helper-free recombinant retrovirus with broad mammalian host range

Transcription

1 Proc. Natl. Acad. Sci. USA Vol. 81, pp , October 1984 Biochemistry High-efficiency gene transfer into mammalian cells: Generation of helper-free recombinant retrovirus with broad mammalian host range ROGER D. CONE AND RICHARD C. MULLIGAN Center for Cancer Research and Department of Biology, Massachusetts Institute of Technology, Cambridge, MA 02139; and Whitehead Institute for Biomedical Research, Cambridge, MA Communicated by Phillip A. Sharp, July 2, 1984 ABSTRACT We have constructed a chimeric retrovirus genome containing ecotropic gag-pol sequences from Moloney murine leukemia virus and envelope sequences derived from the amphotropic virus 4070A. This reconstructed genome, termed pma V-a*-, lacks the af site required for encapsidation of the viral genome. NIH cells transfected with pma V-af-, called aft-am lines, are capable of producing high titer stocks of helper-free recombinant retrovirus with amphotropic host range after transfection with recombinant retroviral vectors carrying the neomycin phosphotransferase gene. Most transfected aft-am cells remain helper-free, even after months in culture. f-am virus stocks infect nearly all human and murine cell lines tested thus far, as assayed by resistance to the neomycin analogue G418. Southern and RNA blot analyses of a-am-infected human cells show that recombinant murine retroviruses integrate randomly into genomic DNA as normal proviruses and express high levels of the subgenomic and genomic viral messages in the expected stoichiometry of 1:1. The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C solely to indicate this fact Recombinant retroviruses, whether created by in vivo recombination events or engineered by DNA cloning, typically are defective for replication and require helper virus to be passed from cell to cell. Recently, we constructed a helper cell line, called ai2, which, upon transfection with recombinant murine retroviral vectors, produces helper-free stocks of recombinant virus (1). Similar helper lines have been created by Watanabe and Temin for use with avian spleen necrosis virus vectors (2). The use of helper-free virus stocks in gene transfer experiments avoids unwanted biological effects of wild-type virus and facilitates efficient gene transfer, because the rapid spread of wild-type virus in tissue culture cells (3) or in whole animals (4) renders the infected cells resistant to superinfection by replication-defective recombinant virus. In addition, the availability of helper-free stocks of transforming retroviruses provides a means of determining the contribution of the helper virus component in retrovirus-mediated tumorigenesis. a/2 was created by transfecting NIH fibroblasts with pmov-a/-, an ecotropic Moloney murine leukemia virus (Mo-MuLV) clone. pmov-a/- expresses all the viral gene products, yet lacks a sequence known as ai, required for encapsidation of the viral genome. The ecotropic viral envelope glycoprotein expressed by pmov-af recognizes a receptor present only on mouse and closely related rodent cells. Hence, the use of 4i2 cells for retrovirus-mediated gene transfer is limited to a small number of species. However, MuLV strains do exist that possess a broad host range (for review, see ref. 5) as a consequence of differences in receptor specificity of the viral envelope gp7o. For example, xenotropic, mink cell focus-forming, and amphotropic MuLV strains each recognize a receptor present on many mammalian cell types, including human cells. Moreover, ecotropic MuLV genomes (such as those we have previously used for the generation of recombinant genomes) can form viral pseudotypes that contain the envelope proteins from such strains and thereby acquire widened host range. Here, we describe the construction and properties of a2- like packaging cell lines, termed a-am, useful for the production of recombinant virus with amphotropic host range. These cell lines contain a modified pmov-a/f genome in which the ecotropic envelope glycoprotein gene has been replaced with envelope sequences derived from the amphotropic virus 4070A. In addition, we have examined the integration and subsequent expression of a retroviral genome containing the neomycin phosphotransferase gene (23) in a number of human cell lines infected with virus stocks generated from the