Site-specific recombination in human cells catalyzed by phage λ integrase mutants12

doi:10.1006/jmbi.2000.3532 available online at http://www.idealibrary.com on

J. Mol. Biol. (2000) 296, 1175±1181

COMMUNICATION

Site-specific Recombination in Human Cells Catalyzed by Phage l Integrase Mutants Elke Lorbach, Nicole Christ, Micha Schwikardi and Peter DroÈge* Institute of Genetics, University of Cologne, Weyertal 121 D-50931 Cologne, Germany

Phage lambda Integrase (Int) is the prototype of the so-called integrase family of conservative site-speci®c recombinases, which includes Cre and FLP. The natural function of Int is to execute integration and excision of the phage into and out of the Escherichia coli genome, respectively. In contrast to Cre and FLP, however, wild-type Int requires accessory proteins and DNA supercoiling of target sites to catalyze recombination. Here, we show that two mutant Int proteins, Int-h (E174 K) and its derivative Int-h/218 (E174 K/E218 K), which do not require accessory factors, are pro®cient to perform intramolecular integrative and excisive recombination in co-transfection assays inside human cells. Intramolecular integrative recombination is also detectable by Southern analysis in human reporter cell lines harboring target sites attB and attP as stable genomic sequences. Recombination by wild-type Int, however, is not detectable by this method. The latter result implies that eukaryotic co-factors, which could functionally replace the prokaryotic ones normally required for wild-type Int, are most likely not present in human cells. # 2000 Academic Press

*Corresponding author

Keywords: site-speci®c recombination; l integrase; mutant recombinase; eukaryotes; genomic engineering

The recent application of site-speci®c recombinases has improved signi®cantly the available techniques for mammalian genome manipulation. Notably, Cre and FLP, both members of the integrase family of conservative site-speci®c recombinases, are used widely in studies elucidating the relevance and function of particular genes of interest (for reviews, see Sauer, 1994; MuÈller, 1999). The phage l-encoded integrase (Int) is the prototype of the integrase family. Int executes both the integration and excision of the phage into and out of the Escherichia coli genome through recombination between pairs of attachment sites, termed attB/attP and attL/attR, respectively (for reviews, see Landy, 1989, 1993). Each att sequence is composed of two inverted 9 bp core Int binding sites and a 7 bp overlap region, which is identical in all E. Lorbach and N. Christ contributed equally to this work. Abbreviations used: Int, phage l-encoded integrase; XIS, phage-encoded exisionase; IHF, integration host factor; FIS, factor for inversion stimulation; XIS, phageencoded exisionase. E-mail address of the corresponding author: [email protected] 0022-2836/00/051175±7 $35.00/0

wild-type att sites. In addition to core sites where strand cleavage and religation occurs, each site except attB contains additional arm Int binding sites. A varying number of recognition sequences for the accessory DNA-bending proteins integration host factor (IHF), factor for inversion stimulation (FIS), and the phage-encoded excisionase (XIS) protein are also present in the ¯anking regions, again with the exception of attB. Int is a heterobivalent DNA-binding protein and, with assistance from the accessory proteins and negative DNA supercoiling, is able to bind simultaneously to core and arm sites within the same att site. Int, like Cre and FLP, executes an ordered, sequential pair of strand exchanges during integrative and excisive recombination (Landy, 1993). The natural pairs of target sequences for Int, attB and attP or attL and attR, can be located either on the same or two different DNA molecules, leading to intra- or intermolecular recombination, respectively. Here, we consistently use the terms integrative and excisive recombination to describe intramolecular recombination between inversely oriented attB and attP, or between attL and attR sequences, respectively. Both reactions thus lead to inversion of the intervening DNA segment. # 2000 Academic Press

