Efficient somatic gene targeting in the lymphoid human cell line DG75

Gene 343 (2004) 91 – 97 www.elsevier.com/locate/gene

Efficient somatic gene targeting in the lymphoid human cell line DG75 Regina Feederlea,b, Henri-Jacques Deleclusea,b, Jean-Pierre Rouaultc, Aloys Schepersa, Wolfgang Hammerschmidta,* a

Department of Gene Vectors, GSF-National Research Center for Environment and Health, Marchioninistr. 25, Munich D-81377, Germany b Department of Virus Associated Tumours, German Cancer Research Center, Im Neuenheimer Feld 242, Heidelberg 69120, Germany c Unite´ INSERM U453, Centre Le´on Be´rard, Lyon Cedex 08 69373, France Received 8 June 2004; accepted 9 August 2004 Available online 25 September 2004 Received by M. Schartl

Abstract Among the different approaches used to define the function of a protein of interest, alteration and/or deletion of its encoding gene is the most direct strategy. Homologous recombination between the chromosomal gene locus and an appropriately designed targeting vector results in an alteration or knockout of the gene of interest. Homologous recombination is easily performed in yeast or in murine embryonic stem cells, but is cumbersome in more differentiated and diploid somatic cell lines. Here we describe an efficient method for targeting both alleles of a complex human gene locus in DG75 cells, a cell line of lymphoid origin. The experimental approach included a conditional knockout strategy with three genotypic markers, which greatly facilitated the generation and phenotypic identification of targeted recombinant cells. The vector was designed such that it could be reused for two consecutive rounds of recombination to target both alleles. The human DG75 cell line appears similar to the chicken DT40 pre B-cell line, which supports efficient homologous recombination. Therefore, the DG75 cell line is a favorable addition to the limited number of cell lines amenable to gene targeting and should prove useful for studying gene function through targeted gene alteration or deletion in human somatic cells. D 2004 Elsevier B.V. All rights reserved. Keywords: Knockout; Epstein–Barr virus; Homologous recombination; Gene targeting

1. Introduction Gene targeting is one of the most powerful approaches to unravel the function of a given gene. It is commonly performed in yeast and in murine embryonic stem (ES) cells (for reviews, see Capecchi, 1989; Muller, 1999). Recently, gene targeting in human ES cells has become feasible through improvement of transfection protocols (Zwaka and Thomson, 2003). Modification or deletion of a gene in somatic cells and cell lines is orders of magnitudes less

Abbreviations: EBV, Epstein–Barr virus; GFP, green fluorescent protein; Hy, hygromycin; HyR, hygromycin-resistant/resistance; ORF, open reading frame; TK, thymidine kinase; TB7, TD-binding protein 7; UTR, untranslated region. * Corresponding author. Tel.: +49 89 7099 506; fax: +49 89 7099 225. E-mail address: [email protected] (W. Hammerschmidt). 0378-1119/$ - see front matter D 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.gene.2004.08.005

efficient but is of obvious interest for reverse genetics to explore and characterize the function of any human gene. Since differentiated cells and cell lines do not support efficient homologous recombination, several techniques have been developed in recent years to facilitate gene targeting in diploid somatic cells (Sedivy and Dutriaux, 1999). The positive–negative selection scheme has dramatically enhanced targeting efficiency and is now a common approach for creating knockouts (Mansour et al., 1988). The use of promoterless targeting constructs has further increased the proportion of homologous vs. illegitimate recombination (Jasin and Berg, 1988; Sedivy and Sharp, 1989), but this strategy cannot be considered when the location of the promoter is unknown or silent in the cell of interest. It has also been reported that heterologous expression of gene products involved in homologous recombination, such as bacterial recA and human rad51,