F-AM lines. MATERIALS AND METHODS NIH cells were grown in Dulbecco's modified Eagle's medium (DME medium) containing 10% calf serum. The adherent HeLa cell line JW36 (provided by P. Sharp) and early passage human foreskin fibroblasts (provided by B. Weinberg) were grown in DME medium containing 10% fetal calf serum. The human histiocytic lymphoma line U937 (6) was grown in RPMI medium containing 10% fetal calf serum. All cell cultures producing recombinant amphotropic viruses were maintained under P3 containment conditions. The plasmid pl1 was kindly supplied by A. Oliff. Plasmid constructions (7), transfections (8, 9), and reverse transcriptase assays (10) were done using standard techniques. Viral stocks were prepared by adding 10 ml of fresh DME medium containing 10% calf serum to a near confluent monolayer of cells and, after 18 hr, filtering the supernatant through a 0.45-pum filter (Milex). Infections were done by incubating 105 cells with 1 ml of virus plus 8 pug of Polybrene (Aldrich) for 2.5 hr. Cell lines were selected for resistance to the drug G418 (GIBCO) at a concentration of 1 mg/ml, except for the U937 line, which required 2 mg/ml. Genomic DNA was prepared as described (7). Total cellular RNA was prepared using a guanidine thiocyanate procedure (11), denatured for 15 min at 550C in sample buffer (50% formamide/2.2 M formaldehyde/1 x running buffer/3% Ficoll/0.001% bromphenol blue), and electrophoresed on a 1% agarose gel containing 2.2 M formaldehyde/i x running buffer [5 mm Na acetate/1 mm EDTA/20 mm 3-(N-morpholino)propanesulfonic acid, ph 7.0]. Nick-translations and RNA blot and Southern transfers were done as described (7). RESULTS MuLVs encode two polyproteins, gag-pol and env (Fig. 1). To insert an amphotropic envelope gene into pmov-af we Abbreviations: MuLV, murine leukemia virus; cfu, colony-forming units.

2 6350 Biochemistry: Cone and Mulligan Pet Bol RI SphI Sphl Portiol NdeI Colp/fte SphI NdeI GAG-POL ENV 1ca! pmov-*~- Ndel Spitl RI ENV Sph HindZ pr2 Sp hi! FIG. 1. Construction of pmav-qi-. pmav-qf was constructed by ligating together fragments from three retroviral clones, pmovif, pl1, and pzipvgpt. pbr322 sequences are represented by thin lines, Mo-MuLV sequences by medium thick lines, Ampho 4070A sequences by thick lines, F-MuLV sequences by dashed lines, and genomic sequences flanking proviruses by wavy lines. The threepart construction was necessitated by one or more Sph I sites in the genomic sequences flanking the 3' long terminal repeat of pmov- 4F. Half of the pbr322 backbone and the 5' portion of the retrovirus was contributed by pmov-iv (Nde I to Sph I), the env region was contributed by the amphotropic virus plasmid pl1 (Sph I to Cla I), and the 3' long terminal repeat through pbr322 to the Nde I site was contributed by pzipvgpt. relied on the observation that the proviruses of amphotropic MuLVs share many common restriction sites with those of ecotropic strains (12). Very little sequence data exist for the amphotropic viruses, yet the accumulated restriction data suggest that these viruses, as a whole, differ from ecotropic strains primarily in the viral envelope glycoprotein, gp7o, which is a cleavage product of the env polyprotein (12). Based on these observations, we chose to insert amphotropic gp7o sequences derived from the 4070A genome into pmov-qi- between a Sph I site -200 amino acids before the carboxyl terminus of gag-pol and a Cla I site 15 amino acids before the carboxyl terminus of pl5e. Both of these sites are found in similar positions in Mo-MuLV and 4070A. The presence of one or more unmapped Sph I sites in the large mouse DNA sequence flanking the 3' long terminal repeat of pmov-qi- necessitated the three-part construction diagrammed in Fig. 1. The recombinant clone, called pmavi/, was cotransfected with the plasmid psv2gpt (13) at a ratio of 10,ug/1,ug into NIH fibroblasts, and 3 days later the cells were split into gpt selective medium (14). Thirtyone gpt+ clones were isolated by using cloning cylinders, and tested for reverse transcriptase activity. Ten clones were found to be positive, and the levels of reverse transcriptase activity varied widely, from levels just above background to levels greater than those found in wild-type virusinfected cells. Six clones with the highest reverse transcriptase activity were expanded for further analysis. Our characterization