1176 As outlined above, an important difference between the Int and Cre/FLP system is that Int requires additional protein factors for integrative and excisive recombination, and negative supercoiling for integrative recombination. It is this complication that has presumably prevented application of the l Int system in eukaryotic gene targeting techniques. As a ®rst step towards the transfer of the Int system to mammalian cells, we have therefore explored the use of two mutant Int proteins, Int-h and its derivative Int-h/218. Both enzymes promote integrative recombination in the absence of accessory proteins in E. coli (Miller et al., 1980; Christ & DroÈge, 1999) and DNA supercoiling in vitro (Lange-Gustafson & Nash, 1984). Likewise, excisive recombination by Int-h (Int-h/218) can occur in the absence of IHF and XIS (Christ & DroÈge, 1999). The potential to perform recombination in the absence of accessory factors has been explained by an enhanced af®nity of Int-h for core recognition sequences (Patsey & Bruist, 1995). In order to test whether Int-h (Int-h/218) is competent to perform integrative and/or excisive recombination inside human cells, it was ®rst necessary to demonstrate that Int-h protein is synthesized. We constructed eukaryotic expression vectors, termed pKEXInt-h and pPGKInt-h, which contain the Int-h coding region under control of the CMV and the PGK promoter, respectively (Figure 1(a)). After transient transfection of pKEXInt-h into two BL60 reporter cell lines, termed B2 and B3 (see below), we could detect both unprocessed and correctly spliced Int-h mRNAs by RTPCR (data not shown). Cell lysates were analyzed 72 hours after transfection of pKEXInt-h by Western blotting using mouse polyclonal antibodies raised against Int. The parental vector pKEX, lacking the Int-h gene, was used as a control. The results show that a protein with the expected molecular mass of Int-h is detectable in both cell lines when pKEXInt-h was introduced (Figure 2(a); compare lanes 2 and 4 with M). This protein is absent from cells containing the control vector (lanes 1 and 3). The Int-h protein can also be detected in HeLa cells after transfection of pPGKInt-h and its derivative pPGKInt-hNLS; the latter carries a nuclear localization signal fused to the C terminus of the Int-h gene (Figure 2(b), lanes 1 and 2, respectively). The protein is absent when the control vector pPGK is used (lane 3). We conclude that presumably full-length Int-h protein is produced by two different human cell lines. In order to test whether the Int-h protein produced in human cells can perform integrative recombination, we constructed substrate vector pGFPattB/attP (Figure 1(b)). The two recombination sites attB and attP are in inverted orientation with respect to each other and ¯ank the gene for green ¯uorescence protein (GFP). The GFP gene itself is in inverted orientation with respect to the CMV promoter. Hence, recombination between attB and attP should lead to its inversion and subsequent expression.

Genomic Engineering by Phage  Integrase Mutants

We co-introduced pGFPattB/attP either with pPGKInt-h or pKEXInt-h to HeLa or BL60 cells, respectively. DNA was isolated 72 hours after transfection and recombination was monitored by PCR employing primer pair p3/p4. These primers should yield a 0.99 kb product if inversion of the GFP gene has occured (compare Figure 1(b)). The results show that the expected product is detectable in both cell lines only when the respective expression vector is present (Figure 3(a), lanes 1 and 4). Control PCRs furthermore con®rmed that the substrate pGFPattB/attP is present in all transfected cell lines (data not shown). In order to test for excisive recombination, we constructed pGFPattL/attR. This vector is identical with pGFPattB/attP, except that attL and attR replace attB and attP, respectively (Figure 1(c)). Recombination assays were performed as described for pGFPattB/attP and the results show that the expected PCR product (1.09 kb) is detectable only when the respective expression vector was introduced together with pGFPattL/attR (Figure 3(b), lanes 1 and 3). The presence of the substrate vector in cell lines was again con®rmed by PCR. DNA sequencing of isolated PCR products that result either from integrative or excisive recombination con®rmed that the strand transfer reactions occured as expected for wild-type Int (data not shown). We conclude that Int-h is pro®cient at performing integrative and excisive recombination on episomal DNA substrates in two different human cell lines. In order to test whether Int-h can also catalyze integrative recombination when the target sites are stably integrated into the host genome, we generated three BL60 reporter cell lines (B1-B3). Southern analysis of their genomic DNA con®rmed that B1 and B3 carry multiple copies of pGFPattB/ attP integrated as tandem repeats, while B2 contains only a single copy (data not shown). Vectors pKEXInt-h and pKEX were transfected separately into these reporter cell lines. Cells were harvested 72 hours after electroporation, genomic DNAs puri®ed, and analyzed by PCR for recombination events. The results revealed that the expected products generated with primer pairs p3/p4 and p1/ p2 (compare Figure 1(b)) are detectable only when pKEXInt-h is introduced into the cells (data not shown). DNA sequencing of isolated PCR products con®rmed that both attL and attR are present, and that the GFP gene has been inverted. Furthermore, RT-PCR analysis con®rmed that the GFP gene is expressed due to recombination (data not shown). We performed these assays three times and could, in each case, detect recombination between attB and attP by genomic PCR and RT-PCR in all three cell lines expressing Int-h. In order to test whether wild-type Int and a second Int mutant, Int-h/218 (Christ & DroÈge, 1999), is pro®cient for integrative recombination in a HeLa reporter cell line, we introduced pCMVSSInt and pCMVSSInt-h/218, as well as pCMVSSInt-h and pCMV as positive and negative

