INTRODUCTION

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For the past 25 years, it has been possible to construct precisely designed DNA molecules in the test tube thanks to the techniques of recombinant DNA. In contrast, the ability to make controlled and efficient alterations in the genomes of living higher cells has been limited. The use of site-specific recombinases such as Cre and FLP provided an important advance (17), but because of the reversibility of these enzyme reactions, their main utility has been for creating deletions. For the integration of new material into the genome, fortuitous integration of transfected DNA is most often used, and it produces integration at random locations at low frequency. Homologous recombination provides site specificity, but at very low efficiency (26).

We began working with another site-specific recombinase, the phage C31 integrase, because it offered the potential for unidirectional integration that would therefore occur at higher net frequencies than the reversible integration directed by recombinases, such as Cre. Cre recombines two identical loxP sites, recreating two identical sites after recombination that can undergo a subsequent round of recombination. In contrast, the attB and attP recognition sites recognized by the C31 integrase are dissimilar in sequence (15). After reaction, the recombined att sites differ from attB and attP and are refractory to further synapsis by the integrase, thus locking in integration reactions (23, 24). We demonstrated that this enzyme, derived from a Streptomyces phage (9), worked well in the human cell environment (7), consistent with its lack of cofactor requirements (23). This feature distinguishes it from better known phage integrases, such as that of phage , which does require cofactors (10). The  integrase is in the family of recombinases that includes Cre and FLP and carries out a tyrosine-mediated strand exchange (4, 13). The C31 integrase is in the other major family of site-specific recombinases that includes many resolvases and invertases and uses a serine-catalyzed reaction mechanism (20). The two site-specific recombinase families are unrelated. The C31 integrase is a member of a recently discovered subclass of the serine recombinase family whose members are especially long and function as phage integrases (9, 15, 23).

By using extrachromosomal plasmids in human cells, we documented that the C31 integrase mediates highly efficient intramolecular integration reactions (>50%) and also efficient intermolecular integration into an Epstein-Barr virus model chromosome (7). These results suggest that the C31 integrase would be useful for mediating integration into mammalian chromosomes, which forms the subject of this study. We created human and mouse cell lines containing att sites inserted at random locations in the chromosomes. These lines were tested for the efficiency of integration of incoming plasmids bearing marker genes and att sites when cotransfected with the C31 integrase gene. We describe attP-containing cell lines that exhibit site-specific integration at appreciable frequencies.

Another opportunity afforded by this integrase system is the possibility of accessing integration at naturally occurring chromosomal sequences. Such reactions would obviate the need to first place a target att site in the genome. This strategy is of particular relevance in applications such as in vivo gene therapy, where a high frequency of integration in unaltered patient tissue is desired. We previously showed that the sizes of the attB and attP target sites recognized by the C31 integrase are 34 and 39 bp, respectively (7). This size range makes it statistically feasible that "pseudo" att sites, sites with degenerate att identity that is still recognizable by the enzyme, may be present in large genomes, such as those of mammals (25). We document here the presence of active pseudo attP sites in the human and mouse genomes that are recognized at significant frequencies by the C31 integrase. This phenomenon gives rise to a strategy for the efficient and precise alteration of the genomes of living cells at predetermined sites.

	MATERIALS AND METHODS

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Plasmids. Plasmids used for the measurement of luciferase activity were made as follows. A fragment carrying the cytomegalovirus (CMV) immediate-early promoter was cloned into the SmaI site upstream of the firefly luciferase gene of pGL3-basic (Promega Corporation, Madison, Wis.) to create pL. Plasmid pL-attB (Fig. 1A) was generated by cloning a 307-bp EcoRI fragment from pTA-attB (7) containing the minimal length C31 attB site and ~270 bp of surrounding C31 sequence into the BamHI site of pL. Plasmid pL-attP was generated by cloning a 250-bp EcoRI fragment from pTA-attP (7) containing the minimal attP site and ~210 bp of surrounding C31 sequence into the BamHI site of pL.

