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Home , Plasmid, Retrovirus

Proc. Nati. Acad. Sci. USA Vol. 81, pp. 7137-7140, November 1984 Developmental

Construction of a capable of transducing and expressing in multipotential embryonic cells (retroviral restriction/embryonal carcinoma celis/neo ) JOHN L. R. RUBENSTEIN, JEAN-FRANIOIS NICOLAS*, AND FRAN(OIS JACOB Unitd de Gdndtique Cellulaire du ColIfge de France et de I'Institut Pasteur, 25, Rue du Dr. Roux, 75724 Paris Cedex 15, France Contributed by Franfois Jacob, July 16, 1984

ABSTRACT Retroviral is inhibited in em- SVtk-neo and neo fragments. These fragments were then in- bryonal carcinoma (EC) cells. We have constructed a recombi- serted between the Sal I and BamHI sites of pB6 (12), a plas- nant retroviral vector that is capable of expressing the neomy- mid derived from pMOV3 (13) (a proviral copy of M-MuLV). cin-resistance (neo) gene in EC cells. The critical modification The Sal I site was constructed by inserting the M13mp8 po- that permits expression of the neo gene is the insertion of a lylinker (14) into the Pst I site at position 563 ofthe M-MuLV composite simian 40 early gene- gRNA (15). The BamHI site, which is at position 6537 of the type 1 gene (SVtk) promoter 3' to the viral M-MuLV gRNA, is in the 5' end of the gene. first intron and 5' to the neo gene. When the SVtk promoter is Procedures used in the construction of these vectors are deleted, the recombinant retrovirus is either unable or ex- referenced in Rubenstein and Chappell (16). The tremely inefficient at expressing the neo gene in EC cells. used in the construction were obtained from Boeh- ringer Mannheim. Microinjection is the only method that has been used suc- Culture, Virus , and G418 Selection. Cell cessfully to introduce DNA into cells of the , and lines. All cells were grown in Dulbecco's modified Eagle's recently proper developmental regulation of microinjected medium (DME medium) containing 10% fetal calf serum, genes has been obtained (1). An alternative to this technique penicillin, and streptomycin. The mouse EC cell lines uti- would be to use viral vectors to transduce genes into embry- lized were LT-1, PCC4AzaR1, and PCC3 HPRT-OuabR (17). onic cells. are an attractive choice for several The mouse differentiated cell lines were q2 (5), NIH 3T3 reasons. The only structures required to produce recombi- (18), and NIH 3T6. nant genomic RNA (gRNA) from proviral DNA are grouped Virus production. 42 cells containing recombinant retro- at the 5' and 3' termini of the . These structures in- viral DNA were placed in 6-cm-diameter culture dishes con- clude: the long terminal repeats (LTR), which are required taining 4 ml of medium without G418. The cell density was for integration (2); ofthe gRNA and polyadeny- approximately at 50% confluency. After incubating the cells lylation (3); and the first intron, which contains the packag- at 37°C for 24 hr, the medium was removed, and then it was ing signal (4, 5). Furthermore, a transcomplementing cell line centrifuged at 3000 x g at 4°C for 15 min. has been constructed that efficiently produces virions con- Virus infection. One milliliter of virus stock, containing 5 taining only the recombinant gRNA (5). Finally, the integrat- ,ug of Polybrene (Sigma) per ml was added to 5 x 105 cells in ed recombinant virus can be reisolated by infecting the cell 6-cm plates. The samples were incubated at 37°C for 2 hr, with a wild-type retrovirus (6). and then the medium was replaced with fresh medium. One However, although retroviruses can infect embryonic day after the viral infection (or DNA transformation, see cells and integrate into the (7), there is a block next section), various numbers of cells (between 5 x 105 and in their gene expression in both (8) and embryonal 2.5 x 104) were transferred to 10-cm plates. One day after carcinoma (EC) cells (9). This block may be at the level of this transfer, the medium was replaced with medium con- transcription (10) or RNA maturation, or both. An approach taining 500 ,ug of G418 (geneticin from GIBCO) per ml and to overcome this block stems from our observation that the was changed every 2 days until only G418-resistant cells simian virus 40 (SV40) enhancer is functional in EC cells were present, at which point the medium was changed every (11). In this paper, we describe the construction of a recom- 3 days. The G418-resistant colonies were counted at 10-14 binant retroviral vector, based on the Moloney murine leu- days after the beginning of the G418 selection. Individual