Genomic Engineering by Phage  Integrase Mutants

1177

Figure 1. (a) Int expression vectors. Relevant genetic elements present on expression vectors for wild-type Int and two mutant Int proteins, Int-h and Int-h/218, are illustrated. CMV, human cytomegalovirus promoter; PGK, phosphoglycerate kinase promoter; NLS, SV40 large tumor antigen nuclear localization signal. The Figure shows representations of substrate vectors for (b) integrative and (c) excisive recombination. Important features of both vectors are explained in the text. Recombination will, in each case, lead to inversion of the GFP gene. The position and orientation of PCR primers used to analyze recombination are indicated by small arrows beneath each vector. Also indicated are relevant restriction sites and the DNA segment used as a probe in Southern analysis. GFP, green ¯uorescence protein gene; neo, neomycin resistance gene; SV40, early SV40 promoter; pA, polyadenylation signal. Expression vectors for Int-h (pKEXInt-h) and Int-hNLS (pKEXInt-hNLS) are derivatives of pKEX-2-XR (Rittner et al., 1991). The Int genes were cloned by PCR using primers (3343) 50 -GCTCTAGACCACCATGGGAAGAAGGCGAAGTCA-30 and (3289) 50 -AAGGAAAGCGGCCGCTCATTATTTGATTTCAATTTTGTCC-30 . pKEXInt-hNLS was generated with primer (3344) 50 -AAGGAAAGCGGCCGCTCATTAGACCTTACGCTTCTTCTTGGGTTTGATTTCAATTTTGTCCCACT-30 instead of (3289). The resulting PCR fragments were inserted into pKEX-2-XR using XbaI and NotI. Int-h and Int-hNLS were ampli®ed from pHN16 (Lange-Gustafson & Nash, 1984). Wild-type Int and Int-h/ 218 were generated from pTrcInt and pTrcInt-h/218, respectively (Christ & DroÈge, 1999). Expression vector pPGKInth is a derivative of Cre expression vector pPGKCrebpA (K. Fellenberg, Cologne). It was constructed from pKEXInt-h by PCR using primers: (Int-N-EU) 50 -AACTGCAGCTCGAGGTCCACCATGGGAAGAAGG-30 and (Int-C-EU) 50 GCTCTAGAGCGGCCGCTCATTATTTGATT-30 . The PCR fragment was inserted into pPGKCrebpA using PstI and XbaI. pPGKInt-hNLS was constructed as pPGKInt-h, except that pKEXInt-hNLS was used as template and primer (Int-C/NLS-EU) 50 -GCTCTAGATCATTAGACCTTACGCTTCTTCT-30 replaced (Int-C-EU). Expression vector pPGKInt-h/218 is a derivate of pCMVInt-h/218 (see below) and was constructed by replacing the Int-h gene with a PstI/XbaI restriction fragment containing Int-h/218. Expression vector pCMVInt-h is a derivative of pPGKInt-h and was constructed by PCR. In pCMVInt-h, the pPGK promoter and intron were replaced by a CMV promoter fragment, that was inserted between EcoRI and PstI sites. pCMVInt and pCMVInt-h/218 are derivatives of pCMVInt-h. The Int genes were generated by PCR as described above, and inserted into pCMVInt-h using XbaI and PstI. The pCMVSS vectors are derivatives of pCMVInt-h and contain a hybrid intron placed between the promoter and the recombinase gene. The Int genes plus intron were ampli®ed by PCR using the respective pPGK vectors as template. Substrate vectors are derivatives of pEGFP-C1 (Clontech). The recombination cassettes are driven by the CMV promoter. pGFPattB/attP was constructed by deleting the GFP gene from pEGFP-C1 using AgeI and BamHI. AttB was inserted as a double-stranded oligonucleotide into the AgeI-cleaved vector using oligos (B1OB) 50 -CCGGTTGAAGCCTGCTTTTTTATACTAACTTGAGCGAACGC-30 and (BOB1) 50 -AATTGCGTTCGCTCAAGTTAGTATAAAAAAGCAGGCTTCAA-30 . The attP site was generated by PCR from pAB3 (Wassarman et al., 1988) using primers (p7) 50 -TCCCCCCGGGAGGGAGTGGGACAAAATTGA-30 and (p6) 50 -GGGGATCCTCTGTTACAGGTCACTAATAC-30 . The PCR fragment was cleaved with XmaI and BamHI and ligated to an AgeI/EcoRI restriction fragment containing the GFP gene. The product was inserted into the MfeI/BamHI-cleaved, attB-containing vector. pGFPattL/attR was generated by recombining pGFPattB/attP in E. coli to yield attL and attR, and deleting the correctly oriented GFP gene through partial restriction using BsiEI and HindIII. In order to insert the GFP gene in inverted orientation with respect to the CMV promoter, it was ampli®ed by PCR using primers (p2) 50 AATCCGCGGTCGGAGCTCGAGATCTGAGTCC-30 and (p3) 50 -AATCCCAAGCTTCCACCATGGTGAGCAAGGG-30 . The PCR fragment was cleaved with HindIII and BsiEI, and ligated to the partially digested, recombined pGFPattB/ attP. Plasmid DNAs were isolated from E. coli strain DH5a using af®nity chromatography (Qiagen, Germany). Expression and substrate vectors, as well as PCR-derived constructs were sequenced using the ¯uorescence-based 373A system (Applied Biosystems). PCRs were performed with 30 cycles using the Master Mix Kit (Qiagen, Germany). The reaction temperatures were calculated based on the respective primer sequences and the length of the expected product.