View larger version (21K): [in this window] [in a new window]   FIG. 1.   Plasmids used to detect chromosomal integration. (A) The pL-attB plasmid carried the C31 attB site and the luciferase gene and was used to measure the level and persistence of luciferase expression in the presence and absence of the C31 integrase, reflecting chromosomal integration at pseudo attP sites. A similar plasmid, pL-attP, carried the attP site and was used to monitor integration at pseudo attB sites. (B) pHZ-attP was used to place attP sites into the genome by using the hygromycin resistance selectable marker. pHZ-attB is a similar plasmid used for placement of attB sites in the genome. (C) pNC-attB, which carried an attB site and the neomycin resistance marker, was used as the incoming plasmid for identification of clones that underwent integration at an attP site or a pseudo attP site. pNC-attP was the parallel plasmid used to detect integration at attB or pseudo attB sites. (D) Zeocin selection scheme. From the top is shown a host chromosome (wavy lines) in which we integrated plasmid pHZ-attP (straight lines) bearing the hygromycin resistance gene for selection, an attP site for integrase-mediated recombination with attB, and a zeocin resistance gene lacking a promoter. The donor plasmid pNC-attB carries a neomycin resistance gene for selection, a green fluorescent protein (GFP) gene for analysis of transfection efficiency, and an attB site for integrase-mediated recombination with attP. Adjacent to attB is the strong promoter from CMV. Upon cotransfection of pNC-attB with pCMV-Int as a source of integrase, site-specific integration leads to the potential for expression of the zeocin resistance gene from the CMV promoter introduced on pNC-attB. The same donor plasmid was used in unmodified 293 and 3T3 cells, where it was selected with neomycin alone.

Cell culture and luciferase assays. 293 human embryonic kidney cells (6) and mouse NIH 3T3 cells (American Type Culture Collection, Manassas, Va.) were grown in Dulbecco's modified Eagle medium (Life Technologies) supplemented with 110 mg of sodium pyruvate/liter and 9% fetal bovine serum.

Construction of att cell lines. Plasmids pHZ-attB and pHZ-attP were treated with HpaI to generate linear molecules. Either 5 or 10 µg of linearized plasmid DNA were electroporated into 293 and NIH 3T3 cells using a Bio-Rad Gene Pulser according to the manufacturer's recommendations. The cells were then plated onto nonselective medium and allowed to recover. After 24 h, selection was started using medium containing 200 µg of hygromycin B (Calbiochem, La Jolla, Calif.)/ml. Single, well-isolated colonies were picked 12 to 14 days after the start of selection and expanded until ready for analysis. Cell lines were screened for the presence of the att site by PCR and Southern analysis. Four attP-containing 293 cell lines (293P1, 293P2, 293P3, and 293P4) and three attP-containing 3T3 cell lines (3T3P1, 3T3P2, and 3T3P3) were selected for further analysis, along with several attB cell lines of each cell type.

Selection assays. Unmodified 293 and 293 att-containing cell lines were grown to 50 to 80% confluency in 60-mm-diameter dishes and transfected with 50 ng of the donor plasmid and 5 µg of either pCMV-Int or pCMVSPORTGal carrier by using Lipofectamine (Life Technologies). At 24 h after transfection, the cells were transferred onto 100-mm-diameter dishes at an appropriate dilution. We found that 5 µg of DNA is near the upper limit for transfection of 293 cells on 60-mm-diameter dishes without appreciable toxicity. At this point, the total number of transfected cells was determined by counting the number of enhanced green fluorescent protein (EGFP)-expressing cells. The transfection frequency ranged from 4 to 7% for 293 cells. A further 24 h after expansion, selection was started with medium containing either 350 µg of Geneticin (G418, a neomycin analog; Life Technologies)/ml or a combination of Geneticin and 200 µg of zeocin (Invitrogen)/ml. Selection was continued for 14 days, and individual colonies were counted. The integration frequency was calculated as the ratio of the number of colonies obtained to the total number of transfected cells and was expressed as a percentage. Similar experiments were performed with NIH 3T3 cells and 3T3 att-containing cell lines, with the following changes: 50 ng of donor plasmid was transfected along with 2 µg of either pCMV-Int or pCMVSPORTGal carrier by using Lipofectamine Plus (Life Technologies). The transfection frequency ranged from 1 to 5% for 3T3. Selection was performed with medium containing either 650 µg of Geneticin/ml or 650 µg of Geneticin/ml and 200 µg of zeocin/ml.

Plasmid rescue and sequence analysis of integrants at pseudo att sites. Unmodified human 293 and mouse 3T3 cells were cotransfected with attB donor plasmid pHZ-attB and C31 integrase expression plasmid pCMV-Int. Transfections were split to three 100-mm-diameter tissue culture dishes 24 h after transfection, and selection with hygromycin (200 µg/ml) was begun 48 h after transfection. After 2 weeks of selection, colonies were trypsinized and redistributed over the plates to generate pools of hygromycin-resistant integrant clones. The pools were grown to confluency, and genomic DNA was prepared using a Blood and Cell Culture DNA Maxi kit (Qiagen, Valencia, Calif.).