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can improve gene targeting efficiency (Yanez and Porter, 1999; Scherbakova et al., 2000), similar to the situation in the DT40 chicken B-cell line (Bezzubova et al., 1997). To date, only a few reports on successful gene targeting in human cell lines are available (for recent reviews, see Yanez and Porter, 1998; Sedivy et al., 1999; Bunz, 2002; Hudson et al., 2002) and there is no evidence for one particular human cell line to be more recombinogenic than another. In this work, we report on a human B-cell line, which we found to support efficient homologous recombination. We generated a homozygous knockout of a cellular gene encoding a protein that interacts with a viral origin of DNA replication. The gene product of the TB7 gene (TDbinding protein 7) binds to the TD domain of the lytic origin of replication (oriLyt) of Epstein–Barr virus (EBV) (Hammerschmidt and Sugden, 1988; Gruffat et al., 1995; Baumann et al., 1999). EBV lytic replication is tightly controlled in human cells, and only a minority of latently infected cell lines fully support virus production. TB7 represses EBV lytic DNA replication (R.F. and W.H., to be published elsewhere), suggesting that a TB7 / cell might be permissive to yield progeny virus. Since EBV has a strict tropism for human cells, analysis of its lytic functions cannot be performed in murine embryonic cells. The EBVnegative Burkitt lymphoma cell line DG75 (Ben Bassat et al., 1977) was shown to be nonpermissive for induction of the lytic cycle (our unpublished data). In order to delete the TB7 gene in DG75 cells, a protocol that is based on our experience with targeting the resident EBV genome in latently infected human B cells was developed (Delecluse et al., 1998). Gene targeting of the TB7 gene by homologous recombination was found to occur with high frequency in DG75 cells. This approach was also successfully applied to two other genetic loci in this cell line (B. Kempkes and A. Kieser, personal communication). Our findings show that genetic manipulation of DG75 cells is relatively easy and extends the number of human cell lines amenable to gene targeting.

2. Materials and methods 2.1. Targeting vector A genomic clone encompassing the TB7 locus was isolated from a human placenta library (Rouault et al., 1992) using a TB7-specific cDNA probe. The TB7 gene locus is 14,646 bp in size (GeneBank accession no. AJ316575). A genomic subclone containing the first 14,285 bp of the TB7 gene was introduced into the pACYC177 cloning vector (New England Biolabs) and sequenced by standard methods. The structure of the TB7 gene locus, together with the targeting vector, is shown in Fig. 1. In an initial step, three oligonucleotides with loxP motifs (Hoess and Abremski, 1985) were cloned in the same relative orientation into the 5V noncoding regions flanking the TB7 gene and into the

second intron of TB7. The negative selection marker, a 1.8kbp XhoI–HindIII fragment containing the herpes simplex virus thymidine kinase cassette (Mansour et al., 1988), was inserted into the NruI site downstream of the 3Vuntranslated region (UTR). Finally, a 3.4-kbp NaeI–NotI fragment of p1919 (Delecluse et al., 1998) containing the hygromycin resistance cassette (Giordano and McAllister, 1990) and an enhanced version of the green fluorescent protein (eGFP; Clontech) was cloned into the single SapI site within the 3V UTR to generate the final TB7 targeting vector (Fig. 1). The targeting vector was linearized at one of two alternative sites prior to transfection. To target the first TB7 allele, the restriction enzyme FspI was used for linearization, whereas AscI was used to target the second allele. 2.2. Cell culture, transfection, and selection DG75, an EBV-negative Burkitt lymphoma cell line (Ben Bassat et al., 1977), was grown in RPMI 1640, 10% fetal calf serum, 2 mM glutamine, and antibiotics (50 U/ml penicillin and 50 Ag of streptomycin) (Life Technologies, Germany). HeLa is a human cervix carcinoma cell line (Jones et al., 1971) and was grown in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal calf serum, 2 mM glutamine, and antibiotics (Life Technologies). For DNA transfections, 3106 DG75 cells were washed in RPMI 1640 without fetal calf serum, resuspended in 250 Al of the same medium, and placed with 3 Ag of the linearized plasmid DNA in 4 mm gap electroporation cuvettes. Cells were transfected by electroporation at 240 V and 960 AF using the BioRad gene pulser (BioRad, Germany) and kept at high density in RPMI 1640 with 10% fetal serum. Two days after transfection, cells were plated onto 96-well plates (104 cells/well) and the medium was exchanged with selection medium containing 200 Ag/ml hygromycin (Calbiochem, Germany) and 10 AM ganciclovir (CymevenR; Hoffmann-La Roche, Switzerland). After 4 weeks, GFPexpressing clones were expanded, genomic DNA was extracted, and the structure of the rearranged locus was analysed by Southern blotting. For Cre-loxP-mediated excision of TB7 gene sequences, 107 cells were transfected with 10 Ag of pBS185 (Sauer and Henderson, 1990), an expression plasmid containing the cre gene under the control of the immediate-early human CMV promoter/enhancer. After 48 h, cells were cloned by limiting dilution in RPMI 1640 and 10% fetal calf serum containing antioxidants (Brielmeier et al., 1998), and gfp-negative cell clones were expanded and analysed individually by Southern blotting and polymerase chain reaction (PCR), using primers specific for sequences flanking the loxP sites. 2.3. DNA analysis Genomic DNA was isolated using a modified Hirt procedure (Hirt, 1967). Cells were lysed in 1% SDS; proteins were digested in proteinase K (Roche Diagnostics,