1178

Figure 2. Western analysis. (a) Int-h protein expressed in two different Burkitt0 s lymphoma reporter cell lines (B2 and B3). (b) Int-h protein expressed in HeLa cells. Note that the Int-h/NLS variant shows a slightly reduced mobility due to the NLS. M, marker lanes containing extracts from E. coli expressing Int-h/NLS. Cell lysates from transiently transfected cells were prepared by boiling the harvested cells in sample buffer for ®ve minutes Proteins were separated through SDS 12.5 % (w/v) polyacrylamide gels and transferred onto a nitrocellulose membrane (Immobilon P, Millipore). The membrane was blocked with 1 % blocking solution and incubated with mouse polyclonal antibodies raised against wild-type Int at a 1:50,000 dilution. Peroxidaseconjugated secondary antibodies were used for detection (Boehringer Mannheim, Germany).

controls, respectively (compare Figure 1(a)). The cell line contains about eight copies of pGFPattB/ attP randomly integrated at different genomic loci (data not shown). Genomic DNA was isolated 72 hours after electroporation, cleaved with NcoI and analyzed by Southern hybridization using the GFP gene as probe (compare Figure 1(b)). The results show that the expected genomic restriction fragment resulting from inversion of the GFP gene is detectable only after transfection with pCMVSSInth and pCMVSSInt-h/218 (Figure 4, lanes 5 and 6). Considering the transfection ef®ciency of about 50 % usually observed for HeLa cells (data not

Genomic Engineering by Phage  Integrase Mutants

shown), a quantitative analysis by phosphorimaging revealed that 16 % and 30 % of the pGFPattB/ attP molecules are recombined by Int-h and Int-h/ 218, respectively. We also tested BL60 reporter cell line B3, which contains about seven copies of pGFPattB/attP as tandem repeats at the same genomic locus, for integrative recombination by Southern analysis. Consistent with our PCR analysis, we found that both mutant Int proteins are also pro®cient to catalyze inversion in B3 cells (data not shown). A quantitative analysis by phosphorimaging revealed that about 6 % of the pGFPattB/attP molecules were recombined by Int-h/218. Recombination by wildtype Int was again not detectable. In summary, we conclude that mutant Int proteins are pro®cient to perform intramolecular, integrative recombination at genomic target sites in two different human cell lines. We have shown above that Int-h catalyzes excisive recombination between attL and attR in transient co-transfection assays. It was therefore interesting to test how Int-h performs on a genomic substrate for excisive recombination. We generated three BL60 and two HeLa reporter cell lines, each carrying a single copy of pGFPattL/attR stably integrated into the host genome. We performed different recombination assays using expression vector pKEXInt-h for BL60 and pPGKInt-h for HeLa cells, exactly as described above. However, we were unable to detect excisive recombination employing PCR at 72 hours after transfection in any of the cell lines. Only genomic DNA isolated from one of the two HeLa cell lines produced a faint PCR signal indicative of excisive recombination at 144 hours post transfection (data not shown). We have demonstrated in this study that mutant l Int proteins are pro®cient to catalyze intramolecular, integrative recombination in transient cotransfection experiments and in human reporter cell lines that carry the respective target sites stably integrated into their genomes. Excisive recombination, however, is detectable only at 72 hours post transfection with episomal substrate vectors. We demonstrated that integrative recombination in reporter cells expressing wild-type Int is not detectable through Southern analysis. The latter result is explained best by the known capacity of mutant recombinases to perform recombination in the absence of their natural protein accessory factors and negative DNA supercoiling. Hence, suf®cient unconstrained supercoiling and/or proteins that could functionally replace the natural protein accessory factors required for wild-type Int are most likely not present in human cells. This ®nding appears to contradict the ®ndings of a recent study, which indicates that wild-type Int encoded by the related phage HK022 can carry out integrative and excisive recombination on episomal substrates without its natural accessory proteins in mouse and monkey cells (Kolot et al., 1999). However, whether HK022 Int-mediated recombination