Analysis of distribution of integration sites. pNC-attB was transfected into the four 293 attP-containing cell lines as described above. After 14 days of neomycin selection, individual colonies were picked and expanded for further analysis of integration. A total of 24 neomycin-resistant clones and 24 neomycin- and zeocin-resistant clones were picked from each cell line, for a total of 96 neomycin-resistant clones and 96 neomycin- and zeocin-resistant clones. Genomic DNA was prepared from each clone using the DNeasy 96 Tissue kit (Qiagen) and screened for the presence of site-specific recombination junctions. These genomic DNA samples were amplified by PCR using primers specific for a junction generated by a site-specific recombination reaction between attP and attB. The remaining neomycin-resistant clones were similarly screened for integration into pseudo attP site human A by using primers specific for that site.

	RESULTS

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Genomic integration detected by stable luciferase expression. We wished to determine whether the C31 integrase could direct measurable integration into the chromosomes of unmodified human and mouse cells. As a first approach, we placed the luciferase gene on an incoming attP or attB plasmid construct and cotransfected with a plasmid expressing the integrase. We then measured the expression of luciferase over a time course. These results revealed a statistically significant increase in long-term luciferase expression in human 293 cells when the gene was transfected on a plasmid bearing attB in the presence of integrase (Fig. 2). The stability of luciferase expression over the 4-week time course, in contrast with its rapid extinction in the absence of integrase and an attB site, was consistent with integrase-mediated integration of the luciferase gene into the chromosomes. The dependence of the reaction on integrase and attB suggested that native sites similar to those of attP were being recognized and accessed in the human genome. A statistically significant increase in luciferase expression was not observed when the incoming plasmid carried attP (Fig. 2). Similar results were seen when these experiments were carried out with mouse 3T3 cells. These data suggested that sites similar to attP were also being recognized in the mouse genome, as confirmed and examined in detail below.

View larger version (19K): [in this window] [in a new window]   FIG. 2.   Luciferase expression after transfection of att-luciferase plasmids into unmodified 293 cells. Shown is the time course of luciferase expression for constructs that carried the luciferase gene and either no att site (pL), attB (pL-attB), or attP (pL-attP) and which were transfected with and without the integrase-expressing plasmid pCMV-Int. Each data point represents the average of three independent experiments, with appropriate standard error bars. A greatly elevated level of luciferase expression indicative of efficient chromosomal integration was seen only when the incoming plasmid carried an attB site and integrase was present. Similar data were obtained for mouse 3T3 cells.

Genomic integration detected by selection. The luciferase studies suggested that attP sites recognized by the C31 integrase were naturally resident in the genomes of human and mouse cells. To examine this possibility more closely, we cotransfected unmodified human 293 and mouse 3T3 cells with a plasmid bearing the neomycin resistance marker and either attB or attP, with and without a plasmid expressing the C31 integrase. Negative controls included parallel transfections with a non-att-bearing plasmid.

Characterization of pseudo attP sites. Efficient integration of plasmids bearing attB in unmodified human and mouse cells in the presence of C31 integrase suggested that sites with significant identity to attP were present in these genomes. In order to test this prediction, we cloned the integration sites by plasmid rescue. Total DNA was prepared from pools of hygromycin-resistant 293 and 3T3 cells that had received pCMV-Int and pHZ-attB, which carried attB and the genes for hygromycin, zeocin, and ampicillin resistance. From the elevations in integration frequency observed above, ~80 to 90% of such integrations were expected to occur at native sites resembling attP. The genomic DNA was cut with sets of restriction enzymes with compatible ends that do not cut in pHZ-attB, was self-ligated, was transformed into E. coli cells, and was selected for the ampicillin and zeocin markers on pHZ-attB. Selection for two intact selectable markers on the plasmid served to limit the recovery of random integrants among the rescued plasmids. Sixty-seven colonies rescued from two independent pools of transfected human cells and 120 colonies rescued from four independent pools of transfected mouse cells were analyzed. Restriction enzyme digests and agarose gel analysis of the rescued plasmids displayed fragments expected for pHZ-attB, as well as various fragments contributed by the flanking genomic DNA for each integration event. This analysis showed that some of the rescued plasmids had the same fragment patterns within a species, consistent with repeated integrations into the same sites, while other plasmids were present as single occurrences.

