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Construction of a recombinant adenovirus for efficient delivery of the I-SceI yeast endonuclease to human cells and its application in the in vivo cleavage of chromosomes to expose new potential telomeres
Introduction
Materials And Methods
Cell culture
Plasmids andtransfection
Viruses
Protein analysis
DNA analysis
In vitro I-SceI activity assay
Results
Construction of AdMSceI encoding the yeast I-SceI endonuclease
Expression of I-SceI from AdMSceI
Functional analysis of I-SceI expressed from AdMSceI
AdMSceI-mediated I-SceI expression leads to cleavage of chromosomal DNA at a specific site exposing a new potential telomere
Discussion
Acknowledgements
References
Construction of a recombinant adenovirus for efficient delivery of the I-SceI yeast endonuclease to human cells and its application in the in vivo cleavage of chromosomes to expose new potential telomeres
Received June 1, 1999; Revised August 5, 1999; Accepted September 8, 1999
ABSTRACT We have constructed a replication-defective adenovirus vector encoding the yeast I-SceI endonuclease under the control of the murine cytomegalovirus immediate-early gene promoter (AdMSceI) for efficient delivery of this enzyme to mammalian cells. We present evidence of AdMSceI-mediated I-SceI protein expression and cleavage activity in replication-permissive 293 cells, and of cleavage of chromosomes in vivo in both 293 cells and in non-permissive human cells. We have exploited this system for the generation of chromosomes capped by artificial telomeric sequences in cells with integrated plasmids containing telomeric DNA arrays adjacent to an I-SceI recognition site. The properties of the AdMSceI virus described here make it a useful tool for studying biological processes involving induction of DNA breaks, recombination and gene targeting in cells grown in culture and in vivo.
INTRODUCTION
Rare cutting endonucleases have been isolated from different organisms (1-5) and used as tools for DNA manipulation and genome analysis. I-SceI is a yeast endonuclease involved in an `intron homing' process (reviewed in 6). Due to its relatively large recognition site (>18 bp) and high specificity (7-9), I-SceI constitutes a powerful tool that has been widely used in cells from many organisms, including mammals (reviewed in 10). To investigate their effects on DNA repair and genome stability, double-strand breaks have been generated in cell genomes through the introduction of artificial I-SceI sites followed by endonuclease expression (11-14). The ability of I-SceI to induce DNA recombination has made this enzyme useful for gene targeting in Xenopus oocytes and in mouse embryonic stem cells (15-18). Moreover, I-SceI has been used in high resolution physical mapping (19,20) and genome organization analysis (21,22).
Delivery of the endonuclease to cells has commonly been by transfection of I-SceI-encoding plasmids, a method which limits the number of I-SceI-positive cells to a subset of the cell population and requires selection of these cells for analysis of the I-SceI products. Efficient delivery of I-SceI to the whole cell population in a synchronous manner would represent a substantial improvement of the system. Recombinant vectors derived from human adenoviruses are particularly suited for efficient transduction of foreign genes into mammalian cells since they can infect a wide variety of cell types from different organisms and induce a high level of transgene expression (reviewed in 23). Typically, adenovirus vectors are rendered replication-defective by deletion of the E1 region that encodes functions required for viral replication. These vectors can be rescued and propagated only in complementing cells, namely 293 cells that constitutively express the viral E1 region. However, they retain the ability to infect non-permissive cells and have been widely used for various purposes in cells in culture and in vivo.
We are interested in the biology of human telomeres, the essential nucleoprotein structures at the ends of chromosomes (reviewed in 24) whose erosion with cell division has been causally associated with the senescence of normal human cells (25,26; reviewed in 27). Direct manipulation of telomere length within a cell is not possible at present, and introduction (`seeding') of artificial telomeres of defined length has been achieved in immortal but not in normal somatic cells (28-31). To overcome these impediments to the evaluation of the role of telomeres in cell senescence, we have explored the use of the I-SceI endonuclease for in vivo cleavage of chromosomes at internally seeded artificial telomeric sequences.