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Fig. 1. Strategy for the disruption of the TB7 gene. (A) Schematic of the genomic locus of the wild-type TB7 gene and the linearized targeting vectors used for genetic alteration of the first and second alleles. (B) Structure of the targeted TB7 gene after homologous recombination and the two disrupted alleles of the TB7 gene. Thin lines represent locations and sizes of flanking sequences appropriate for homologous recombination; grey boxes represent the seven exons and the 3VUTR as indicated. Black arrowheads represent loxP sites; an arrow indicates the putative translational start signal. The two alleles were targeted in two consecutive rounds of recombination with the same targeting vector. To target the first TB7 allele, the targeting vector was linearized with FspI-generating homologous flanking regions of 4.3 and 1.8 kbp suitable for anticipated homologous recombination. To target the second allele, the targeting vector was linearized with AscI-generating flanks of 3.2 and 1.8 kbp for homologous recombination. (B) The predicted structure of the targeted alleles before and after Cre-loxP-mediated excision of the two alleles is shown and the sizes of the expected DNA fragments after PsiI (P) digestion and Southern blot hybridization with the probe (nos. 14285–14646) are indicated (arrows). The locations of restriction sites for PsiI, FspI, and AscI are given. Hy/GFP: hygromycin phosphotransferase and green fluorescent protein expression cassette; TK: HSV thymidine kinase expression cassette.

Germany) for 3 h at 50 8C (50 Ag/ml final concentration); and the extraction medium was diluted to 0.3 M NaCl, thoroughly mixed, and spun at 7000 rpm for 30 min. Supernatants were precipitated with 2 vol of ethanol, and pellets were washed in 70% ethanol and resuspended in TE (10 mM Tris/5 mM EDTA). For Southern blot analysis, 10 Ag of genomic DNA was digested with PsiI restriction enzyme, separated on a 0.8% agarose gel, and transferred to

Hybond N+ Nylon Membrane (Amersham Pharmacia, Germany) after depurination in 0.25% HCl using an alkaline transfer procedure. Blots were hybridized with a 32Pradiolabelled probe, which comprised 360 bp of the TB7 3VUTR sequence, which was not part of the targeting vector. The hybridisation was performed in dChurch bufferT (7% SDS/1 mM EDTA/0.5 M Na2 HPO4) at 65 8C overnight. After hybridisation, the membranes were washed in 1%

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SDS/0.1 SSC and exposed to X-ray films in combination with intensifying screens (Kodak). 2.4. RNA analysis Total RNA was isolated from 107 cells using an RNA isolation kit (Qiagen, Germany) and mRNA was obtained using a PolyATractRmRNA isolation system (Promega, USA). One microgram of mRNA was separated on a 1% formaldehyde gel, blotted onto Hybond N+ Nylon membrane (Amersham Pharmacia), and hybridized with a 32Pradiolabelled probe. A 608-bp cDNA fragment of the TB7 3V UTR was used as a probe for Northern blot analysis.