kemia virus (M-MuLV), in which the gag, (polymerase), clones were obtained by sucking them up into a 2-ml pipette and env (envelope) genes are replaced by the Tn5 - and transferring them into 1-cm culture wells. resistance (neo) gene, under the transcriptional control of a DNA Transformation. Plasmid DNA was transfected into composite SV40 early gene-herpes simplex type 1 (HSV-1) cells by the technique of Graham and van der Eb (19). The thymidine kinase gene (tk) promoter (SVtk) (11). We report calcium phosphate precipitate was formed with 20 ,ug of that this retroviral vector can lead to the production of high DNA in a volume of 500 ,ul. In acute and in titers of a virus that is capable of transmitting the neo gene most of the stable transfections, 6.6 ,tg of the plasmid into EC cells. Furthermore, the neo gene-encoded amino- pCH110 was added to the other DNA as an internal control glycoside 3'-phosphotransferase is stably expressed. for the efficiency of . This plasmid expresses f3- galactosidase (20). The DNA precipitates were added to a 6- MATERIALS AND METHODS cm plate containing 5 x 105 cells. Then, after incubating the Construction of Retroviral Vector. Description of the con- struction of the retroviral vectors will be presented else- Abbreviations: LTR, ; EC, embryonal carcino- where. Briefly, pSVtk-neo3 (11) was used to obtain the ma; neo, neomycin-resistance gene; M-MuLV, Moloney murine leu- kemia virus; HSV-1, herpes simplex virus type 1; gRNA, genomic RNA; env, envelope gene; pol, polymerase gene; SV40, simian virus The publication costs of this article were defrayed in part by page charge 40; tk, thymidine kinase gene; SVtk, composite of SV40 early gene payment. This article must therefore be hereby marked "advertisement" promoter-HSV-1 tk promoter. in accordance with 18 U.S.C. §1734 solely to indicate this fact. *To whom reprint requests should be addressed.

7137 Downloaded by guest on September 26, 2021 7138 Developmental Biology: Rubenstein et al. Proc. Natl. Acad ScL USA 81 (1984) cells for 30 min at 220C, 5 ml of medium was added. One day tional control of only the LTR, whereas in pM-MuLV-SVtk- later, the medium was changed; then, after an additional 24 neo, the neo gene is expected to be under the transcriptional hr, extracts were prepared from the cells. For stable control of the LTR, the SV40 early gene promoter, and the transformation experiments, the cells were diluted into 10- HSV-1 tk promoter. cm plates containing fresh medium after the initial 24 hr (see The Retroviral Vector Containing the Internal SVtk Pro- the previous section). moter Expresses the neo Gene in EC Cells. Transient expres- Assay of Protein Extracts for Aminoglycoside 3'-Phospho- sion. To test whether unintegrated copies of a retroviral vec- and 8-Galactosidase. Cells were washed twice us- tor can express the neo gene, we transfected pM-MuLV- ing TS buffer (138 mM NaCI/5 mM KCI/0.7 mM Na2HPO4/ SVtk-neo DNA into fibroblast cells (NIH 3T6) and EC cells 25 mM Tris base/0.7 mM CaCl2/1.0 mM MgCl2, pH 7.4). (LT-1). The recombinant retroviral DNA led to the produc- Then, after adding 200 p.l of lysis buffer (85% TS buffer/15% tion of detectable quantities of aminoglycoside 3'-phospho- glycerol containing 10 mM dithiothreitol), the cells were transferase in both cell lines, although pM-MuLV-SVtk-neo scraped off of the plates and sonicated (Ultrasonics W-375) was expressed 5-fold more efficiently in the NIH 3T6 cells for 2 min at 40C at 100% power output and 50% duty cycle than in the LT-1 cells (Fig. 2). settings. Next, 1/10th vol of 0.5% deoxycholate/1.0% Noni- Stable transformants. Next we tested whether the recom- det P-40 was added. The cellular debris was pelletted by cen- binant retroviral vectors could stably express the neo gene in trifugation for 10 min in a Beckman Microfuge, and the su- EC cells (presumably after chromosomal integration). We pernatant was saved. The protein concentration of the ex- transfected LT-1 cells with pM-MuLV-neo or pM-MuLV- tract was assayed by the Bradford procedure (21). The SVtk-neo DNA. Table 1 reports the efficiency of producing activities of f-galactosidase and of aminoglycoside 3'-phos- G418-resistant clones and shows that only the retroviral vec- photransferase were assayed as described, respectively (22, tor containing the SVtk composite promoter had the ability 23). to produce G418-resistant LT-1 cells, although both plas- mids were equally efficient at transforming differentiated cells (see next section). Next, to learn whether adjacent ret- RESULTS roviral