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

Vicente DíazDagger §, Fernando Rojo, Carlos Martínez-ADagger , Juan C. Alonso, and Antonio BernadDagger parallel

From the Dagger  Departamento de Inmunología y Oncología and the  Departamento de Biotecnología Microbiana, Centro Nacional de Biotecnología, Consejo Superior de Investigaciones Científicas, Campus de la Universidad Autónoma de Madrid, Cantoblanco, E-28049 Madrid, Spain

    ABSTRACT
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Abstract
Introduction
References

The development of new strategies for the in 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 (beta -recombinase), which catalyzes only intramolecular recombination, is active in eukaryotic environments. beta -Recombinase, encoded by the beta  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 beta -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 beta -recombinase protein. In stable NIH/3T3 clones bearing different number of copies of the target sequences integrated at distinct chromosomal locations, transient beta -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.

    INTRODUCTION
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Abstract
Introduction
References

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 beta  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). beta -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 bp1 (see Fig. 1) and is composed of two subsites, termed I and II, to which the recombinase binds (19, 21). beta -Recombinase is encoded by the beta  gene of the Gram-positive broad host range plasmid pSM19035 (22, 23).

In this study, we have explored the use of the prokaryotic site-specific beta -recombinase for the manipulation of mammalian genomes. We describe the cloning and expression in eukaryotic cells of the gene coding for beta -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 pSVbeta 2 was constructed by PCR amplification of the coding sequence for the beta  gene from plasmid pBT233 (22). The primers used for PCR were as follows: betaUP, 5'-GAGAGAAAGCTTGGTTGGTTGAAAATGGCT-3'; and betaDO, 5'-GAGAGATGATCAGTACTCATTAACTATCCC-3'. 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-) 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 beta -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 DEAE-dextran 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 × 106 NIH/3T3 cells at 220 V and 960 microfarads with 20 µg of pCB8 DNA and pLXSN DNA at a 10:1 ratio. Transfected cells were selected with 1 mg/ml G418 (Sigma) for ~2 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 beta -recombinase were obtained by conventional techniques (26). beta -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 -20 °C for 5 min, air-dried, and rehydrated with phosphate-buffered saline. Cells were then incubated with rabbit polyclonal anti-beta -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 MgCl2, 1 mM CaCl2, 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-beta -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 MgCl2, 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. beta -Recombinase detection was performed on Western blots as described. A monoclonal anti-beta -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 µg 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 Tm). A new pair of primers was thus designed: pBT338UP158, 5'-CCGGCTCGTATGTTGTGTGGAAT-3'; and pBT338DO802, 5'-TGGCGAAAGGGGGATGTGCTG-3'. These primers are hereafter referred to as a' and b', 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 1× 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 beta -recombinase, a recombination-dependent gene expression system was constructed, as depicted in Fig. 5, for the reporter gene lacZ. For analysis of beta -galactosidase expression, plasmids were transiently transfected in a cell line constitutively expressing beta -recombinase activity,2 and 48 h after transfection, the proteins were extracted according to the protocol from Luminescent beta -galactosidase detection kit II (CLONTECH). lacZ gene expression was measured in a scintillation counter for each condition.

    RESULTS

Expression of the Prokaryotic beta -Recombinase in Mammalian Cells-- The coding sequence for beta -recombinase was cloned in the pSV2 vector under the control of the SV40 early promoter. A control plasmid that does not contain the beta -gene was also generated during this process (pSVc). The resulting constructs, pSVbeta 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 pSVbeta 2) is amplified.


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Fig. 1.   Schematic representation of the site-specific recombination mechanism mediated by beta -recombinase and the plasmids used in this study. A, beta -recombinase (shown as a dimer; open circles) interacts, in the presence of a host factor (HMG1, shown as a monomer; open polygons), with two identical copies of the six site (shaded and open arrows) to form a synaptic complex. If the reaction occurs within the host genome, this complex is resolved, giving rise to two recombination products. One of those, a circular intermediate harboring the intervening sequences, would be lost. The other, containing one full six site, would remain intact in the host cell. B, the essential features of plasmid pSVbeta 2 and pCB8 and pBT338 DNAs are indicated. The six site is shown schematically, and sites I and II are highlighted. Orientation of the six sites is denoted by the direction of the shaded arrows. The coding region of the xylE gene, initially designed for expression in bacteria, is indicated. Also indicated are the hybridization sites of the specific primer pairs a/b and a'/b' used for PCR amplification to detect the recombination products and of the probe used for specific hybridization controls.