3. Results 3.1. Experimental design Our aim was to delete the two alleles of an entire gene in a diploid human cell line. We had identified the previously unknown TB7 gene, which is expressed in B cells, in a yeast one-hybrid screen with the lytic origin of DNA replication of EBV as a bait (Hammerschmidt and Sugden, 1988; Baumann et al., 1999). Since the TB7 gene product was characterized as negatively regulating DNA replication (unpublished), we speculated that it might be indispensable for in vitro proliferation of human cells. To confirm this, we designed a conditional knockout strategy with a single targeting vector to facilitate the individual steps for a genetic knockout in diploid cells. The targeting construct can be used in consecutive rounds of recombination and is based on an intact allele flanked with loxP sites. In addition, the targeting vector was designed to minimise the inherent risk of genetic revertants. As a proof of principle of our targeting strategy, we first chose to delete the novel TB7 gene in the human cell line DG75. Sequence analysis of TB7-specific cDNAs and the corresponding TB7 gene locus of 14,646 kbp identified the intron/exon structure of the TB7 gene (Fig. 1; R.F. and W.H., to be published). The construction of the targeting vector that is suitable for two consecutive rounds of recombination is depicted in Fig. 1. The targeting vector encompassed the TB7 gene locus with its putative promoter and seven exons, but lacked the last 361 bp of the 3VUTR region (see Materials and Methods for details). Three artificial loxP sites, which are the binding motifs of Cre recombinase, were added at convenient restriction sites. One loxP site was placed upstream of the first noncoding exon of TB7, the second loxP site was positioned downstream of the translation start signal within the second intron, and the third loxP site was incorporated into the last noncoding exon of TB7 (Fig. 1A). An expression cassette encoding hygromycin phosphotransferase and enhanced GFP (Hy/GFP) was added upstream of the third loxP site within the UTR of TB7. An expression cassette for thymidine kinase of herpes

simplex virus (TK) was incorporated into the nonhomologous plasmid backbone of the targeting vector to counterselect a random chromosomal integration of the entire targeting construct. The additional DNA sequence elements were positioned such that they would not interfere with the coding capacity of TB7. As shown in Fig. 1A, linearization of the TB7 targeting vector at the single FspI site provided left and right flanks suitable for homologous recombination, which are 4.3 and 1.8 kbp in length, respectively. Successful homologous recombination in diploid cells was expected to yield one allele of TB7 bracketed with loxP sites and one remaining wild-type allele. Transient expression of Cre recombinase in cells that carry this modified allele of TB7 would excise the DNA fragment in between the two most distal loxP sites to yield the genetic knockout of the first allele and concomitantly remove both phenotypic marker genes, Hy and GFP (Fig. 1B). As a consequence, one wild-type allele and one knockout allele will both be present in heterozygous cells, which will become negative for GFP and hygromycin resistance after Cre-mediated deletion of the dfloxedT locus. In order to avoid revertants of the first knockout TB7 allele, the targeting vector used in the second round of homologous recombination was linearized with AscI to exclude homologous sequences upstream the distal 5VloxP site. About 3.2 and 1.8 kbp at the left and right flanks, respectively, were available for homologous recombination in the second targeting round (Fig. 1A). 3.2. TB7 targeting in DG75 cells After transfection of 3106 DG75 cells with the FspIlinearized targeting construct and coselection with hygromycin B and ganciclovir, we obtained more than 200 GFPpositive clones under conditions of limiting dilution that favour outgrowth of single cell clones. Twenty-nine clones were considered for further analysis. DNA Southern blotting demonstrated that 20 of 29 clones (69%) scored positive for homologous recombination (Table 1). PCR analysis was performed to confirm the presence of the three loxP sites within the targeted allele. Out of 14 clones tested, six had Table 1 Summary of TB7 targeting frequency in DG75 cells Assays

Positive/tested clones

First allele

Second allele

Southern blot hybridization PCR confirmation

Targeted/GFPpos HyR clones loxP+-confirmed/ targeted clones Cre-deleted/loxP+confirmed clones

20/29a

7/21

6/14b

2/7

10/12

12/12

PCR, Southern blot hybridization

A total of 3106 cells were stably transfected with 3 Ag of linearized targeting vector. These data were derived from two separate experiments. a Only 29 out of more than 200 GFPpos HyR clones were tested. b Only 14 targeted clones out of a total of 20 were tested in PCR analysis.

