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Proc. Natl. Acad. Sci. USA Vol. 76, No. 8, pp. 3987-3990, August 1979

Molecular analysis of -mediated gene transfer (thymidine of herpes simplex /Southern blot hybridization/gene rearrangement/) GEORGE A. SCANGOS*, KENNETH M. HUTTNERt, SAUL SILVERSTEIN*, AND FRANK H. RUDDLE*t * Department, Kline Biology Tower, and tDepartment of Genetics, Yale University, New Haven, Connecticut 06520; and tDepartment of Microbiology, Columbia University, College of Physicians and Surgeons, New York, New York 10032 Contributed by Frank H. Ruddle, May 9, 1979

ABSTRACT Metaphase isolated from a was decreased in stable derivatives. The transgenomes in all four line carrying the thymidine kinase (TI) gene of herpes simplex transformants were greater than 17 kb. Finally, we found that virus type 1 were used to transform the TK-deficient cell line line LH7 possessed an increased number of copies of the TK LMTK- to the TK+ . Four independent transformants were isolated, all of which expressed virus-specific TI. Each fragment after prolonged cultivation in selective medium. of the four transformant cell lines initially became TK- at a rate of 12% per day. All four transformants possessed multiple copies MATERIALS AND METHODS of the TI gene and in one ofthe four a rearrangement occurred adjacent to the TK sequences. Stable TK+ derivatives of each . The cell lines LMTK- and LH7 were line, isolated after prolonged cultivation, retained fewer copies maintained in monolayer culture at 370C under 10% C02/90% of the TI gene than did their unstable . The transferred air in Dulbecco's modified Eagle's medium (GIBCO) supple- chromosomal fragment was larger than 17 kilobases in each mented with 5% fetal bovine serum (Flow Laboratories, line. McLIean, VA). Cell lines were periodically tested for myco- Previous work has shown that isolated metaphase chromosomes plasma contamination as described by Barile et al. (12). can be used as vectors for the transfer of mammalian genes from Chromosome-Mediated Gene Transfer. LH7 was grown one cell line to another (1-5). Recipient cells (transformants) as a Fuspension culture to a density of 4 X 105 cells per ml in isolated from chromosome-mediated gene transfer experiments DulVecco's modified Eagle's medium plus 5% fetal bovine usually possess a subchromosomal donor fragment (transge- serum. Colcemid was added to a 1-liter culture to a final con- nome), which often is lost at a rapid rate in the absence of se- centration of 0.1 Lg/ml. Cells were incubated for 8 and lection (unstable expression). Upon continuous cultivation of harvested by centrifugation, and chromosomes were isolated unstable transformants, subclones arise that retain the trans- and applied to recipient LMTK- cells as previously described ferred marker stably. Previous data provide strong evidence (3). TK+ recipient cells were selected in the hypoxanthine/ that in stable transformants the transgenome is integrated into amioopterin/thymidine (HAT) medium (13). a chromosome (6-9). Stability Tests. The cell line to be tested was maintained in Several questions remain unanswered regarding the structure nonselective medium. Periodically, 400 cells were plated in and function of the transgenome. How many copies exist per HAT selective medium and in nonselective (hypoxanthine/ cell? What is their size range? How are such transgenomes or- thymidine) medium. Ten days after plating, surviving colonies ganized in the unstable state and in the stable state, and what were stained with Wright's strain and counted. The ratio of is the molecular basis of stabilization? We felt that these ques- colonies in selective to nonselective medium was taken as the tions could be answered most directly by the use of a selectable fraction of the that retained the transferred phe- marker for which a probe existed. For this purpose notype at the time of plating. we used the