of these i-am cell lines was aimed toward determining which clones produced the highest viral I Proc. NatL Acad ScL USA 81 (1984) titers after transfection with recombinant retroviral vectors, and also whether any of these clones produced detectable levels of wild-type amphotropic virus. After more than a month in culture, 4-AM clones 22b, 224, and 227 were found to be negative for wild-type virus production by reverse transcriptase assay of NIH cells infected with supernatants from these lines (data not shown). A more important test, however, would be to assay for wild-type virus production after stable transfection of these lines with retroviral transducing vectors that contain a qi site for viral encapsidation. The cells would contain high levels of two viral transcripts, which, if copackaged into virions, might be expected to recombine at high frequency upon subsequent infection of cells, to regenerate fully wild-type amphotropic virus. Virus Titers of Transfected to-am Lines. The method used for characterizing H-AM clones is outlined in Fig. 2. H-AM lines were transfected with 10 Ag of either pzipneosv(x)1 (15) or pmsvneo. Three days later, the cells were split into G418 selection. After 10 days to 2 weeks, G418r clones were isolated by using cloning cylinders, and plates containing clones were pooled (henceforth referred to as q,- AM populations). After continuous culture of clones and populations for >4 weeks post-transfection, virus harvests were taken as described. NIH cells and JW36 HeLa cells were infected with various dilutions of these virus stocks and then split into G418 medium 72 hr later. The titers of 4-AM virus stocks are comparable whether assayed on the murine NIH line or the human HeLa line (Fig. 3). Generation of Wild-Type Virus in H-AM Lines. The G418r titers produced by individual H-AM clones varies widely. Titers are commonly as high as 104 colony-forming units (cfu)/ml, and occasionally we have isolated clones such as q-am2275, which produces 2 x 105 G418r cfu/ml. To determine whether any H-AM clones or H-AM populations were producing wild-type amphotropic virus, supernatants from NIH cells infected with 1 ml of undiluted virus from each clone or population were assayed for reverse transcriptase activity (Fig. 2, first passage). The values shown are in arbitrary units, with a corresponding H-AM clone set at a value of 100. In addition, 1 ml of these same supernatants was assayed for G418r cfu by reinfection of new NIH and HeLa cells (Fig. 2, second passage). From >20 experiments of this type, we have shown that in most cases it is possible to transfect H/-AM lines and generate G418r cell populations or clones that continuously produce high titer stocks of helper-free recombinant virus, even after many months in culture. Nevertheless, we have also observed two different types of apparent recombinational events. For instance, NIH cells infected with virus from the pzipneosv(x)1- transfected O-AM22b population produce a very low titer of amphotropic G418r cfu (Fig. 2, row 1). However, these cells have no detectable reverse transcriptase activity, even after long-term culture. As expected, and HeLa cells infected with supernatants from first passage cells show no reverse transcriptase activity (Fig. 2, second passage) and do not produce any G418r cfu (data not shown). Furthermore, the reverse transcriptase activity and titer produced by these first passage cells remain stable over time. A second recombinational event yielded wild-type virus with amphotropic host range. After transfection of 4-AM224 and i-am222 with the vector pmsvneo, both G418r clones (Fig. 2, rows 17-19, 21) and populations (Fig. 2, rows 14 and 15) produced wild-type virus, as shown by reverse transcriptase assay of infected NIH cells and the subsequent ability of first passage cells to produce a titer of G418' cfu comparable to that of the parent H-AM cells. Second passage cells were also reverse transcriptase positive. Kinetics of Virus Spread Suggests That the Generation of Wild-Type Virus Is Rapidly Detectable. In general, when testing C/-AM cells for wild-type virus production, we have