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retained all loxP sites (Table 1), confirming that homologous recombination had taken place distal from the introduced loxP sites. One of the correct cell clones was

Fig. 2. Analysis of the disrupted TB7 gene. (A) Southern blot analysis of chromosomal DNA from parental, wild-type DG75 cells are indicated with +/+; DNAs from cell clones with alterations of the first and second alleles are indicated with dtarg. 1stT and dtarg. 2ndT; clones after Cre recombination are indicated with +/ or / . Ten micrograms of PsiI-digested DNA was separated on a 0.8% agarose gel. After Southern blotting, DNA was hybridized with a 32P-radiolabelled probe, encompassing a 360-bp sequence of the 3V UTR of the TB7 gene (see Fig. 1). As shown schematically in Fig. 1, the 3Vflanking TB7 genomic probe confirmed the expected genetic modifications in the individual steps. (B) RNA from wildtype DG75 cells and a TB7 / mutant clone were examined by Northern blot analysis. One microgram of polyA+ mRNA was separated on a 1% formaldehyde gel (left panel) transferred to Nylon N+ membranes and hybridized with a probe encompassing a 680-bp sequence of the 3VUTR of the TB7 gene after Northern blotting (right panel). The probe detects a 4.4kb transcript in the parental DG75 cells as expected (data not shown), whereas in the TB7 / homozygous knockout clone, the probe detects only a faint signal of about 2.4 kb in length (arrowhead), which represents the truncated, noncoding TB7 transcript. mRNA isolated from HeLa cells was used as an internal control for the detection of TB7 mRNA.

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chosen for consecutive manipulations. Transient transfection of an expression plasmid encoding Cre recombinase into the chosen clone led to the immediate excision of the TB7 gene and the Hy/GFP cassette in 10 of 12 clones as attested by the loss of GFP fluorescence, Southern blot hybridization, and PCR analysis (Table 1, Fig. 2A; data not shown). In order to delete the remaining second wild-type TB7 allele, the identical steps as described above were performed with one of the heterozygous TB7 +/ cell clones. After transfection of the AscI-linearized vector and coselection with hygromycin B and ganciclovir, 21 GFP-positive cell clones were isolated and the structure of the TB7 locus analysed by Southern blot hybridization. Seven cell clones (33%) showed the expected restriction pattern, two of which carried both loxP sites (Table 1). One of these clones was again transiently transfected with an expression plasmid encoding Cre recombinase and 16 single cell clones were analyzed as described before. Southern and Northern blot analyses confirmed the TB7 double knockout (TB7 / ) in all cell clones (Fig. 2A and B). The homozygous TB7 / cells did not show any obvious cell growth phenotype in culture. Unexpectedly, genetic targeting of the TB7 gene proved that the DG75 cell line is appropriate and quite efficient to serve as a novel tool for gene targeting experiments in human somatic cells. Consequently, two colleagues employed the parental DG75 cell line to generate additional somatic knockouts to elucidate the function of two other genes following exactly the same strategy as described for our initial TB7 knockout. In contrast to our findings, the targeting frequency varied between 1% and 20%, but isolation of modified cell clones was always successful. In parallel, the same strategy and targeting vectors, which had been successful in the DG75 cell line, were employed to target the identical genetic loci in the human cell lines BJAB, BL2, and HeLa. No correct recombinant could be obtained (B. Kempkes and A. Kieser, personal communication). These findings show that DG75 is a cell line with unusual recombinogenic characteristics.

4. Discussion We report the successful and rather straightforward generation of a homozygous TB7 / knockout in the human DG75 cell line of B-cell origin (Goldblum et al., 1990). We used a targeting strategy that included a conditional knock-in approach as it was initially unknown whether deletion of the TB7 gene might have adverse effects on cell proliferation. The conditional knock-in step required insertion of loxP sites into the TB7 gene locus without interfering with its expression (data not shown). It was necessary to identify those cell clones in which homologous recombination had taken place to introduce the loxP sites as planned. The screening step was mandatory because legitimate recombi-

