data not shown). The regenerated six site (see Fig. 1A) obtained after recombination was unaltered. These data indicate that ␤-recombinase can catalyze site-specific recombination in mammalian genomes. It therefore seems that the chromatin structure provides superhelical torsion suitable for ␤-recombinase-mediated recombination. DISCUSSION The common genome manipulation techniques, including transgenesis and gene targeting, have opened a new path for the understanding of a wide variety of mechanisms involving diverse genetic functions. The utility of these systems becomes limited, however, when the overexpression or inactivation of a given gene has fatal effects on embryo development (as an example, see Refs. 11 and 30) or when the lack of gene function can be bypassed or compensated by redundant mechanisms (31,32). Moreover, the effects of gene inactivation outside the tissue or cell lineage of interest are usually unknown and uncontrollable (33). These problems have been overcome to some extent by the development and application of the site-specific recombination techniques (reviewed in Ref. 7) that allow spatiotemporal control of the targeting event. This is the case of the Cre-loxP and Flp-FRT systems (reviewed in Refs. 3 and 4).
We show that the prokaryotic ␤-recombinase, which belongs to the resolvase/invertase family of enzymes, can be functionally expressed in eukaryotic cells and can promote the deletion of DNA sequences located between directly oriented target sites in mammalian cells. ␤-Recombinase appears to have high avidity for the nuclear compartment since, following transfection, it was detected mainly in the nuclear region, forming a very condensed and speckled pattern on indirect immunofluorescence. This point was reassessed in subcellular fractionation experiments (see "Results" and Fig. 3). This behavior is similar to that observed for the Cre enzyme (13). Cre and ␤-recombinase do not present a canonical or bipartite nuclear localization motif in their primary sequence (34,35). Since they have access to the nuclear compartment, it is assumed that this localization occurs by diffusion through the nuclear membrane or following the transient disorganization of this membrane during mitosis.
Transient ␤-recombinase expression by plasmid pSV␤2 promoted site-specific recombination between the two directly oriented six sites in the substrate plasmid pCB8 when both plas-FIG. 5. Recombination-activated gene expression mediated by ␤-recombinase activity. A shows the schematic structure of the plasmids used to transfect a cell line constitutively expressing ␤-recombinase. The six sites (triangles) and the genes pac and lacZ (rectangles) are indicated. Each transfection was performed in triplicate. After 48 h, the proteins were extracted, and ␤-galactosidase activity was measured as described under "Experimental Procedures." B shows the representation of the mean cpm Ϯ S.D. from each triplicate condition. mids were cotransfected in mammalian cells. As a result, the sequences between the two target sites were deleted from the DNA substrate. The site-specific recombination product was detected by PCR amplification of the Hirt extracts and reassessed by Southern hybridization of the amplified products. The presence of this recombination product was strictly dependent on the cotransfection of plasmids pSV␤2 and pCB8; no recombination products were observed when plasmids pSV␤2 and pCB8 were transfected separately. It therefore seems that ␤-recombinase can promote strand exchange of an extrachromosomal DNA (pCB8 DNA) in the mammalian environment, with no detectable spontaneous recombination. Similar results were obtained in recombination-activated ␤-galactosidase expression experiments. This reporter gene was designed to be expressed only upon recombination due to ␤-recombinase (plasmid Recombiner; see Fig. 5). As expected, high expression of ␤-galactosidase was obtained compared with the negative controls. This experiment not only provides additional evidence of recombination due to ␤-recombinase in mammalian cells independent of PCR detection, but also confirms the possibility of designing experiments to activate the expression of genes of interest with an analogue approach.
Since in vitro recombination requires a chromatin-associated protein (28), we assume that this factor is provided by the host. Indeed, it is known that the mammalian HMG1 chromatinassociated protein can efficiently stimulate in vitro ␤-mediated recombination (20,28). It has recently been observed that chromatin-associated proteins from plants can also assist ␤-recombinase in mediating DNA recombination (29), suggesting that ␤-recombinase might be also suitable for manipulation of plant genomes.
We have also studied the ability of ␤-recombinase to act on chromatin-integrated target substrates. Several stable NIH/ 3T3 clones were established bearing different copy numbers (5-75) of the substrate plasmid pCB8 randomly integrated in the host chromatin. Transient ␤-recombinase expression led to the excision of the sequences between the two directly oriented six sites; the recombination product was detected by PCR amplification from purified genomic DNA and Southern hybridization, and its identity was confirmed by direct DNA sequencing of the amplified product (data not shown).
We provide the first evidence, in eukaryotic cells, for the activity of a DNA recombinase belonging to the prokaryotic resolvase/invertase family. Enzymes of this family promote DNA recombination through a mechanism different from that of DNA integrases. Integrases such as Cre or Flp promote intramolecular as well as intermolecular recombination, whereas recombinases of the resolvase/invertase family are highly specialized in intramolecular recombination. If confirmed in animal models, the availability of a tool such as ␤-recombinase will expand the possibilities for the programmed modification of eukaryotic genomes currently under use. ␤-Recombinase, used alone or in combination with the already existing recombination systems, will allow a more specific spatiotemporal control of the recombination events. Researchers would have the opportunity to design several independently controlled recombination events in the same animal or cell, thus providing new, more flexible solutions to general research. In this respect, different approaches to assess whether all these recombination systems can work simultaneously will be of great interest for further investigations.