sequences have an effect on the expression of the Structural Features of the Retroviral Vectors. Fig. 1 shows SVtk-neo gene in EC cells, we compared the efficiency of the structure of two M-MuLV-derived retroviral vectors. transformation of pM-MuLV-SVtk-neo to that of pSVtk- There are a number of features of these molecules that war- neo,3, a nonretroviral plasmid that also uses the SVtk pro- rant description. (i) There are two 492-base-pair LTRs that moter to express the neo gene. These two were es- are essential for transcription of the genomic RNA and inte- sentially equally efficient at conferring G418 resistance to gration of the provirus (2, 3). (ii) 3' to the 5' LTR are 418 LT-1 cells (Table 1). Therefore, it is unlikely that a base pairs (up to position 563 of the gRNA; see ref. 15) that is required for pM-MuLV-SVtk-neo to be transcriptionally contain M-MuLV's first intron (15) and the putative packag- active in EC cells. ing signal of the genomic RNA (5). (iii) The following Sal I Production of from a Recombinant Retroviral Plas- recognition site is derived from the M13mp8 bacterial phage mid Containing an Internal SVtk-neo Transcription Unit. To polylinker (14). (iv) Between this Sal I site and the BamHI produce virions carrying gRNA encoded by pM-MuLV-neo site (position 6537 in the M-MuLV gRNA) is the 1100-base- or pM-MuLV-SVtk-neo, we transfected these plasmids into pair neo gene. The neo gene can confer G418 resistance to q2 cells, a retrovirus transcomplementing cell line that does mammalian cells (11, 24). One of the plasmids, pM-MuLV- not produce M-MuLV but can package recombinant retro- SVtk-neo has the SV40 early gene promoter and the HSV-1 viruses (5). After the plasmid transfection, cells expressing tk promoter [600 base pairs (11)] 5' to the neo gene. (v) Fol- the neo gene were selected by adding G418 to their culture lowing the neo gene are 1,237 base pairs corresponding to medium. The efficiency of obtaining G418-resistant clones the 3' end ofthe M-MuLV env gene, followed by the 3' LTR, was approximately 10-3 for both plasmids (Table 1). which contains the polyadenylylation signal (15). (vi) Flank- The transformed clones were mixed, and their supernatant ing both LTRs are sequences derived from the mouse chro- was collected. We tested whether this supernatant contained mosome (200 base pairs 5' and 800 base pairs 3') (12). (vii) retroviruses carrying the neo gene by exposing NIH 3T3 The remaining 4.4 kilobases of the plasmid are sequences cells to the supernatant and then selecting for cells capable corresponding to a rearranged copy of pBR322, which ex- of growing in G418. The supernatants from the 4v2 cells car- presses the ampicillin-resistance gene. Therefore, the neo rying the pM-MuLV-neo or the pM-MuLV-SVtk-neo plas- gene in pM-MuLV-neo is expected to be under the transcrip- mids had a titer of -8 x 105 G418-resistant colony-forming tj R SaIl LTR SailI BEmHI

FIG. 1. Diagram of the two retroviral vectors that express the neo gene. m, M-MuLV LTR; o, M-MuLV sequences; m, neo gene (the arrow corresponds to the orientation of the coding strand of the gene); gm, mouse chromosomal DNA; mn, pBR322 sequences, W, SVtk promoter; *, Sal I site in pM-MuLV-neo was destroyed during the construction of this molecule. Downloaded by guest on September 26, 2021 Developmental Biology: Rubenstein et aL Proc. Natl. Acad ScL USA 81 (1984) 7139

pM-MuLV-SVtk-neo pSVtk-neolB M-Mu LV- SVtk- neo M-MuLV-neo

Vi V -\ .,L,,4\+ £qv( ,q qC.iC $'4%Ec, \ *'b ^ 1. ' .e - _ i nIm 4. MD I 9.6 3.7 B gal B A

FIG. 2. Autoradiograph of the in situ assay of aminoglycoside 3'-phosphotransferase [APH (3')II; the neo gene-encoded phosphotransfer- ase] after electrophoretic separation from the endogenous phosphotransferase activity. The arrow points to the position of the APH(3')II activity. (A) Extracts from NIH 3T6 and LT-1 cells were assayed from APH(3')II 48 hr after cotransfection with pM-MuLV-SVtk-neo and pCH110 DNA. We used the level of t3-galactosidase (,gal) expressed for the pCH110 as an internal control for the efficiency of transfection. The numbers 9.6 and 3.7 are the number of f-galactosidase units in the NIH 3T6 and LT-1 extracts, respectively. The definition of l3-galacto- sidase units can be found in ref. 25. (B) APH(3')II assay of extracts from G418R LT-1, PCC3, PCC4, and NIH 3T3 cells, which had received the neo gene by DNA transformation (pSVtk-neo,8) or by viral infection (M-MuLV-SVtk-neo and M-MuLV-neo). Individual clones (A, B, C, and F) or mixtures of many clones (M) were assayed.