Transiently transfected cells were stained with rabbit polyclonal anti-beta -recombinase antibodies. Fluorescence microscopy of the pSVbeta 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 pSVbeta 2-transfected cells incubated first with preimmune rabbit serum instead of the polyclonal anti-beta -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 beta -protein (Fig. 2F, c lane), was developed by the anti-beta -recombinase antibodies when COS-1 cells were transfected with the pSVbeta 2 plasmid (+ lane), but not in the mock-transfected cells (- lane). Definitive evidence for the preferential nuclear location of beta -recombinase was provided by subcellular fractionation experiments. As shown in Fig. 3, the specific band corresponding to beta -recombinase appeared only on the nuclear enriched fraction of the pSVbeta 2-transfected cells.


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Fig. 2.   beta -Recombinase expression in mammalian cells detected by indirect immunofluorescence and Western blotting. COS-1 cells, transfected with the indicated plasmids, were cultured on glass coverslips. After 48 h, cells were fixed and stained as indicated. A and B correspond to mock- and pSVc-transfected cells, respectively, incubated with polyclonal anti-beta -recombinase antibodies and developed with fluorescein-conjugated anti-rabbit IgG. C-E show pSVbeta 2-transfected cells. In C, the pSVbeta 2-transfected cells were incubated first with preimmune rabbit serum and then developed as described above. In D and E, showing two different fields of the pSVbeta 2-transfected cells, they were incubated with polyclonal anti-beta -recombinase antibodies and developed as described above. In F, COS-1 cells, transfected as indicated above, were harvested after 48 h and lysed as described under "Experimental Procedures." The whole cell extract proteins were resolved by SDS-polyacrylamide gel electrophoresis, blotted onto nitrocellulose, and analyzed by Western blotting. Shown is the autoradiograph of a Western blot incubated first with polyclonal anti-beta -recombinase antibodies and then developed with peroxidase-conjugated anti-IgM. - lane, mock-transfected COS-1 cells; + lane, pSVbeta 2-transfected COS-1 cells; c lane, puriied beta -recombinase (10 ng). The molecular mass of the band of interest is indicated.


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Fig. 3.   Nuclear localization of beta -recombinase detected by subcellular fractionation of nuclei and cytoplasm. COS-1 cells were transfected with the indicated constructs. After 48 h, cells were harvested and lysed as described under "Experimental Procedures" to prepare the nuclear (N) and cytoplasmic (C) fractions. The proteins from each fraction/condition were resolved by 10% SDS-polyacrylamide gel electrophoresis (15 µg/lane) and blotted. Shown are three autoradiographs of the Western blots corresponding to anti-beta -recombinase, anti-beta -actin, and anti-histones primary antibodies, incubated separately using the same membrane (stripped after each blotting) and developed with the appropriate peroxidase-conjugated anti-IgM antibodies. Lane 1, no DNA; lane 2, transfection with the pSVc plasmid; lane 3, transfection with pSVbeta 2; + lane, purified beta -recombinase protein (10 ng). The sizes of the bands of interest are indicated.

These results indicate that beta -recombinase can be expressed in eukaryotic environments, showing strong avidity for the nuclear compartment. Additional experiments with stable beta -recombinase-expressing clones showed the same cellular distribution, without affecting cellular viability.3

beta -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), beta -recombinase catalyzes intramolecular recombination and has a strict requirement for a chromatin-associated protein to mediate DNA recombination (19, 20). beta -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 chromatin-associated 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 beta -recombinase was first tested by transient cotransfections in COS-1 cells with plasmids pSVbeta 2 (bearing the beta -recombinase gene) and pCB8 (the substrate DNA containing two target sites for beta -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 >2.7 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 pSVbeta 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 (+))and in the pCB8/pSVbeta 2 cotransfection sample. In lanes corresponding to transfections containing the pSVbeta 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 beta -recombinase is active in a eukaryotic environment, using the machinery/factors provided by the host cell.