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nation can take place anywhere within the homologous region of the targeting construct (i.e., in between two loxP sites). Screening can be done via PCR analysis or by introducing diagnostic enzyme restriction sites in close vicinity to the loxP sites that allow unequivocal detection by Southern blot hybridisation. Transient transfection of a Creexpressing vector into cell clones carrying the anticipated genetic alteration efficiently led to the excision of the sequences flanked by the distal loxP sites. To identify cells that had undergone Cre-mediated deletion, it was helpful to screen phenotypically for the loss of GFP expression. Finally, the same targeting vector could be reused to target the second unmodified allele in a consecutive round. The molecular basis for the unusual high targeting efficiency in DG75 cells is unclear. Although introducing an efficiently expressed hygromycin gene and the gfp gene as selectable and phenotypic markers, respectively, augmented our strategy, this approach failed in other human cell lines tested. Many parameters influence the efficiency with which eukaryotic cellular genes can be manipulated. Cell lineage and differentiation stage of the cell likely play a major role in this regard. The propensity to recombine dforeignT DNA fragments with the genomic locus is highest in pluripotent cells such as ES cells or cell lines derived from mouse embryos (Gardner and Brook, 1997). Lymphoid precursor cells are also likely to efficiently carry out recombination or gene conversion, as these cells need to rearrange the immunoglobulin or the T-cell receptor locus in the course of their differentiation. In line with this assertion, the chicken pre-B-cell lymphoma cell line DT40 shows a high recombinogenic activity (Buerstedde and Takeda, 1991). Similarly, the DG75 cell line used in this work has been established from an immature human B-cell lymphoma (Ben Bassat et al., 1977). Lymphoma cells show a differentiation block that might be responsible for their enhanced recombinogenic activity. Other cell types have been used for targeting chromosomal loci via homologous recombination, indicating that many cells, if not all, maintain a certain recombinogenic potential (Williams et al., 1994; Waldman et al., 1995; Brown et al., 1997; Itzhaki et al., 1997; Sedivy et al., 1999 and references therein). It is obvious from these published data that somatic gene targeting is nevertheless a very laborious and inefficient process, which requires the screening of hundreds of single cell clones. In addition to its exceptional recombinogenic characteristics, several practical parameters might contribute to the efficiency of gene targeting in the DG75 cell line. For example, selection efficiency is essential for straightforward identification of genetic mutants in the process of homologous recombination. A sustainable expression level of a drug resistance gene allows higher drug concentrations during the selection process, thereby enhancing its efficiency. The hygromycin phosphotransferase gene confers resistance against hygromycin B and has been previously used in gene targeting, but showed a low degree of

efficiency compared to other selection systems, such as the neomycin resistance gene (Hanson and Sedivy, 1995; Waldman et al., 1995). Using an SV40 promoter-driven hygromycin resistance gene, which itself was also optimised for expression in eukaryotic cells (Giordano and McAllister, 1990), we obtained recombinant clones with an efficiency comparable or even better than the one reported for neomycin. One of the more widely used methods favouring homologous vs. illegitimate recombination in ES cells is the promoterless strategy. Although this strategy requires a precise knowledge of the promoter structure and functional cis-acting elements, combining the promoterless strategy with our more general approach should even yield a higher proportion of positive clones. In our approach, the large size of the flanking regions appeared to favour homologous vs. nonhomologous recombination events. Targeting frequency has been reported to increase exponentially with the length of homologous sequences before reaching a plateau at about 14 kbp (Deng and Capecchi, 1992). The use of large flanking fragments proved to be very efficient in the construction of mutants of EBV (Delecluse et al., 1998). Therefore, we decided to follow a similar strategy for cellular genes. In fact, the linear targeting construct used for ablation of the second genomic TB7 allele was smaller than the one used for the first TB7 allele, which proved to be less efficient (Table 1). Numerous cosmid and bacterial artificial chromosome libraries in which complete gene loci are contained are easily available now and allow construction of targeting vectors with large homologous flanking arms. In general, it appears that DG75 cells are particularly well suited to support genetic manipulation. The karyotype of DG75 cells is stable and diploid (our unpublished observations) and the cells are easy to handle under various cell culture conditions. Additionally and in contrast to many B-cell lines of human origin, single cell clones of the parental DG75 cell line can be obtained at ease without special precautions. Furthermore, isogenic DNA in the targeting vector is not required for efficient targeting, as appears to be the case in human somatic cells. Lastly, DG75 cells can be transfected with DNA by various methods and could therefore become an important tool for cell biologists to analyze gene function by conditional genetic knockouts in human somatic cells.

Acknowledgements We thank Bernhard Neuhierl for helpful discussions and Wolfgang Wurst for providing us with the Cre expression plasmid. We are grateful to Bettina Kempkes and Arnd Kieser for sharing their results prior to publication, and Pierre Debs for reading the manuscript. This work was supported by Public Health Service grant CA70723, grants Ha 1354/3 and SFB 455 from the Deutsche Forschungsgemeinschaft, and by a grant from the Deutsche Krebshilfe.

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