units (Table 1). Therefore, /,2 cells containing these retro- ferase. The neo gene product was expressed in all of the viral vectors are efficient producers of virions that can trans- G418-resistant cells infected with the M-MuLV-SVtk-neo vi- fer the neo gene into differentiated cells. Furthermore, the rus (Fig. 2). On the other hand, the G418-resistant PCC3 internal SVtk promoter does not substantially change the ef- cells derived from infection with the M-MuLV-neo virus ficiency of producing these recombinant retroviruses (Table contained at least 40-fold less aminoglycoside 3'-phospho- 1). Finally, both M-MuLV-neo and M-MuLV-SVtk-neo ex- transferase than did EC cells infected with the M-MuLV- pressed the same level of aminoglycoside 3'-phosphotrans- SVtk-neo virus or differentiated cells infected with either the ferase in infected NIH 3T3 cells (Fig. 2). M-MuLV-neo or M-MuLV-SVtk-neo virus. Viral of the neo Gene into EC Cells. We next Therefore, the addition of the internal SVtk promoter 5' to tested whether the M-MuLV-neo and the M-MuLV-SVtk- the neo gene confers upon retroviruses the ability to trans- neo viruses could confer G418 resistance to EC cells. Three duce and stably express the neo gene in nullipotent and mul- EC cell lines were studied, each having different develop- tipotent EC cells. Furthermore, the few G418-resistant EC mental properties. LT-1 is a nullipotent cell line, whereas cells transduced by M-MuLV-neo produced less amino- PCC3 and PCC4 are multipotent (17). These EC cells were glycoside 3'-phosphotransferase by a factor of 40 than did grown in the virus-containing medium, and the cells were M-MuLV-neo-transduced differentiated cells. then transferred into medium containing G418. In contrast to the result obtained with differentiated cells (NIH 3T3), only DISCUSSION the M-MuLV-SVtk-neo virus was capable of conferring sta- ble G418 resistance to all three types of EC cells. The M- Retroviral gene expression is inhibited in EC cells due to a MuLV-neo virus was either completely unable to transduce block in transcription (10, 26-28) or RNA maturation, or an active neo gene (when infecting LT-1 cells) or very ineffi- both. We have constructed a recombinant retrovirus that cient relative to the pM-MuLV-SVtk-neo virus (when infect- overcomes this restriction. By inserting the SVtk composite ing PCC4 or PCC3 cells) as shown in Table 1. promoter after the M-MuLV first intron, and 5' to the neo We isolated individual G418-resistant EC clones, all of gene (Fig. 1), the retrovirus (M-MuLV-SVtk-neo) was capa- which maintained their typical EC morphology. To prove ble of introducing and expressing the neo gene in all EC cell that the neo gene is actually expressed in these cells, we as- lines tested. Each of these EC cell lines has characteristics of sayed cellular extracts for aminoglycoside 3'-phosphotrans- different stages of embryonic development (17), suggesting Table 1. Efficiency of producing G418-resistant cells by using the recombinant retroviruses Virus infection, G418R cfu/ml DNA transfection* Cells M-MuLV-neo M-MuLV-SVtk-neo pM-MuLV-neo pM-MuLV-SVtk-neo pSVtk-neoc iI2 - 11 x 10-4 8 x 10-4 NIH 3T3 9 x 105 6.5 x 105 LT-1 -1 2 x 102 52 x 10-6 3 x 10-5 6 x 10-5 PCC4 10 2.8 x 102 PCC3 4 x 102 2 x 103 Differentiated (NIH 3T3) or EC (LT-1, PCC4, and PCC3) cells were infected with virus produced by 4i2 cells containing either pM-MuLV-neo or pM-MuLV-SVtk-neo. The table reports the number of G418-resistant (G418R) colony-forming units (cfu)/ml of q2 supernatant. Efficiency of producing G418R cells was also measured in differentiated (4f2) or EC (LT-1) cells that were transfected with pM-MuLV-neo, pM-MuLV-SVtk-neo, or pSVtk-neo/B DNA. Note that the data are not corrected for the efficiency of the EC cells, which is approximately 1/10 that of the NIH 3T3 cells (unpublished results). *The efficiency of producing G418R colonies is reported as the ratio of the number ofG418R colonies divided by the number of cells initially transfected. Downloaded by guest on September 26, 2021 7140 Developmental Biology: Rubenstein et aLPProc. NatL Acad ScL USA 81 (1984) that this retroviral vector may be suitable for introducing and Fondation Andre Meyer. J.