View larger version (53K): [in this window] [in a new window]   FIG. 3.   DNA sequence characterization of pseudo attP sites and recombination junctions in human and mouse cells. (A) The wild-type C31 attP site is shown on top. The 3-bp TTG crossover region is marked, and arrows delineate the imperfect palindromes. Beneath are the DNA sequences for four pseudo attP sites. The GenBank accession numbers for these pseudo attP sites are in parentheses below. Human A (AF333429) is the primary site of C31 integrase-mediated integration in unmodified human cells, and human C (AF333430) and D (AF333431) are two secondary human integration sites. Mouse A (AF333432) is a site used repeatedly in mouse cells. A 100-bp DNA sequence centered on the attP core is presented here, with identities with attP shown filled in. (B) The sequences of the crossover region between the human A pseudo attP site (lowercase letters) and the attB site on the incoming plasmid (uppercase letters) for eight representative rescued events from neomycin-resistant pools of 293 cells. Colons represent bases missing in the novel joint. These small deletions ranged from 0 to 11 bp among 120 junctions sequenced. The bases affected by deletion in one or more junctions are highlighted in the composite junction sequence at the bottom of the diagram. (C) The sequences of the crossover region between an inserted wild-type attP site and the attB site on the incoming plasmid, which were derived from representative neomycin-resistant or neomycin- and zeocin-resistant colonies that were shown by PCR to have integrated at inserted attP site. The contribution from attP is shown in lowercase letters, while the contribution from attB and the 3-bp crossover region are in uppercase letters. The crossover junctions were perfect in each case (8 out of 8).

Integration at inserted att sites. In order to determine whether the C31 integrase could also mediate efficient integration at wild-type attB and attP sites placed into the context of mammalian chromosomes, we created human and mouse cell lines carrying attB or attP recognition sites and then cotransfected the lines with two plasmids, one expressing the C31 integrase and one carrying the complementary att site along with the neomycin resistance selectable marker.

Distribution of integration events. Because the overall integration frequency in human cell lines in which we had inserted an attP site was within twofold of the frequency in unmodified 293 cells, we expected that the integration events would be distributed between the attP site and the pseudo attP sites. To examine this point, we determined where the incoming attB plasmid was integrated by PCR analysis of DNA extracted from independent colonies derived from each of the four attP 293 cell lines. We examined 96 of the colonies selected with neomycin alone and 96 of the colonies selected with both neomycin and zeocin. In each case, 24 colonies were chosen at random from each of the four attP cell ines. The neomycin- and zeocin-resistant group represented 2 to 6% of the number of colonies seen with neomycin selection. They were expected to be located at the inserted attP sites because the incoming attB plasmid provides a promoter for the zeocin resistance gene (Fig. 1D). As expected, essentially all (95 out of 96) neomycin- and zeocin-resistant colonies showed a PCR band indicative of site-specific integration into the inserted attP site.

	DISCUSSION

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This study demonstrates that site-specific integration into the chromosomes of living mammalian cells can be obtained by using the C31 phage integrase, which carries out precise recombination between attP and attB recognition sites of minimum lengths of 30 to 40 bp (7). In cell lines containing an inserted attP site, we detected integration into the genome at frequencies approximately 10- to 20-fold above the spontaneous background frequency of random integration in mouse and human cells. Furthermore, in the absence of an inserted attP site, we detected integrase-mediated recombination at endogenous sites in the genome at frequencies ~5- to 10-fold above the background of random integration. These integration events were shown to occur at sets of native sequences having partial sequence identity to attP, which were termed pseudo attP sites.

These integration frequencies compare favorably to other site-specific integration systems described to date. Our frequencies are ~2 to 3 orders of magnitude higher than those typically involved in targeting by using homologous recombination. The frequency of integration by homologous recombination appears to be in the vicinity of 10⁶ for most mammalian cells (2, 26), though it has been reported that this frequency can be increased up to 20-fold by using completely isogenic DNA (22). By making a double-strand break at the target site, homologous recombination efficiency can be improved by ~100-fold or more (3, 14). However, a means to generate such a break in endogenous sequences is currently lacking. The integration frequency mediated by the C31 integrase is approximately 10- to 100-fold higher than Cre-mediated integration at an inserted wild-type loxP site (18) or integration mediated by FLP at an inserted FRT site (12). Use of specially designed loxP cassettes designed to limit the reverse reaction has resulted in higher integration frequencies (5, 16, 17). However, this strategy is not applicable when using endogenous sequences as targets. The C31 integrase appears to be more efficient in mammalian cells than phage integrases of the tyrosine-catalyzed site-specific recombinase family, such as integrases from phages  and HK022 (8, 11). Integration mediated by retroviruses and some transposases can be efficient (1, 28), but it takes place at random, leading to mutagenesis and inconsistency of gene expression.