3 L- vice~ TRANSFECT WITH io/g pzipneosv(x)i or 10og pmsvneo 22b itorl ~~~~~~~~~~~22 Biochemistry: Cone and Mulligan Proc. NatL Acad ScL USA 81 (1984) 6351 (ULATIO4S \ HARVEST INFECT 6418 INFECT SUPERNATANTS HELA CELLS 6418 HARVEST and 6418 SELECTION SUPERNATANTS HELA CELLS SELECTION CLONE (/[ HalASSAY 2:> REVERSE la TRANSCRIPTASE 0 ASSAY REVERSE TRANSCRIPTASE FIRST PASSAGE SECOND PASSAGE *-AM TITER 48r HELPER CFU * RqEVERSE t J/ml TRANSCSRIPTASE ACTIVITY TITERREVERSE TITERH TRANSCRIPTASE ACTIVITY LINE I HLa HoLe $ b I 5 x 10" x x 04 I x ea 0 ezipmoosv~x1 e 22b47 X 104 2x a 0 0 tp~~~ip~eosv(x)ii ~~~22b6 x 102 \/ x xl4 2.5b x04 4 x b 0 0 por x x b 0 0 p8r322 ~~~~~ ~~~~2276 1x1063 TRANSFECTION WITH logg OF: 22b population aia 103 l.3xlo c 227 population 3l x g b p S'V population 21 5 sited >i ~~~~~~~~~~~22b population 3 x c 0 0 plice 224 population 55x x C 3 x 103 si x 10 + S$t TR 222population 6x x I C ).I x103 I x population 5 x 3x C 0 0 siteei x10 + LTR AUIr 2224 ~ 3XI04 + pmsvnfo I it 14 + JpR o2 + "~~~Tantiqsn 2246 ~ 3x00 Polyoma--j-, 22b1 110 Early Region ori ori 22b2 5.6xzO _ 0 22b3 I x102 22b5 ' x 102 U22b6 IXI2, FIG. 2. Summary of I-AM transfection experiments. I-AM lines were transfected with two different retroviral vectors containing G418r markers as shown. G418r clones and populations ( clones) were isolated, and viral harvests were used to infect NIH and HeLa cells (first passage). After several weeks in culture, supernatants from these G418' cells, previously infected with 1 ml of undiluted virus, were assayed for reverse transcriptase activity and used to infect new NIH and HeLa cells (second passage). *, Titers were determined by counting the G418F colonies found after infection with the greatest dilution of virus still yielding colonies. t, Reverse transcriptase activity is in arbitrary units with (a) O-AM22b, (b) O-AM227, or (c) I6-AM222 set at a value of /- determinations were made by autoradiography after spotting the reverse transcriptase assay mix onto DE52 paper. SOURCE OF Vi RUS q'-am22b population Ak- 4AM 227 populationi CELL LINE IN FECTED ;( HeLa t ( HeLa VIRUS DILUTiON IO Q '. FIG. 3. Viral titering of pmsvneo-transfected I-AM cell lines. l-am lines were transfected with a retroviral vector, pmsvneo, and G418r populations were expanded. Approximately 5 x 10' HeLa or NIH cells were infected with 1 ml of virus diluted as shown. After 3 days, -5 x 10' cells were put into medium containing G418. Selective medium was changed every 3 days, and after 10 days to 2 weeks the cells were stained with crystal violet. maintained the cells in culture 2-4 weeks post-transfection before taking a viral harvest, and then maintained the subsequently infected cells in culture, again for 2-4 weeks, before assaying for reverse transcriptase or G418' cfu. To show that the spread of wild-type virus through a culture could be detected in this period of time, we have conducted a number of reconstitution assays, an example of which is shown in Fig. 4. NIH cells infected with 1 ml of undiluted virus from pzipneosv(x)1-transfected 4/-AM227 and 4i- AM22b populations were cultured for 17 days. Then, 105 of each of these cells were split into 10 ml of normal medium on a 100-mm Petri dish with 10, 1000, or 100,000 wild-type ecotropic Mo-MuLV, or amphotropic 1504A virus-producing cells. The cultures were split 1:20 every 2-3 days, and 18-hr harvests from confluent plates were assayed for reverse transcriptase activity. Reverse transcriptase activity plateaus at wild-type levels in 7-11 days and is easily detectable at 3-4 days, even if only 10 wild-type virus-producing cells are added. Expression of Recombinant Murine Retrovirus in qi-am-infected Human Cell Lines. A number of investigators have reported on the variety of non-murine cells infectable with the amphotropic viruses (16, 17). In general, the immunofluorescence focus assays used to detect infection indicate that at least some viral antigens must be expressed in the non-murine cell types infected. However, little is known about transcription of murine retroviruses in human cells. To ensure