-F.N. is "Maitre de Recherche" at Insti- expressing genes in embryos. tut National de la Sante et de la Recherche Medicale. J.L.R.R. is a On the other hand, the retrovirus without the SVtk pro- Fellow of the Fondation pour la Recherche Medicale. moter (M-MuLV-neo) is unable or extremely inefficient at 1. Brinster, R. L., Ritchie, K. A., Hammer, R. E., O'Brien, expressing the neo gene in EC cells. For instance, by either R. L., Arp, B. & Storb, U. (1983) Nature (London) 306, 332- DNA transformation or viral infection (Table 1), pM-MuLV- 336. neo was unable to confer stable neo gene expression to LT-1 2. Panganiban, A. T. & Temin, H. M. (1983) Nature (London) cells. In contrast to LT-1 cells, PCC4 and PCC3 cells were 306, 155-160. transformed to G418 resistance by the M-MuLV-neo retrovi- 3. Weiss, R. A., Teich, N., Varmus, H. E. & Coffin, J. M., eds. rus, although at an extremely low efficiency (10 and 400 (1982) The of Tumor Viruses, Part III: RNA G418R cfu/ml, respectively, Table 1). These G418-resistant Tumor Viruses (Cold Spring Harbor Laboratory, Cold Spring cells maintain EC morphology and multipotency. Although Harbor, NY). they are resistant to G418, their level of aminoglycoside 3'- 4. Watanabe, S. & Temin, H. M. (1982) Proc. Natl. Acad. Sci. USA 79, 5986-5990. phosphotransferase expression is lower by at least a factor of 5. Mann, R., Mulligan, R. C. & Baltimore, D. (1983) Cell 33, 153- 40 than in the EC cells that had been infected with M-MuLV- 159. SVtk-neo (Fig. 2). Therefore, the retroviral promoter and 6. Perkins, A. S., Kirschmeier, P. T., Gattoni-Celli, S. & Wein- splicing are probably functioning in these cells but at a re- stein, I. B. (1983) Mol. Cell. Biol. 3, 1123-1132. duced efficiency. One of the characteristics of PCC3 cells is 7. Jaenisch, R., Fan, H. & Croker, B. (1975) Proc. Natl. Acad. their ability to differentiate in culture. For this reason, it is Sci. USA 72, 4008-4012. likely that within the PCC3 cell population, cells at different 8. Jaenisch, R., Jahner, D., Nobis, P., Simm, I., Lohler, J., states of differentiation coexist (29). We hypothesize that a Harbers, K. & Grotkopp, D. (1981) Cell 24, 519-529. subpopulation of PCC3 cells have the ability to express, al- 9. Peries, J., Alves-Cardoso, E., Canivet, M., Debons Guillemin, M. C. & Lasneret, J. (1977) J. Natl. Inst. 59, 463-465. though inefficiently, genes under the transcriptional control 10. Linney, E., Davis, B., Overhauser, J., Chao, E. & Fan, H. of the retroviral LTR. (1984) Nature (London) 308, 470-472. The fact that the internal SVtk promoter did not signifi- 11. Nicolas, J. F. & Berg, P. (1983) Teratocarcinoma Stem Cells, cantly change the amount of virus produced by the qi2 cells in Cold Spring Harbor Conferences on Cell Proliferation, eds. containing pM-MuLV-SVtk-neo (Table 1) shows that a sec- Silver, L. M., Martin, G. R. & Strickland, S. (Cold Spring ond promoter, which is in tandem with the LTR, is not nec- Harbor Laboratory, Cold Spring Harbor, NY), Vol. 10, pp. essarily deleterious to the propagation of a retrovirus. Other 469-485. workers also have been able to propagate retroviruses con- 12. Yermanian, P. (1984) Dissertation (Universit6 Paris XII, Paris, taining internal promoters (30-32). We have extended this France). 