In this report we describe the construction and the characterization of a recombinant adenovirus vector, AdMSceI, in which expression of I-SceI is driven by the murine cytomegalovirus (MCMV) immediate-early gene promoter, and illustrate the application of this vector in the generation of chromosomes with new telomeric termini. To this end, we modified a strategy used in yeast (32) for the generation of naked chromosome ends that involved construction of a strain with telomeric DNA arrays preceded by the recognition site for the rare cutting HO endonuclease (1). Cleavage by HO generated a telomere-less chromosome. In our system, the recognition site for a different endonuclease, I-SceI, was placed at the 3[prime]-end of the telomeric array so that, after integration of the telomeric plasmid and cleavage by I-SceI in vivo, a new telomere may be formed.
MATERIALS AND METHODS
Cell culture
The cell line 293 (33) was grown in MEM-F11 supplemented with antibiotics and 10% fetal calf serum (Gibco). The human foreskin diploid fibroblast strain NSF5A and human embryonic kidney cell strain (HEK) were grown in [alpha]-MEM supplemented with antibiotics and 10% fetal calf serum.
Plasmids andtransfection
Restriction and modification enzymes were purchased from Boehringer Mannheim and Pharmacia. Plasmids were constructed by standard protocols and grown in Escherichia coli DH5[alpha]. Plasmid DNA was extracted by the alkaline lysis method (34) and purified by CsCl density gradient centrifugation. pCMVn-I-SceI (a gift from Maria Jasin, Sloan-Kettering Institute, New York, NY) contains the I-SceI ORF driven by the human cytomegalovirus immediate-early gene promoter, in-frame with a Kozak consensus sequence, a nuclear localization signal and the HA epitope tag (pCMVn-I-SceI is a slightly modified version of pCMV-I-SceI; 35; P.Rouet and M.Jasin, unpublished results). pMH4 (36) is a shuttle plasmid which contains the left end of the Ad5 genome with the E1 region replaced by the promoter of the MCMV immediate-early gene, a polylinker and the SV40 polyadenylation signal. pJM17 carries the entire Ad5 genome with a deletion/substitution in E3 and an insertion of pBR322 in E1 (37,38). pMA1 and pMA2 (Fig. 1B) contain a synthetic I-SceI recognition site, GTTACGCTAGGGATAA/CAGGGTAAT (7), the neor selectable marker, and the SV40 large T antigen gene. The latter was introduced into the seed plasmids to induce lifespan extension of normal cell strains (39). In pMA1 and pMA2, the I-SceI recognition site is 3[prime] to T2AG3 telomeric repeat arrays of 1600 and 800 bp, respectively. Transient transfection of plasmids was performed by the calcium phosphate protocol (40) and plasmid DNA was extracted from transfected cells according to Serghini et al. (41). For stable transfections, 5 × 106 cells in 500 µl PBS were electroporated with 2 pg plasmid DNA/cell using a BTX-300 transfector (Biotechnologies and Experimental Research Inc.) and a single pulse of 400 V, 200 µF, and seeded in 10 cm plates (5 × 105 per plate) in non-selective medium. After 24 h the cells were re-fed with medium supplemented with G418 (400 µg/ml). Clones were isolated after 10-15 days.
Figure 1. (A) Construction of AdMSceI. An 853 bp EcoRI-SalI fragment containing the I-SceI ORF was inserted into the unique EcoRI (E) and SalI (S) sites of the shuttle plasmid pMH4, and the resulting pMH4SceI plasmid was co-transfected with pJM17 into 293 cells. Thin lines represent plasmid sequences and black boxes viral sequences. The location of the MCMV promoter, SV40 poly(A) tract and ampicillinr gene are indicated. Plasmids are not drawn to scale. (B) Plasmids pMA1 and pMA2. The telomeric arrays T2AG3 (Tel) (1600 bp in pMA1 and 800 bp in pMA2) are oriented in the direction of the arrow. The locations of the I-SceI site, neomycinr gene (Neo), ampicillinr gene (Amp) and SV40 large T antigen gene (TAg) are indicated.
Viruses
Addl70-3 is a recombinant adenovirus with a deletion in the E1 region (42). Recombinant AdMSce-I adenovirus was constructed, grown and titrated in replication-permissive 293 cells as described (43) and shown in Figure 1A.