thymidine kinase (TK) gene of herpes simplex virus Biochemical Procedures. [125I]Iododeoxycytidine was a type 1 (HSV). This gene resides on a 3.4-kilobase (kb) BamHI generous gift of W. Summers and J. Smiley. restriction endonuclease fragment of HSV. Wigler et al. (10) of the iododeoxycytidine by TK was assayed as described demonstrated that this fragment could transform the TK-de- (14). ficient mouse cell line LMTK- to the TK+ phenotype. In such DNA was isolated by a modification of the method of Pellicer TK+ cell lines, the HSV TK fragment was found to be inte- et alt (11). BamHI and EcoRI restriction endonucleases were grated into high molecular weight DNA (11). prepared as described by Greene et al. (15). Xba I, Kpn I, We have used one such line, LH7, as a donor in chromo- HindIII, and HincII were purchased from New England some-mediated gene transfer experiments. We describe four Bio1abs, and all digestions were performed under conditions transformants that initially lost the transgenome at a rate of 12% specified by this supplier. per cell per day. We have used the TK fragment, labeled in Filter Hybridization. An equal amount of DNA (30-50,g) vitro with 32p, as a probe to characterize the state of the trans- from each cell line was applied to a 1% agarose (Seakem) gel in each of the lines. We show that a rearrangement of in a horizontal apparatus. Electrophoresis was carried out in 160 the region containing the TK sequences occurred in one of the mM Tris-HCl (pH 8.0)/80 mM NaOAc/80 mM NaCI/5 mM unstable transformants and that stable derivatives of each of EDTA at 370 mA until marker dye had migrated 11-12 cm the four lines had restriction endocnulease patterns indistin- (approximately 22 hr). DNA was denatured and transferred guishable from those of their unstable parents. In addition, we to nitrocellulose filters by using published procedures (16). I4 demonstrate that each of the unstable transformants possessed vitro labeling of the TK probe was performed as described multiple copies of the transgenome, whereas the copy number by Maniatis et al. (17). We routinely obtained specific activities of 1-5 X 108 dpm/,gg of DNA. The publication costs of this article were defrayed in part by page Initially, the TK fragment was obtained directly from HSV charge payment. This article must therefore be hereby marked "ad- vertisement" in accordance with 18 U. S. C. §1734 solely to indicate Abbreviations: TK, thymidine kinase; HSV, herpes simplex virus type this fact. 1; kb, kilobase; HAT, hypoxanthine/aminopterin/thymidine. 3987 Downloaded by guest on September 24, 2021 3988 Genetics: Scangos et al. Proc. Nati. Acad. Sci. USA 76 (1979) strain 43+ (a gift of W. Summers). Virus was grown and DNA was isolated as described (18). Viral DNA was cleaved with BamHI and electrophoresed, and the 3.4-kb fragment was eluted according to Tabak and Falvell (19). More recently, we have used as a probe the TK fragment cloned in pBR322 (20). This , termed pTK1, was grown in strain LE392 under P2-EK1 containment conditions. Plasmid pTK1 DNA was isolated according to Guerry et al. (21). Filter hybridization was performed by a modification of the procedure of Jeffreys and Flavell (22). TK activity in vitro activity was assayed by a modification of published procedures (23). Reactions were stopped by cooling on ice, and samples were applied to Whatman DE81 DEAE- cellulose discs. Filters were washed three times in 1 mM am- monium formate, once in distilled H20, and once in 95% eth- anol. RESULTS Recipient Cell Lines Possessed Viral TK. Chromosomes from LH7 were applied to LMTK- cells and four independent colonies were isolated in HAT selective medium. No TK+ revertants of LMTK- have ever been identified in our labora- tory. Furthermore, two lines of evidence confirm the viral or- igin of the TK activity in these cells. First, these lines-termed LHM1, LHM21, LHM22, and LHM23-were able to grow in