4 , Biochemistry: Cone and Mulligan _ X _ I'~~~~~~~~~~~. X,~~~~~/.I 10 it) 0 I -, I I I 7 Ū0D 60 22b 50 -J Proc. NatL Acad ScL USA 81 (1984) U937 HeLa HOF a b c d e f g h... kb i I I I I I t Virus producing cells added DAYS ( 17 days post infection) FIG. 4. Analysis of the kinetics of spread of wild-type Mo- MuLV and 1504A virus in NIH cultures. NIH cells were infected with 1 ml of undiluted virus from populations of pzip- NeoSV(X)1-transfected qi-am227 or O-AM22b lines. Seventeen days after infection 0 (o), 10 (), 1000 (o), or 100,000 (-) Mo-MuLV (broken lines) or 1504A (solid lines) producer cells were added to 105 NIH cells. Cultures were maintained for 12 days, and the cells were split 1:20 every 2-3 days. Just before confluence, 18-hr viral harvests were collected for reverse transcriptase assays. The logarithm of the total counts incorporated into high-molecular-weight material during a standard reverse transcriptase reaction is plotted against number of days after addition of virus-producing cells. that OAM-packagepd retroviral vectors will be useful for FIG. 5. RNA blot analysis of O-AM infected cells. Cells were infected with virus from pzipneosv(x)1-transfected R-AM clones or populations. After selection in G418 medium, and cloning in the case of the human diploid fibroblasts (HDF), total cellular RNA was :8 0 electrophoresed on a formaldehyde gel, transferred to a nylon-type t s, s membrane (Zetabind), and probed with a nick-translated pbr derivative containing the neomycin phosphotransferase gene (pxlneo). Thirty micrograms was loaded per lane except for lanes a (6 Ag), g (5 pg), and h (3,Ag). Size markers were 18S rrna, 2 kilobases (kb); 28S rrna, 5.3 kb; and polio RNA, 7.5 kb. Cells were infected with virus from mock control (lane a), u.-am22b (lanes b, gene transductioh iflto human cells, we examined the integration and expression of H-AM pzipneosv(x)l virus in three hurman cell types, JW36 HeLa cells, U937 (a histiocytic lymphoma line), and early passage human diploid fibroblasts. The Mo-MuLV promoter is a moderately strong one in murine cells, because the level of viral mrna in infected cells is estimated to be 5-10% of the total cellular mrna (18, 19). We analyzed the levels of viral RNA present in infected human cells by RNA blot analysis of total cellular RNA, using the neo gene as a probe (Fig. 5). Surprisingly, we found a higher level of viral RNA in all three human lines than in NIH cells. The slight difference in migration between the human and murine RNAs appears to be an artifact, because the bands comigrated on some gels and in mixing experiments. The vector pzipneosv(x)1 constructed by Cepko et at. (15) contains -400 nucleotides between the splice donor and splice acceptor. The two bands in Fig. 5 correspond to the 4.7-kilobase full-length viral message and the 4.3-kilobase spliced subgenomic message. Since the two messages always appear to be present in equimolar amounts, this suggests that the unusual partial splice of retrovirus is also regulated properly in human cells. This fulfills an impord, and f), 4i-AM227 (lanes c and e), O-AM22bl (lane g), and 0- AM2274 (lane h). tant requirement for many of the vectors constructed in our laboratory, which depend on the production of a spliced transcript for expression of a selectable gene such as neo, and production of a genomic RNA for transmission of the recombinant genome and expression of nonselectable DNA sequences. As expected from our RNA blot data, the proviral genome in infected human cells appears to reside in the high-molecular-weight DNA in the form of full-length proviruses (Fig. 6). In addition, there do not appear to be any preferred integration sites in the human genome, at least none that occur more than -10%o of the time, because no discrete flanking fragments are detectable when the DNA is cut with the endonuclease Bgl II (which cleaves the recombinant proviral genome once) and probed with the neo gene. DISCUSSION The construction of H-AM packaging lines extends the versatility of murine retroviral vectors in allowing the infection of a broad range of mammalian cell types. With the exception of some lymphoid cell lines, nearly all human cell lines tested in our laboratory have been infectable as assayed by G418r. Some examples include the HeLa, U937, HepG2, and K562 cell lines, several secondary diploid fibroblast cultures, and secondary keratinocyte cultures (unpublished work). The monkey cell lines CV1 and Cos are also infectable as are a variety of murine and rat cell lines. Notably, we have been unable to infect Chinese hamster ovary cell lines at high efficiency with 4V2 or H-AM virus, although G418r colonies do appear at low frequency with some lines. The H/-AM line is also useful for introducing retroviral vectors into murine cells refractory to ecotropic virus as a result of previous infection (e.g., murine erythroleukemia cells).