This study demonstrated that in the presence of the C31 integrase, a plasmid bearing attB will be efficiently integrated into mammalian genomes. In unmodified human cells, nearly 90% of the integration events will be integrase mediated and will be distributed among a set of pseudo attP sites. By sequencing the junctions at 67 rescued integration events, we identified a hierarchy of pseudo attP sites, some of which were used repeatedly. The pattern of single and recurring sites in this collection of 31 different pseudo attP sites suggests that the total number of pseudo attP sites may be between 10² and 10³, with some sites significantly preferred over others. While integration is occurring at multiple sites, the level of specificity is still dramatically increased over that of random integration. Since the genome contains approximately 3 × 10⁹ bp, many of which are presumably available for random integration, restriction to approximately 10² integration sites represents a gain of several orders of magnitude in specificity.

In mouse cells, where 120 integration events were sequenced, we identified 57 pseudo attP sites, 21 of which were recurrent, creating a similar picture of pseudo attP site frequency. Since the identity of these sites to the wild-type attP was <60%, one would expect a similar number of sites in any mammalian genome. The number of pseudo attP sites found in mammalian genomes suggests that pseudo attP sites for the C31 integrase also exist in smaller genomes, such as those of important model organisms like Caenorhabditis elegans, Drosophila, and Arabidopsis. This integrase may became a valuable tool for genetic manipulation of those organisms. Because the C31 integrase requires no cofactors, it is expected to work well in a broad range of species, including plants, mammals, and other vertebrates and invertebrates, such as insects and worms. Indeed, it is likely that endogenous sites exist for many recombinases. We have documented the occurrence of pseudo loxP sites for Cre in mammalian cells (25), and it appears that these sites can be used in vivo in mice, at least under conditions of continuous high-level expression of Cre (19).

In human and mouse cell lines in which we inserted a wild-type attP site, the randomly placed attP sites competed with the set of native pseudo attP sites. From a sample of 96 colonies from four human cell lines carrying attP, we found that about 15% of the integrations occurred at the inserted attP site, compared to 5% at the predominant human A pseudo attP site. Most of the other integrations were expected to be distributed over the other ~100 pseudo attP sites. The integration frequency at a given attP site is presumably the result of the DNA sequence of the site and its chromosomal context, which may influence gene expression and integrase access. It will be interesting to measure the relative integration frequency at attP and human A when the chromosomal context of both sites is kept constant. We did not observe any multiple integrations in the same cell in this study, but we are not in a position to rule it out.

We have found that integration of a plasmid bearing attB into a chromosomally placed attP site is invariably precise, yielding the expected recombination event at the DNA sequence level. This precision reflects effective operation of the enzyme at its attB and attP recognition sites in many different contexts in mammalian chromosomes. Integration at the pseudo attP sites is, in contrast, slightly imprecise at the sequence level, presumably reflecting a less exact reaction when one of the att sites differs from the wild-type sequence. This result is expected from what is known of the interactions between the C31 integrase and its att recognition sites (24). The sequence of the att site seems to influence the nature of the complex formed, which in turn determines whether or not the reaction will proceed. It is conceivable that with imperfect sites, mammalian repair enzymes may participate in completing the reaction. We observed that when the incoming plasmid bore an attP site instead of an attB site, we detected less-efficient reactions, both with pseudo attB sites or with an inserted attB site. We do not have an explanation for this lack of symmetry. It may reflect the order of formation of the complex and the ability of a complex to form on att sites located on abundant and exposed incoming plasmid DNA versus att sites buried in the chromosomes.

The reaction involving integration of a vector bearing attB into a wild-type attP site previously inserted in the genome has many applications in genetics and biotechnology, such as positioning incoming genes at the same integration site repeatedly in a given cell line. If a selection scheme is employed, such as the zeocin selection used here, then close to 100% of the selected events will be precisely positioned at the chromosomal attP site. For species that possess endogenous sequences that are recognized at appreciable frequencies by the C31 integrase, integration into unmodified genomes is also feasible. This reaction may be valuable in in vivo gene therapy and other applications involving unmodified cells and tissues. This integration reaction would be more valuable if it occurred at an even higher frequency and if the number of target sites were more limited. More stringent target sequence requirements may be characteristic of related integrases found in nature. Additionally, it may be possible to increase enzyme efficiency and change target specificity by directed evolution (21).