the presence of 2 mM thymidine, indicative of HSV TK rather than cellular TK (14). Second, the cells were capable of phos- phorylating iododeoxycytidine and incorporating it into cellular DNA, an enzymatic activity characteristic of HSV TK and not of murine TK (24). Transformants Were Initially Unstable. To initiate stability testing, each of the transformant lines was transferred to non- selective medium. Periodically, equal numbers of cells were plated in selective and nonselective medium, and the number of surviving colonies was counted (Fig. 1). All four transformed lines initially lost the TK+ phenotype at similar rates of ap- Time, days proximately 12% per cell per day. Line LHM1 underwent a rapid conversion to stability; a stable subclone was carried FIG. 1. Stability test of transformant lines. Lines LHM21, through subsequent analyses. LHM22, and LHM23 lost the ability to survive in HAT medium at Rearrangement Can Occur during Transfer. DNA from rates of 10-12% per cell per day. Each line was maintained in nonse- each of the recipient lines was isolated and digested with a lective hypoxanthine/thymidine (HT) medium for 30 days and then battery of restriction . We used BamHI, Xba I, and subcloned in selective medium. One derivative of each line, indicated HindIII, none of which cuts within the TK by the suffix A, was retested. Each retained the TK+ phenotype sta- fragment. Equal bly. LHM1 was a mixed population of stable and unstable cells. A amounts of DNA were electrophoresed and subjected to filter stable derivative was subcloned and carried through subsequent hybridization analysis with the BamHI-derived TK fragment analysis. of HSV as probe. In each case, the state of the TK fragment in lines LHM1, LHM21, and LHM22 was indistinguishable from that of the donor, whereas that of LHM23 differed (Fig. 2). Stable Derivatives Have Fewer Copies. Each of the three EcoRI cleaves twice within the 3.4-kb TK fragment to yield unstable lines was grown in nonselective medium for 30 days an internal fragment of 2.2 kb and two termini. The prominent and then replated in selective medium. Stable TK+ subelones internal band as well as an 8.6-kb terminus (one end of the 3.4 of each were isolated (Fig. 1). The stable derivatives of each kb fragment linked to cellular DNA) was seen in lines LH7, line-termed LHM21A, LHM22A, and LHM23A-were an- LHM1, LHM21, and LHM22 (Fig. 2 bottom). In line LHM23 alyzed by Southern blot No were seen the internal band was no longer present and was replaced by hybridization. changes a band at 13.5 kb, indicating that one of the internal EcoRI sites in the mobility of the TK gene-containing bands in any of the had been lost during transfer into this line. After digestion with lines. However, the intensity of the bands decreased after sta- Kpn I, a single band migrating at greater than 20kb was found bilization, as if the stable derivatives had fewer copies of the in LH7, LHM1, LHM21, and LHM22, whereas the fragment TK gene than their unstable parents (Figs. 2 and 3). Unexpec- in LHM23 was smaller. Parenthetically, cleavage of cellular tedly, the intensity of the bands in the stable donor LH7 ap- DNA with Xba I yielded a fragment of 17 kb in LHM23, larger peared to be similar to that of the unstable lines, and to be than that found in the other lines. An alteration in the mobility reproducibly greater than that of the stabilized derivatives. of the TK band also appeared after digestion with HindIII (Fig. We reasoned that multiple copies of the TK gene might lead 3). Digestion with HincII, which cleaves once within the TK to the synthesis of more and so determined the in vitro fragment, gave one fragment identical to that in LH7 and one TK specific activities of the stable and unstable lines. The levels with altered mobility (data not shown). These changes are all of activity correlated with the intensity of the TK bands (Fig. consistent with a single deletion event that occurred to one side 4), supporting our hypothesis that multiple copies of the TK of the TK gene. gene exist in the unstably transformed lines. Downloaded by guest on September 24, 2021 Genetics: Scangos et al. Proc. Natl. Acad. Sci. USA 76 (1979) 3989 A B C D E F G H 1 e; A B C D EF G H U Em~ -_ E _ oI*