5 Biochemistry: Cone and Mulligan HeLa U937 b c d e Ml a b c d e a b c M2 XbaI Bgll XbaI pzipneosv (X) FIG. 6. Southern analysis of a-am infected cells. Genomic DNA was isolated from aliquots of the same cells used in Fig. 5 that were infected with virus from mock supernatants (lanes a), afi-am22b (lanes b and c), and a-am227 (lanes d and e). DNA (10 Ag) was cut with Xba I (lanes b and d) or Bgl II (lanes c and e), run on a 1% agarose gel, transferred to a nylon-type filter, and probed with pxlneo. Markers are 10 pg of pzipneosv(x)1 plasmid cut with Xba I (lane Ml) or Bgl II (lane M2). kb, Kilobases. Since a-am- and *2-generated virus each recognize unique receptors, both of which are present on NIH cells, we can also rapidly shuttle retroviral genomes between /2 and /-AM to change the host range of the virus or to produce virus stocks containing two different genomes. The titer produced by /-AM lines appears, on average, to be -1/10th that produced by 42 cells transfected with the same retroviral vectors. Since pmav-a/- contains primarily ecotropic sequences, except for gp7o and pl5e, the amphotropic envelope proteins may be less efficient in mediating infection than are their ecotropic counterparts. Nevertheless, one can readily isolate lines such as i-am2275 that produce >105 recombinant virus per ml. These titers are high enough to facilitate the nonselective introduction of genes into 100% of a population of cells at high enough cell numbers to allow rapid analysis of DNA, RNA, or protein. We have observed two different events that lead to the ability of NIH cells infected with virus from transfected a-am cells to produce G418r cfu. In the first instance, the pzipneosv(x)1-transfected afram22b population appears to pass the pma V-4/- genome without subsequent recombination to generate wild-type amphotropic virus. Since retroviral recombination of copackaged genomes occurs at frequencies estimated as high as 10% (20, 21), our data suggest that the frequency of passage of the pmav-a- genome and/or the frequency of its copackaging with pzipneo- SV(X)1 must be extremely low. It is noteworthy that retroviruses are known to package nonviral RNAs at low frequency (22). The second type of event is demonstrated by the pmsvneo-transfected a-am222 and /-AM224 populations, which both produce wild-type amphotropic virus. Individual Proc. Natl. Acad Sci USA 81 (1984) 6353 clones from these transfections also produce wild-type virus, suggesting that a recombinational event occurred soon after transfection, allowing virus to spread throughout the culture. Ordinarily, wild-type ecotropic virus-infected cells are refractory to further infection by a factor of 104 relative to ininfected cells. However, f2 cells are only 1/100th as infectable as NIH cells (unpublished observations), and this is likely to be true for O-AM cells as well. Therefore, any wildtype virus, whether generated by DNA-DNA recombination or viral recombination, should spread rapidly, even through a culture of f-am cells, during the 2-4 weeks required to isolate stably transfected clones and populations. The occasional generation of wild-type virus after transfection of H-AM cell lines suggests that transfected *i-am clones or populations should be tested for such activity prior to use. Interestingly, the recombinational events that lead to regeneration of fully wild-type amphotropic virus have only been observed soon after transfection, while the majority of H-AM producers are helper-free after transfection and remain helper-free even after long-term culture. We thank Alan Oliff for supplying us with cloned 4070A proviral DNA. We also thank members of the laboratory for helpful discussion, Devon Young for preparing the manuscript, and Claire Pritchard for technical assistance. This work was supported by grants from the National Cancer Institute and the MacArthur Foundation. R.D.C. was supported by National Institutes of Health Training Grant GM Mann, R., Mulligan, R. C. & Baltimore, D. B. (1983) Cell 33, Watanabe, S. & Temin, H. M. (1983) Mol. Cell. Biol. 3, Rubin, H. (1960) Proc. Natil. Acad. Sci. USA 46, Stuhlmann, H., Cone, R. D., Mulligan, R. C. & Jaenisch, R. (1984) Proc. Natl. Acad. Sci. USA 81, in press. 5. Weiss, R. (1982) in RNA Tumor Viruses, eds. Weiss, R., Teich, N., Varmus, H. & Coffin, J. (Cold Spring Harbor Laboratory, Cold Spring Harbor, NY), pp Sundstrom, C. & Nilsson, K. (1976) Int. J. Cancer 17, Maniatis, T., Fritsch, E. F. & Sambrook, J. (1982) Molecular Cloning: A Laboratory Manual (Cold Spring Harbor Laboratory, Cold Spring Harbor, NY). 8. Graham, R. & Van der Eb, A. 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