Genomic Engineering by Phage  Integrase Mutants

Figure 3. PCR analysis of recombination in transient co-transfection experiments. (a) Test for integrative recombination. Substrate vector pGFPattB/attP was cointroduced with eukaryotic expression vectors to either BL60 or HeLa cells, as indicated. Recombination was assayed by PCR employing primer pair p3/p4. (b) Test for excisive recombination employing substrate vector pGFPattL/attR. Transient expression (Figure 2) and recombination assays were performed with human Burkitt0 s lymphoma (BL60) (Wolf et al., 1990) or HeLa cells. BL60 cells were cultured in RPMI1640 medium (Life Technologies, Inc.) supplemented with 10 % (w/v) fetal calf serum (PAA), 2 mM L-glutamine, streptomycin (0.1 mg/ml), and penicillin (100 units/ml). HeLa cells were cultured in D-MEM containing 10 % FCS, 2 mM Lglutamine, streptomycin (0.1 mg/ml), and penicillin (100 units/ml). Transient recombination assays through electroporation using BL60 cells were performed as described for Figure 4. Cells were transfected with 20 mg expression and 20 mg substrate vector. Cells were harvested 72 hours after electroporation and DNA isolated as described below. Transient recombination assays in HeLa cells were performed by introducing supercoiled expression and substrate vectors at a molar ratio of about 1:1 through lipofection (FuGene; Boehringer Mannheim, Germany). About 2  105 cells were transfected with 2 mg of mixed plasmid DNA. Cells were harvested 72 hours after transfection, and DNA extracted as described below. In order to analyze products of integrative and excisive recombination through PCRs in reporter cell lines or in transient co-transfection assays, 0.4 or 1.0 mg of isolated DNA was ampli®ed by PCR using 20-50 pmol of the following primers:

1179 will also be detectable by Southern analysis in reporter cells is so far not known. While excisive recombination by Int-h and Int-h/218 is reproducibly detected in transient co-transfection experiments, it is perhaps surprising that this reaction is not observed, even by PCR, at 72 hours after transfection in ®ve different reporter cell lines carrying pGFPattL/attR. We consider three explanations for this discrepancy. First, reporter cell lines differ from each other in the site of target vector integration. Excisive recombination may not occur simply because the genomic region into which pGFPattL/attR has been integrated is not accessible by the recombinase. However, the fact that integrative recombination is reproducibly detected in ®ve reporter lines, two of them contain only a single copy of the target vector, may indicate that target site inaccessibility is not the only reason for our failure to detect excisive recombination. If so, a second possible explanation is that an unknown structural or topological feature of human chromatin speci®cally prevents excisive recombination by Int-h or Int-h/218. Binding to attL of histone dimer H2A-H2B together with Int-h, for example, may result in a nucleoprotein complex that is refractory to excisive recombination (Segall et al., 1994). A third possibility is that excisive recombination occurs at a signi®cantly reduced rate compared to integrative recombination. In fact, our ®nding that a faint PCR signal indicative of excisive recombination was detectable 144 hours post transfection, with one HeLa reporter cell line may indicate that excisive recombination exibits much slower kinetics than integrative recombination. Further experimentation employing reporter cell lines that carry a copy of either pGFPattB/attP or pGFPattL/attR at the same genomic locus is required to determine which, if any, of these possibilities will resolve this discrepancy. Our ®nding that excisive recombination between genomic attL and attR seems to be either prohibited or at least signi®cantly impaired may nevertheless offer a possibility for future controlled recombination events through the additional employment of the prokaryotic accessory protein XIS. An important question with respect to potential applications of the Int recombination system concerns the ef®ciency of the reaction. We tried