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Fig. 4.   Functional expression of beta -recombinase assessed in transient transfection experiments. COS-1 cells were transfected with the indicated combinations of plasmids: pSVc, pSVbeta 2, pCB8, pSVc + pCB8, and pSVbeta 2 + pCB8. Hirt extracts were obtained 48 h after transfection. The upper panel shows the result of PCR amplification from the Hirt extracts using the a/b primer pair indicated in Fig. 1B. The mock lane shows the negative controls of the PCR amplification, and the pBT338 (+) lane shows the positive control (100 pg of plasmid). The size marker lane (kilobases) corresponds to BstEII-digested lambda  DNA (500 ng). The lower panel shows the Southern blot analysis of the agarose gel presented in the upper panel using a probe specific for the six site (see Fig. 1B). The position of the band of interest is highlighted. kb, kilobases.

To provide further experimental evidence of the beta -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 beta -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 beta -recombinase and further isolation and characterization.


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Fig. 5.   Recombination-activated gene expression mediated by beta -recombinase activity. A shows the schematic structure of the plasmids used to transfect a cell line constitutively expressing beta -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 beta -galactosidase activity was measured as described under "Experimental Procedures." B shows the representation of the mean cpm ± S.D. from each triplicate condition.

Upon transfection of these plasmids in a stable beta -recombinase-expressing cell line, the whole protein fraction was extracted from each condition and assayed for beta -galactosidase activity. As shown in Fig. 5B, transfection of the Recombiner construct promoted beta -galactosidase expression several orders of magnitude higher than the mock and pPursixgal transfections, indicating that recombination had occurred on that substrate. Equivalent transfection experiments on the parental cell line not expressing beta -recombinase rendered no detectable beta -galactosidase activity, demonstrating beta -recombinase dependence of the measured activity.

However, the beta -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 beta -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, beta -galactosidase accumulation is reduced.

beta -Recombinase Promotes Recombination in Structured Chromatin-- The need of supercoiled DNA has been described as a critical condition for beta -recombinase-mediated deletions between two directly oriented six sites (20). To explore whether beta -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 beta -recombinase expression plasmid pSVbeta 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 a' and b', 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 (~660 bp) was detected in all cases in the pSVbeta 2-transfected samples (Fig. 6, + lanes). This DNA fragment did not appear when plasmid pSVbeta 2 was not included in the transfection (mock transfection; - 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 pSVbeta 2 plasmid were carried out routinely, and no amplified band of 660 bp was detected (Fig. 6, lanes c and d).


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Fig. 6.   beta -Recombinase-mediated site-specific recombination in chromatin-associated targets. Five NIH/3T3 clones (lanes 1-5) harboring several copies (~75, 72, 12, 18, and 5, respectively) of pCB8 DNA integrated in different chromosomal locations were isolated and characterized. These clones were transiently transfected, either with (+ lanes) or without (- lanes) plasmid pSVbeta 2. Genomic DNA was purified 48 h after transfection, and 500 ng of each sample were analyzed by PCR under the conditions described using the a'/b' primer pair (see Fig. 1B). One-tenth of each sample was electrophoresed on 0.8% agarose gel, blotted onto nylon membrane, and hybridized with an appropriate probe. C +,positive controls pBT338 (100 pg) (lane a) and pBT338 (100 pg) + 500 ng of NIH/3T3 genomic DNA (lane b); C -, negative controls of NIH/3T3 cells without integrated pCB8 DNA transfected with no DNA (lane c) and pSVbeta 2 (lane d). The size (0.66 kilobases) and position of the band of interest are indicated.