Protein analysis
Cells were lysed in NP-40 lysis buffer (50 mM Tris pH 7.2, 150 mM NaCl, 1% NP-40) with 1 mM aprotinin. Proteins were separated by electrophoresis on SDS-10% polyacrylamide gels and transferred to PVDF membrane (Boehringer Mannheim). Western blotting was carried out according to Towbin et al. (44), using an anti-HA rat monoclonal antibody (clone 3F10; Boehringer Mannheim) at a concentration of 100 ng/ml in Tris-buffered 2% blocking reagent (Boehringer Mannheim) and goat anti-rat IgG coupled to alkaline phosphatase as secondary antibody, at a dilution of 1:20 000 in Tris-buffered 2% blocking reagent. The phosphatase reaction was monitored using CSPD chemiluminescent reagent (Boehringer Mannheim) and X-ray films (NENTM, Life Science Products). The membranes were then stained with Fast Green FCF (45) for determination of amount loaded.
DNA analysis
DNA was digested with restriction enzymes, separated on 0.8% agarose gels and transferred to a nylon membrane (Boehringer Mannheim). DNA probes were labeled with [[alpha]-32P]dCTP by random priming (Boehringer Mannheim) and hybridization was carried out overnight in Church buffer at 60°C (46). High stringency washes were: twice in 1× SSC, 0.1% SDS at 68°C and once in 0.2× SSC, 0.1% SDS at 68°C. Detection of the signal was by autoradiography on X-ray films (NENTM, Life Science Products or Kodak) or by exposure to PhosphorImager screens (Molecular Dynamics). Hybridization signals were quantified using ImageQuant software (Molecular Dynamics).
In vitro I-SceI activity assay
Cell pellets were stored at -70°C or directly lysed in I-SceI storage buffer (Boehringer Mannheim) with 0.5% CHAPSO, 0.25 mM deoxycholate, 1 mM aprotinin. Protein aliquots were incubated with 200 ng of NdeI-digested pMA2 plasmid, containing the target sequence for the I-SceI nuclease. The incubation was carried out in working buffer (Boehringer Mannheim) for 30 min at 37°C. The reaction was stopped by phenol/chloroform extraction and the DNA was precipitated. Control digestions with the commercial I-SceI enzyme (Boehringer Mannheim) were also performed. The DNA was then separated by electrophoresis, transferred to nylon membranes and analyzed by Southern hybridization.
RESULTS
Construction of AdMSceI encoding the yeast I-SceI endonuclease
Construction of the recombinant adenovirus AdMSceI is shown in Figure 1A. The 853 bp EcoRI-SalI fragment from pCMVn-I-SceI, containing the I-SceI ORF, a Kozak consensus sequence, nuclear localization signal and HA-epitope tag, was cloned in the EcoRI and SalI sites of pMH4 adjacent to the MCMV promoter. The resulting plasmid pMH4SceI contains the left end of the adenovirus genome with the E1 region replaced by the I-SceI expression cassette. pMH4SceI was co-transfected with pJM17, which contains the entire Ad5 genome, into E1-complementing 293 cells and homologous recombination between the two plasmids yielded the recombinant adenovirus (43). Several plaques were isolated and viral DNA was analyzed to verify the structure of the recombinant. Finally, one such recombinant, AdMSceI, was expanded in 293 cells to produce high titer viral stocks.
The structure of the pMA1 and pMA2 plasmids, containing an I-SceI recognition site at the 3[prime]-end of telomeric repeat arrays of 1600 and 800 bp, respectively, is shown in Figure 1B.
Expression of I-SceI from AdMSceI
Expression of the I-SceI protein in cells infected by AdMSceI was analyzed by western blotting using an antibody against the HA tag. 293 cells were infected at a multiplicity of infection (MOI) of 10 p.f.u./cell with AdMSceI or with the control Addl70-3 virus or were mock infected, and harvested 12-40 h thereafter (Fig. 2A). In AdMSceI-infected cells the I-SceI protein was detectable as early as 16 h after infection and its amount remained essentially constant up to 40 h. Two forms of the protein were produced due to a second in-frame start codon 90 bp downstream of the first one. The full-length 30 kDa product was more abundant, likely because of the Kozak consensus sequence at its translation start site. No expression was observed in mock-infected cells or in cells infected with Addl70-3. In a second assay, 293 cells were infected at different MOI (10-100 p.f.u./cell) with AdMSceI or with Addl70-3 or were mock infected, and harvested after 16 h (Fig. 2B). Expression of I-SceI after infection with AdMSceI increased as a function of MOI.