13. Harbers, K., Schnieke, A., Stuhlmann, H., Jahner, D. & Jaen- result to a retroviral vector containing the isch, R. (1981) Proc. Natl. Acad. Sci. USA 78, 7609-7613. lacZ gene under the transcriptional control of the SV40 early 14. Messing, J. & Vieira, J. (1982) Gene 19, 269-276. promoter. Furthermore, it is likely that the internal SVtk 15. Shinnick, T. M., Lerner, R. A. & Sutcliffe, J. G. (1981) Na- composite promoter is the only active promoter when the ture (London) 293, 543-548. provirus is integrated in EC cells. In that case, by replacing 16. Rubenstein, J. L. R. & Chappell, T. G. (1983) J. Cell Biol. 96, the SVtk promoter with the 5' region of developmentally reg- 1464-1469. ulated genes, one can use this system to introduce new genes 17. Nicolas, J. F., Jakob, H. & Jacob, F. (1981) in Functionally into embryonic cells in order to study the control of gene Differentiated Cell Lines, ed. Sato, G. (Liss, New York), pp. expression during embryogenesis. 185-210. 18. Fan, H. & Paskind, M. (1974) J. Virol. 14, 421-429. We wish to transduce an antiparallel copy of a gene into a 19. Graham, F. L. & van der Eb, A. J. (1973) 52, 456- cell. We (25) and another group (33) have presented evidence 467. that antiparallel mRNA can inhibit the expression of the 20. Hall, C. V., Jacob, P. E., Ringold, G. M. & Lee, F. (1983) J. complementary mRNA in eukaryotic cells. The inhibition of Mol. Appl. Genet. 2, 101-109. a developmentally important gene by expressing an antipar- 21. Bradford, M. (1976) Anal. Biochem. 72, 248-254. allel copy of that gene has the potential of being a powerful 22. Miller, J. H. (1972) Experiments in Molecular (Cold technique to study development. Spring Harbor Laboratory, Cold Spring Harbor, NY). 23. Reiss, B., Sprengel, R., Will, H. & Schaller, M. (1984) Gene 80, 217-223. Note Added in Proof. Cocultivation of 5 x 104 LT-1 cells with 2 x 105 24. Southern, P. J. & Berg, P. (1982) J. Mol. Appl. Genet. 1, 327- mitomycin-treated (overnight with 2 ,tg/ml) M-MuLV-SVtk-neo- 342. producing /i2 cells for 1 week transforms at least 10%o of the LT-1 25. Rubenstein, J. L. R., Nicolas, J. F. & Jacob, F. (1984) C.R. cells to G418 resistance. Furthermore, PCC3 cells transduced with Hebd. Seances Acad. Sci. Ser. III 299, 271-274. the M-MuLV-SVtk-neo virus stably express the neo gene product 26. Stewart, C. L., Stuhlman, H., Jahner, D. & Jaenisch, R. (1982) when grown in medium without G418 for 70 generations. Proc. Natl. Acad. Sci. USA 79, 4098-4102. 27. Gautsch, J. W. & Wilson, M. C. (1983) Nature (London) 301, 32-36. 28. Niwa, O., Yokota, Y., Ishida, H. & Sugahara, T. (1983) Cell We thank Patrick Yermanian and I. Bernard Weinstein for the gift 32, 1105-1113. of pB6 and pMVGT, respectively, and Richard Mulligan and Mar- 29. Nicolas, J. F., Dubois, P., Jakob, H., Gaillard, J. & Jacob, F. tine Canivet for 4'2 and NIH 3T3 cells. We are also grateful to R. (1975) Ann. Microbiol. (Paris) 126A, 3-22. Sprengel, who made the assay of aminoglycoside 3'-phosphotrans- 30. Shimotohno, K. & Temin, H. (1981) Cell 26, 67-77. ferase available to us, and are indebted to Didier Rocancourt for 31. Tabin, C. J., Hoffmann, J. W., Goff, S. P. & Weinberg, R. A. excellent technical assistance. This work was supported by the Cen- (1982) Mol. Cell. Biol. 2, 426-436. tre National de la Recherche Scientifique (LA 269), the Fondation 32. Joyner, A. L. & Bernstein, A. (1983) Mol. Cell. Biol. 3, 2180- pour la Recherche Medicale, the Ligue Nationale contre le Cancer, 2190. the Ministere de la Recherche et de l'Industrie (82V1388), and the 33. Izant, J. H. & Weintraub, H. (1984) Cell 36, 1007-1015. Downloaded by guest on September 26, 2021