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 beta -recombinase can catalyze site-specific recombination in mammalian genomes. It therefore seems that the chromatin structure provides superhelical torsion suitable for beta -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 beta -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. beta -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 beta -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 beta -recombinase expression by plasmid pSVbeta 2 promoted site-specific recombination between the two directly oriented six sites in the substrate plasmid pCB8 when both plasmids 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 pSVbeta 2 and pCB8; no recombination products were observed when plasmids pSVbeta 2 and pCB8 were transfected separately. It therefore seems that beta -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 beta -galactosidase expression experiments. This reporter gene was designed to be expressed only upon recombination due to beta -recombinase (plasmid Recombiner; see Fig. 5). As expected, high expression of beta -galactosidase was obtained compared with the negative controls. This experiment not only provides additional evidence of recombination due to beta -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 chromatin-associated protein can efficiently stimulate in vitro beta -mediated recombination (20, 28). It has recently been observed that chromatin-associated proteins from plants can also assist beta -recombinase in mediating DNA recombination (29), suggesting that beta -recombinase might be also suitable for manipulation of plant genomes.

We have also studied the ability of beta -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 beta -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 beta -recombinase will expand the possibilities for the programmed modification of eukaryotic genomes currently under use. beta -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.

    ACKNOWLEDGEMENTS

We thank Drs. Juan Ortín and Inés Canosa for providing strains and plasmids and Drs. Juan Ortín and Miguel Torres for critical reading of this manuscript. Automated fluorescent sequencing was done by A. Varas and M. A. Gallardo. We also thank Coral Bastos and Catherine Mark for secretarial and editorial support, respectively.

    FOOTNOTES

* This work was supported in part by Grant 08.6/0021/1997 from the Comunidad Autónoma de Madrid, Grant SAF95-1548-CO2-02 from the Comisión Interministerial de Ciencia y Tecnología, and European Project RE:BIO4-CT95-0284 (to A. B.) and by Grant PB96-0817 from the Comisión Interministerial de Ciencia y Tecnología (to J. C. A.). The Department of Immunology and Oncology was founded and is supported by the Consejo Superior de Investigaciones Científicas and Pharmacia & Upjohn.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§ Supported by a fellowship from the Ministerio de Educación y Ciencia.

parallel To whom correspondence should be addressed. Tel.: 34-91-585-4562; Fax: 34-91-372-0493; E-mail: abernad{at}cnb.uam.es.

2 V. Díaz, F. Rojo, C. Martínez-A., J. C. Alonso, and A. Bernad, manuscript in preparation.

3 V. Díaz, F. Rojo, C. Martínez-A., J. C. Alonso, and A. Bernad, unpublished results.

    ABBREVIATIONS

The abbreviations used are: bp, base pair(s); PCR, polymerase chain reaction.