Figure 2. The I-SceI endonuclease encoded by AdMSceI is highly expressed in human cells. Proteins extracted from AdMSceI-infected 293 cells were separated on an SDS-10% polyacrylamide gel, transferred to a PVDF membrane and the I-SceI protein was detected with an anti-HA monoclonal antibody. Molecular weights (in kD) are on the left. Protein staining with Fast Green FCF, shown below each panel, was used for loading control. (A) 293 cells were infected with AdMSceI or Addl70-3 at an MOI of 10 p.f.u./cell or were mock infected and harvested at the indicated times post-infection (h). (B) 293 cells were infected with AdMSceI or Addl70-3 at the indicated MOI or mock infected and harvested 16 h after infection.
Functional analysis of I-SceI expressed from AdMSceI
In a functional assay for enzymatic activity, extracts from 293 cells infected with AdMSceI or control virus or mock infected were tested on the target plasmid pMA2. In vitro activity assays were performed as a function of time post-infection and of MOI (Fig. 3). Protein extracts were incubated with NdeI-digested pMA2 DNA which was then analyzed by Southern hybridization with the neo probe. NdeI digestion of pMA2 was predicted to yield a fragment of 5.7 kb, while a fragment of 4.3 kb was expected after additional cleavage with I-SceI (Fig. 3A). Efficiency of cleavage was determined by the ratio of these two fragments. As shown in Figure 3B, essentially complete cleavage was achieved with the commercial I-SceI nuclease (95%, lane mock+). Cleavage from cell extracts infected with 10 p.f.u./cell of AdMSceI was partial but increased with time up to 16 h of infection. Extracts prepared at later times of infection had decreased activity in this assay, although the amount of I-SceI protein present in the cells remained constant over this period (Fig. 2A). No cleavage was detected upon incubation of pMA2 with extracts from mock-infected or Addl70-3 infected cells. Extracts prepared from cells infected with increasing MOI of AdMSceI and harvested after 16 h showed increasing cleavage activity with a maximum of 45% of cleaved molecules at an MOI of 100 (Fig. 3C). Again, no activity was observed with extracts from mock-infected or Addl70-3-infected cells.
Figure 3. I-SceI expressed from AdMSceI can cleave at its recognition site in vivo and in vitro. I-SceI cleavage of pMA2 DNA was detected by Southern hybridization with a neo probe. (A) Linearized pMA2 with NdeI (N) and I-SceI (S) sites is shown, with the region detected by the neo probe in black. Arrows represent the telomeric array in its orientation. The products detected after cleavage with NdeI and I-SceI (4.3 kb) as well as with NdeI alone (5.7 kb) are indicated. (B) In vitro I-SceI activity assay: efficiency of cleavage as a function of length of infection. 293 cells were infected with AdMSceI or Addl70-3 at an MOI of 10 p.f.u./cell or were mock infected and harvested at the indicated times post-infection (h). Aliquots of 200 ng of NdeI-digested pMA2 were incubated with 15 µg of total protein from the cell extracts. Molecular weights (kb) are on the left. Mock+ refers to plasmid cleaved with the commercial I-SceI and mock- to plasmid incubated with protein from mock-infected cells. The cleavage activity in percent cleaved molecules is shown below each lane. (C) In vitro I-SceI activity assay: efficiency of cleavage as a function of MOI. 293 cells were infected with AdMSceI or Addl70-3 at the indicated MOI or were mock infected and harvested 16 h after infection. Incubation, molecular weights, mock samples and cleavage activity are as in (B). (D) In vivo I-SceI activity assay. 293 cells were transiently transfected with pMA2 and infected with AdMSceI at the indicated MOI or were mock infected. Plasmid was rescued 16 h after infection and cut with NdeI. Mock- refers to the plasmid DNA from mock-infected cells cleaved with NdeI. Molecular weights and cleavage activity are as in (B).
To test I-SceI activity in vivo, 293 cells were transiently transfected with pMA2 and infected with AdMSceI at increasing MOI or mock infected. Plasmid DNA was rescued at 16 h post-infection, cut with NdeI and analyzed by Southern hybridization. In agreement with the in vitro results, cleavage efficiency increased with MOI to reach 62% at an MOI of 100 (Fig. 3D). These values may be an underestimate of the actual enzymatic activity since in this assay plasmid molecules retained in the cytoplasm would not be accessible to the enzyme.