(p1) 50 -GGCAAACCGGTTGAAGCCTGCTTTT-30 ; (p2) 50 -AATCCGCGGTCGGAGCTCGAGATCTGAGTCC-30 ; (p3) 50 -AATCCCAAGCTTCCACCATGGTGAGCAAGG G-30 ; (p4) 50 -AACCTCTACAAATGTGGTATGG-30 ; (p5) 50 -TACCATGGTGATGCGGTTTTG-30 ; (p6) 50 -GGGGAT CCTCTGTTACAGGTCACTAATAC-30 ; (p7) 50 -TCCCCC CGGGAGGGAGTGGGACAAAATTGA-30 ; (p8) 50 -CTCA CATGTTCTTTCCTGCGT-30 . Control PCRs to con®rm the presence of substrate vectors in transient transfection experiments (BL60) employed primer pair p5/p6. Control PCRs using HeLa cells were performed with primers (p3) and (p8) 50 -CTCACATGTTCTTTCCTGCGT-30 .

1180

Figure 4. Analysis of integrative recombination by Southern hybridization. Genomic DNA was incubated with NcoI, separated through agarose gel electrophoresis, and transferred onto nylon membrane. Recombination was analyzed by hybridization with a 32P-labelled probe comprising the GFP gene. Lanes 1 and 2, unrecombined and recombined pGFPattB/attP digested with NcoI, respectively. Note that the signal indicative of recombination is detectable when mutant Int proteins are expressed in HeLa cells (lanes 5 and 6), but not with control vector pCMV (lane 3) or wild-type Int (lane 4). Recombination assays using HeLa reporter cell lines were performed by introducing 20 mg of expression vector to approximately 1  107 cells at 280 V and 960 mF using a Bio-Rad Gene pulser. Cells were harvested 72 hours after transfection, and DNA was extracted using QIAamp Blood Kit (Qiagen, Germany). To test for recombination using BL60 reporter cell lines, about 2  107 cells were electroporated with 40 mg of expression vector, exactly as described above. Cells were harvested by centrifugation, and DNA extracted from the resulting cell pellet. About 15 mg of genomic DNA was incubated overnight with NcoI, separated through 0.8 % (w/v) agarose gel electrophoresis in TBE buffer, and transferred overnight onto a nylon membrane. The 32P-labelled probe was prepared from a PCR product obtained with primers p2/p3 (compare Figure 1(b)). Blots were quanti®ed using a Fuji BAS 1000 phosphorimager. Reporter BL60 cell lines containing either pGFPattB/attP or pGFPattL/attR stably integrated into their genomes were constructed as follows: about 20 mg of either vector was linearized with ApaLI, and mixed with approximately 2  107 cells at 260 V and 960 mF using a Bio-Rad Gene pulser. Stable cell lines were selected with G418 (300 mg/ml). Reporter HeLa cell lines were constructed by introducing 10 mg of each ApaLI-cleaved vector through lipofection using FuGene (Boehringer Mannheim, Germany). Selection of stable transformants was performed in a-MEM containing G418 (500 mg/ml). Reporter cell lines employed in this study were characterized by PCR, DNA sequencing, and Southern hybridization.

to address this question employing two methods. First, we analyzed, through FACS, the fraction