    REFERENCES
Top
Abstract
Introduction
References
  1. Brandon, E. P., Idzerda, R. L., and McKnight, G. S. (1995) Curr. Biol. 5, 625-634[Medline] [Order article via Infotrieve]
  2. Brandon, E. P., Idzerda, R. L., and McKnight, G. S. (1995) Curr. Biol. 5, 758-765[Medline] [Order article via Infotrieve]
  3. Kilby, N. J., Snaith, M. R., and Murray, J. A. (1993) Trends Genet. 9, 413-421[CrossRef][Medline] [Order article via Infotrieve]
  4. Sauer, B. (1993) Methods Enzymol. 225, 890-900[Medline] [Order article via Infotrieve]
  5. Dymecki, S. M. (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 6191-6196[Abstract/Free Full Text]
  6. Abremski, K., and Hoess, R. (1984) J. Biol. Chem. 259, 1509-1514[Abstract/Free Full Text]
  7. Nash, H. A. (1996) in Escherichia coli and Salmonella Cellular and Molecular Biology (Neidhart, F. C., Curtis, R. I., Ingraham, J. L., Lin, E. C. C., Low, K. B., Magasanik, B., Reznikoff, W. S., Riley, M., Schaechter, M., and Umbager, H. E., eds), pp. 2363-2376, American Society for Microbiology, Washington, D. C.
  8. Sauer, B., and Henderson, N. (1990) New Biol. 2, 441-449[Medline] [Order article via Infotrieve]
  9. Fukushige, S., and Sauer, B. (1992) Proc. Natl. Acad. Sci. U. S. A. 89, 7905-7909[Abstract]
  10. DiSanto, J. P., Muller, W., Guy, G. D., Fischer, A., and Rajewsky, K. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 377-381[Abstract]
  11. Gu, H., Marth, J. D., Orban, P. C., Mossmann, H., and Rajewsky, K. (1994) Science 265, 103-106[Medline] [Order article via Infotrieve]
  12. Kuhn, R., Schwenk, F., Aguet, M., and Rajewsky, K. (1995) Science 269, 1427-1429[Medline] [Order article via Infotrieve]
  13. Orban, P. C., Chui, D., and Marth, J. D. (1992) Proc. Natl. Acad. Sci. U. S. A. 89, 6861-6865[Abstract]
  14. van Deursen, J., Fornerod, M., van Rees, B., and Grosveld, G. (1995) Proc. Natl. Acad. Sci. U. S. A 92, 7376-7380[Abstract]
  15. Medberry, S. L., Dale, E., Qin, M., and Ow, D. W. (1995) Nucleic Acids Res. 23, 485-490[Abstract]
  16. Osborne, B. I., Wirtz, U., and Baker, B. (1995) Plant J. 7, 687-701[CrossRef][Medline] [Order article via Infotrieve]
  17. Pichel, J. G., Lakso, M., and Westphal, H. (1993) Oncogene 8, 3333-3342[Medline] [Order article via Infotrieve]
  18. Grindley, N. D. F. (1994) Nucleic Acids Mol. Biol. 8, 236-267
  19. Rojo, F., and Alonso, J. C. (1994) J. Mol. Biol. 238, 159-172[CrossRef][Medline] [Order article via Infotrieve]
  20. Alonso, J. C., Weise, F., and Rojo, F. (1995) J. Biol. Chem. 270, 2938-2945[Abstract/Free Full Text]
  21. Rojo, F., and Alonso, J. C. (1995) Nucleic Acids Res. 23, 3181-3188[Abstract]
  22. Ceglowski, P., Boitsov, A., Chai, S., and Alonso, J. C. (1993) Gene (Amst.) 136, 1-12[CrossRef][Medline] [Order article via Infotrieve]
  23. Rojo, F., Weise, F., and Alonso, J. C. (1993) FEBS Lett. 328, 169-173[CrossRef][Medline] [Order article via Infotrieve]
  24. Miller, A. D., and Rosman, G. J. (1989) BioTechniques 7, 980-982[Medline] [Order article via Infotrieve], 984-986, 989-990
  25. Mulligan, R. C., Howard, B. H., and Berg, P. (1979) Nature 277, 108-114[Medline] [Order article via Infotrieve]
  26. Maniatis, T., Fritsch, E. F., and Sambrook, J. (1982) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY
  27. Hirt, B. (1967) J. Mol. Biol. 26, 365-369[Medline] [Order article via Infotrieve]
  28. Alonso, J. C., Gutiérrez, C., and Rojo, F. (1995) Mol. Microbiol. 18, 471-478[CrossRef][Medline] [Order article via Infotrieve]
  29. Grasser, K. D., Ritt, C., Krieg, M. E., Fernández, S., Alonso, J. C., and Grimm, R. (1997) Eur. J. Biochem. 249, 70-76[Abstract]
  30. Ioffe, E., and Stanley, P. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 728-732[Abstract/Free Full Text]
  31. Schorle, H., Holtschke, T., Hunig, T., Schimpl, A., and Horak, I. (1991) Nature 352, 621-624[CrossRef][Medline] [Order article via Infotrieve]
  32. Kuhn, R., Rajewsky, K., and Muller, W. (1991) Science 254, 707-710[Medline] [Order article via Infotrieve]
  33. Dranoff, G., Crawford, A. D., Sadelain, M., Ream, B., Rashid, A., Bronson, R. T., Dickersin, G. R., Bachurski, C. J., Mark, E. L., Whiisett, J. A., and Mulligan, R. C. (1994) Science 264, 713-716[Medline] [Order article via Infotrieve]
  34. Nath, S. T., and Nayak, D. P. (1990) Mol. Cell. Biol. 10, 4139-4145[Medline] [Order article via Infotrieve]
  35. Robbins, J., Dilworth, S. M., Laskey, R. A., and Dingwall, C. (1991) Cell 64, 615-623[Medline] [Order article via Infotrieve]


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