FIG. 3. Analysis of line LHM23. Track A, molecular weight markers; tracks B, C, and D represent LH7, LHM23A, and LHM23, respectively, digested with Xba I; tracks E, F, and G represent LH7, LHM23A, and LHM23 digested with HindIII; track H, LMTK-, A B C D E F G H digested with HindIII. The band in LH7 is 14.3 kb when digested with Xba I and 19.5 with HindlIH. Lines LHM23 and LHM23A have a band of 17.2 kb with both enzymes. LHM23 appears to have more copies of the TK fragment than LHM23A. The probe was plasmid pTK1. DISCUSSION We have transferred the TK gene of HSV into LMTK- cells by using isolated metaphase chromosomes as the vector. The initial rate of loss of the TK+ phenotype was approximately 12% per A. cell per day in each of the transformed lines. This rate is char- acteristic of a class of transformants that we have seen previ- ously in which the transgenomes are almost always below the 0.1% of the ge- A B C D E F G H limit of cytological detection (roughly haploid a~.. nome or 2 X 103 kb).§ It was of interest to determine a mini- mum size estimate for such transgenomes in both the unstable and stable states. We have demonstrated that all four inde- pendently derived transgenomes characterized in this study are greater than 17 kb. Previous studies on chromosome-mediated gene transfer have shown that, in the stable state, transgenomes are associated with recipient cell chromosomes, probably covalently linked to recipient cell DNA (6-9). Unstable transformants possibly maintain the transgenome in an unintegrated, autonomous state. We have detected no change in the mobility of the bands containing the TK fragment in any of the lines after stabiliza- tion. Thus, if the unstable transgenomes are autonomous, at- tachment to a recipient chromosome has involved a fragment greater than the 17-kb interval analyzed. The copy number of the TK fragment decreased after sta- bilization of the transgenome, as judged by autoradiographic intensity. The diminished band intensity is not due to mixed of TK+ and TK- cells because each stable deriva- Fiw. 2. (Top) Xba I digest of transformant cell DNA. Track A, tive plates with equal efficiency in selective and nonselective HindlII digest of A phage DNA used as molecular weight markers; media (Fig. 1). We can also rule out artifacts of electrophoresis H, LHM23A; C, LHM22A; D, LHM21A; E, LMTK-; F, LH7; G, and filter hybridization, because the same band patterns were LHM23; H, LHM22; I, LHM21; J, LHM1. The probe was plasmid seen with all three pairs of stable and unstable lines on different Lines LHM1, LHM21, and LHM22 and the donor LH7 have p)TKI. gels and regardless of the arrangement of the on the gel a band at 14.3 kb. Line LHM23 and its stable derivative LHM23A or of the enzyme used to cleave the DNA. band in- possess a band of 17.2 kb. The unstable lines appear to have more Although intense bands than their stable derivatives (denoted by the suffix tensity differences are difficult to quantitate, it is clear that the A). stable derivatives have fewer copies of the TK fragment than (Middle) HindIII digestion of transformant cell DNA. Track A, their unstable parents. Moreover, the level of TK enzyme ac- lHM21A; B, LHM21; C, LHM22A; D, LHM22; E, LHM23; F, LH7. tivity in the unstable lines is greater than that found after sta- ( and H contain mixtures of LMTK- DNA and plasmid pTK1. Track bilization. H contains 5 times as much plasmid as track G. The probe was intact Unstable transformants that overexpressed the transferred plasmid pTK1. LH7, LHM21, LHM22, and LHM23 have a single band of 19.5 kb. In line LHM23, the