Genomic Engineering by Phage  Integrase Mutants

of reporter BL60 cells expressing GFP as a result of integrative recombination. While we were able to detect GFP expression by RT-PCR, these attempts proved unsuccessful (data not shown). We consider two explanations for this negative result. First, FACS controls performed with recombined pGFPattB/attP transiently introduced into BL60 and HeLa cells revealed that GFP expression is ®ve to ten times weaker than that observed with the parental vector pEGFP (data not shown). It is possible that attL positioned between the CMV promoter and the GFP gene interferes to some extent with translation of the GFP mRNA. A second explanation results from the fact that cell lines B1 and B3, and the HeLa cell line analyzed through Southern blotting contain multiple copies of pGFPattB/attP integrated into their genomes. Because only a fraction is recombined per cell (compare Figure 4), transcription from unrecombined copies would result in antisense GFP mRNA. This may also inhibit translation of the GFP message. The same reasoning applies to transient co-transfection assays. Using GFP expression as a reporter, we are therefore not yet able to provide a precise quantitative analysis of the fraction of cells in which recombination has occured. This fraction, however, can be signi®cant, since a quantitative analysis by phosphorimaging of Southern blots revealed that up to 30 % of pGFPattB/attP copies present in HeLa reporter cells expressing Int-h/ 218 are in fact recombined within 72 hours (Figure 4). Hence, even if we assume that all eight copies of pGFPattB/attP are recombined per cell expressing Int-h/218, at least 30 % of those cells must contain recombined pGFPattB/ attP. If the l Int recombination system described here with human cell lines can be con®rmed in animal models and re®ned, for example, through the use of accessory proteins such as XIS, the currently available possibilities for controlled in vivo and ex vivo manipulations of eukaryotic genomes will be expanded. Int mutants alone or in combination with already existing recombinases will allow a more speci®c spatiotemporal control of recombination events.

Acknowledgments Very special thanks go to R. Tirumalai, J. Liu, and A. Landy for antibodies against wild-type Int. Special thanks are due to H. Nash for plasmids pHN1 and pHN16, to K. Rajewsky and A. Tarakhovsky, who provided access to cell culture facilities, and to A. Landy and B. MuÈller-Hill for discussions and encouragement. These studies were ®nanced through a Boehringer Ingelheim Fonds predoctoral fellowship (N.C.), and through SFB 274 grant B10 and DFG grants Dr187/10-1/-10-2 to P.D., who is a DFG Heisenberg fellow (Dr187/8-1/-8-2).

Genomic Engineering by Phage  Integrase Mutants

References Christ, N. & DroÈge, P. (1999). Alterations in the directionality of l site-speci®c recombination catalyzed by mutant integrases in vivo. J. Mol. Biol. 288, 825836. Kolot, M., Silberstein, N. & Yagil, E. (1999). Site-speci®c recombination in mammalian cells expressing the Int recombinase of bacteriophage HK022. Mol. Biol. Rep. 26, 207-213. Landy, A. (1989). Dynamic, structural and regulatory aspects of lambda site-speci®c recombination. Annu. Rev. Biochem. 58, 913-949. Landy, A. (1993). Mechanistic and structural complexity in the site-speci®c recombination pathways of Int and FLP. Curr. Opin. Genet. Dev. 3, 699-707. Lange-Gustafson, B. J. & Nash, H. A. (1984). Puri®cation and properties of Int-h, a variant protein involved in site-speci®c recombination by bacteriophage l. J. Biol. Chem. 259, 12724-12732. Miller, H. I., Mozola, M. A. & Friedman, D. I. (1980). int-h: an int mutation of phage l that enhances sitespeci®c recombination. Cell, 20, 721-729. MuÈller, U. (1999). Ten years of gene targeting: targeted mouse mutants, from vector design to phenotype analysis. Mech. Dev. 82, 3-21.

1181 Patsey, R. L. & Bruist, M. L. (1995). Characterization of the interaction between the Lambda intasome and attB. J. Mol. Biol. 252, 47-58. Rittner, K., StoÈppler, H., Pawlita, M. & Sczakiel, G. (1991). Versatile eukaryotic vectors for strong and constitutive transient and stable gene expression. Methods Mol. Cell. Biol. 2, 176-181. Sauer, B. (1994). Site-speci®c recombination: developments and applications. Curr. Opin. Biotechnol. 5, 521-527. Segall, A. M., Goodman, S. D. & Hash, H. A. (1994). Architectural elements in nucleoprotein complexes: interchangeability of speci®c and non-speci®c DNA binding proteins. EMBO J. 13, 4536-4548. Wassarman, S. A., White, J. H. & Cozzarelli, N. R. (1988). The helical repeat of double-stranded DNA varies as a function of catenation and supercoiling. Nature, 334, 448-450. Wolf, J., Pawlita, M., Bullerdiek, J. & zur Hausen, H. (1990). Suppression of the malignant phenotype in somatic cell hybrids between Burkitt's lymphoma cells and Epstein-Barr virus-immortalized lymphoblastoid cells despite deregulated c-myc expression. Cancer Res. 50, 3095-3100.

Edited by M.Yaniv (Received 1 November 1999; received in revised form 10 January 2000; accepted 17 January 2000)