AdMSceI-mediated I-SceI expression leads to cleavage of chromosomal DNA at a specific site exposing a new potential telomere
We completed the functional characterization of the AdMSceI-encoded I-SceI by assessing its ability to cleave I-SceI recognition sites integrated into the cell genome. In initial experiments, pMA2 was stably transfected in permissive 293 cells and DNA from clonal cell populations was analyzed for plasmid integration and presence of the I-SceI site by digestion with HindIII and with the commercial I-SceI enzyme, followed by Southern hybridization. Clone 293.1 had a single insertion of pMA2 and was chosen as suitable for further studies. Cells were infected with AdMSceI at increasing MOI or infected with Addl70-3 at an MOI of 100 p.f.u./cell or mock infected, and after 22 h the DNA was extracted, cut with HindIII and analyzed by Southern blotting (Fig. 4). HindIII digestion generated a fragment of 3 kb, while additional cleavage with the commercial I-SceI resulted in the expected fragment of 2 kb. Cleavage was observed in AdMSceI-infected cells with an efficiency of 10, 21, 31 and 43% of cleaved molecules at MOI of 10, 20, 50 and 100 p.f.u./cell respectively, revealing its direct dependence on the MOI as suggested by the results of in vitro assays.
Figure 4. AdMSceI-encoded I-SceI can efficiently cleave an intrachromosomal recognition site in vivo in replication-permissive cells. Genomic DNA, extracted from AdMSceI-infected 293.1 cells, Addl70-3-infected cells or mock-infected cells at 22 h after infection was digested with HindIII and analyzed by Southern hybridization with a neo probe. (A) Structure of integrated pMA2. Solid bars represents plasmid DNA with the region detected by the neo probe in black. Thin lines represent chromosome sequences and arrows indicate the telomeric array in its orientation. The products of cleavage with HindIII (H) and I-SceI (S) (2 kb) or HindIII alone (3 kb) are shown. (B) MOI and molecular weights (in kb) are indicated. The cleavage activity in percent cleaved molecules is shown below each lane. Mock- refers to DNA from mock-infected cells digested with HindIII, while mock+ refers to the same DNA digested with HindIII and commercial I-SceI.
Viral replication in permissive 293 cells ultimately results in cell lysis which prevents long-term analysis of the fate of the modified chromosome and its effects on cell growth. To assess whether infection of non-permissive cells would similarly result in sufficient production of active I-SceI and chromosomal cleavage, pMA1 and pMA2 were stably transfected in diploid HEK cells and NSF5A fibroblasts. A total of 26 clones from both cell types were screened for plasmid integration by HindIII digestion and Southern hybridization, and 15 were also analyzed for the presence of the I-SceI site by cleavage with the commercial I-SceI. Of these, 12 clones retained the site. Pilot experiments were performed with these clones by infection with AdMSceI at an MOI of 200 p.f.u./cell for 48 h. Widely different efficiencies of cleavage were obtained for different clones with a maximum of 85% of cleaved molecules (data not shown). We focused on the NSF5A clone 6B22, because its DNA was cleaved in vivo with the highest efficiency. Further characterization of this clone revealed that two copies of pMA2 were inserted in the cell genome in a head-to-head manner such that the I-SceI site was flanked on both sides by telomeric sequences (Fig. 5A). Cells were infected with AdMSceI at an MOI of 20, 50, 100 and 200 p.f.u./cell or with Addl70-3 at an MOI of 200 or mock infected, and DNA was extracted after 48 h and analyzed by Southern hybridization (Fig. 5B). Fragments of 4 or 8.6 kb were expected after DNA digestion with HindIII or NdeI, respectively, and additional cleavage with I-SceI should reduce these fragments to doublets of 2 and 4.3 kb, respectively (Fig. 5A). In agreement with the results obtained with 293 cells, in AdMSceI-infected 6B22 cells cleavage efficiency increased as a function of MOI (Fig. 5B). Due to the particular orientation of the integrated plasmids, cleavage with I-SceI generated two chromosome ends both having terminal telomeric DNA sequences, of which one will be lost with the acentric chromosome arm upon cell division. The long-term functionality and the fate of the artificial telomere associated with the centromeric chromosome arm were not assessed in this study.
Figure 5. AdMSceI-encoded I-SceI can efficiently cleave an intrachromosomal recognition site in vivo in non-permissive cells. (A) Structure of integrated pMA2. HindIII (H), NdeI (N) and I-SceI (S) sites are indicated. Solid bars represent the plasmid DNA and thin lines chromosomal DNA. The region detected by the neo probe is indicated in black. Arrows indicate the telomeric array in its orientation. The products detected after digestion with HindIII and I-SceI (2 kb), with HindIII alone (4 kb), with NdeI and I-SceI (4.3 kb) and with NdeI alone (8.6 kb) are shown. (B) 6B22 cells were infected with AdMSceI or with Addl70-3 at the indicated MOI or mock infected, and DNA was extracted at 48 h post-infection, cleaved with HindIII (left) or with NdeI (right) and analyzed by Southern hybridization. Molecular weights (in kb) are indicated on the side and cleavage activity in percent cleaved molecules at the bottom of each panel. Mock+ refers to DNA from mock-infected cells, cleaved with HindIII or NdeI and the commercial I-SceI, and mock- refers to the same DNA cleaved with HindIII or NdeI alone.
DISCUSSION
The yeast I-SceI endonuclease is a powerful tool for cleaving DNA and has been used in studies on DNA repair, gene targeting and genome analysis (13,14,18,22). To achieve efficient delivery of I-SceI to human cell populations we have constructed the recombinant adenovirus AdMSceI encoding the endonuclease under control of the MCMV promoter. Infection of cells with AdMSceI resulted in the expression of I-SceI protein in amounts increasing with MOI. Detection of the protein in transfected cells has not been reported to date (35). Thus, the easiness of detection and abundance of the I-SceI protein in infected cells is further demonstration of the efficiency of the viral delivery system. Functional characterization of the AdMSceI-encoded I-SceI indicated that the protein was active and specifically cleaved I-SceI sites on plasmids or integrated into the cell genome. Enzymatic activity increased with MOI but declined at late times post-infection in permissive cells. The reason for this decline, which was not observed in non-permissive cells (data not shown), is unclear but might be related to the length of infection and the onset of cell lysis. This may also contribute to the partial activity of the enzyme detected in these cells on both types of DNA substrates (plasmid and genomic).
Higher cleavage efficiency (up to 85%) was obtained in clones of non-permissive cells bearing a chromosomal I-SceI site. Analysis of this system, however, revealed great variability in the susceptibility of integrated recognition sites to cleavage, including the existence of uncleavable sites, suggesting that chromatin conformation may affect accessibility of the site to the enzyme. Selection for the cleavage event through addition, distal to the I-SceI site, of a marker whose loss confers resistance to a drug (e.g. HSV-TK) should counteract the generation of heterogeneous cell populations while still maintaining the advantages of delivering the enzyme by infection rather than transfection and of rescuing the majority of the cells without the need for clonal selection. Interestingly, no dependence on chromosomal location was detected in previous studies (47) assessing the ability of adenovirus-encoded Cre recombinase to cleave chromosomally integrated loxP sites, pointing to differences in the potency and structural requirements for the activity of Cre and I-SceI.
We have successfully used AdMSceI infection for the generation of chromosomes capped by artificial telomeric sequences in mortal human cells, resulting in a method for direct manipulation of telomere length in vivo that should allow us to investigate the role of a critically short telomere on cell growth and viability (reviewed in 27). The human diploid mortal cells used as recipient of telomeric constructs have no mechanism for telomere maintenance (49; reviewed in 27) but, given their derivation from newborn or fetal tissues, contain telomeres that are still 8-10 kb in length (49,50). We expect therefore that the short telomeric sequences exposed by the I-SceI cleavage will be progressively lost with cell division at the same rate as those of the longer natural telomeres, ultimately creating a single telomere-less chromosome. The combination of AdMSceI infection and of plasmids bearing telomeric sequences can also be used to generate chromosome truncations for gene mapping, in a modification of the telomere-associated chromosome fragmentation system (29,48). We envisage a variety of additional useful applications of the AdMSceI delivery system ranging from the induction of double-strand DNA breaks for repair studies to gene targeting and gene manipulation in vivo (reviewed in 10).
ACKNOWLEDGEMENTS
We are grateful to Maria Jasin for the pCMVn-I-SceI plasmid, and to Frank L. Graham and Cristiana Guiducci for critical reading of the manuscript. This work was supported by a grant (no. 7251) form the National Cancer Institute of Canada (NCIC). S.B. was a Terry Fox Cancer Research Scientist of the NCIC.
REFERENCES
*To whom correspondence should be addressed. Tel: +1 905 525 9140; Fax: +1 905 546 9940; Email: bacchett{at}fhs.mcmaster.ca
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