single band is 17.2 kb. marker have been reported previously (25). In addition, we (Bottom) EcoRI digestion of transformant cell DNA. Tracks A and have recently isolated several transformants that express ele- 13 contain mixtures of LMTK- DNA and plasmid pTK1. Track A vated levels of transferred cellular markers after chromo- contains 5 times as much plasmid as track B. C, LH7; D, LHM23; E, some-mediated gene transfer. § Although no probe is available IHM22; F, LHM22A; G, LHM21; H, LHM21A. Note the loss of the to characterize the number of gene copies in these additional in appearance of a band at 2.2-kb internal fragment LHM23 and the cases, we believe that gene amplification subsequent to transfer 1:3.5 kb. One terminus can be seen as an 8.5-kb fragment in all lines. may account for the increased activity. The unstable lines LHM21 and LHM22 show more intense bands t han their stable derivatives LHM21A and LHM22A. The probe was i nitact plasmid pTK 1 DNA. § L. Klobutcher, C. L. Miller, and F. H. Ruddle, unpublished data. Downloaded by guest on September 24, 2021 3990 Genetics: Scangos et al. Proc. Nati. Acad. Sci. USA 76 (1979) altered sites in cellular DNA adjacent to the TK fragment for the enzymes Xba I, Kpn I, BamHI, HindIII, and HincIl. In addition, one internal EcoRI site in the TK fragment has been deleted, indicating the rearrangement lies very close to or within the TK gene itself. These data are consistent with a deletion or with breakage and rejoining of the TK fragment to a second, previously unlinked, fragment. In either case, the rearrangement and translocation events that occur in the transgenome should make chromosome-mediated gene transfer a useful tool for the analysis of mammalian cell genetics. We thank Drs. R. Axel, A. Pellicer, and J. Smiley and Mr. L. Klo- butcher for helpful discussions and criticism. We thank Ms. Clemencia Colmenares for excellent technical assistance, Ms. Suzy Pafka for photography, and Ms. Marie Siniscalchi for the preparation of the manuscript. We thank Drs. J. Smiley and W. Summers for the generous gifts of [il25]iododeoxycytidine and of viral strains. This work was supported by National Institutes of Health Grant GM09966. G.A.S. was a Postdoctoral Fellow of the Jane Coffin Childs Memorial Fund for Medical Research and was subsequently supported by National Insti- tutes of Health Postdoctoral Fellowship GM06528-01. K.M.H. is a Predoctoral Trainee of the Medical Scientist Training Program. 1. McBride, 0. W. & Ozer, H. L. (1973) Proc. Natl. Acad. Sci. USA 70, 1258-1262. 2. Willecke, K. & Ruddle, F. H. (1975) Proc. Nati. Acad. Sci. USA 72, 1792-1796. 3. Miller, C. L. & Ruddle, F. H. (1978) Proc. Natl. Acad. Sci. USA 75,3346-3350. 4. Ruddle, F. H. & McBride, 0. W. (1976) in The of the Mammalian Genetic Apparatus, ed. Ts'o, P. O. P. (Else- vier/North-Holland, Amsterdam), pp. 163-169. 5. McBride, 0. W. & Athwal, R. S. (1976) In Vitro 12,777-786. Time, min 6. Athwal, R. S. & McBride, 0. W. (1977) Proc. Natl. Acad. Sci. USA 74,2943-2947. FIG. 4. Determination of TK activity in lysates of transformant 7. Willecke, K., Mierau, R., Kruger, A. & Lange, R. (1976) Cyto- lines. All curves represent the average of two independent assays, each genet. Cell Genet. 16, 405-408. performed in duplicate. The relative amounts of activity correlate with 8. Willecke, K., Mierau, R., Kruger, A. & Lange, R. (1978) Mol. the intensity of the TK bands visualized on the autoradiograms. Lines Gen. Genet. 161, 49-57. LHM23 and LHM23A possess less TK activity than the other lines. 9. Fournier, R. E. K. & Ruddle, F. H. (1977) Proc. Natl. Acad. Sci. This may reflect fewer copies of the gene or an alteration of expression due to the breakage within the TK fragment in these lines. USA 74,3937-3941. 10. Wigler, M., Silverstein, S., Lee, L., Pellicer, A., Cheng, Y. & Axel, R. (1977) Cell 11, 223-232. LH7, as characterized by Pellicer et al. (11), possessed one 11. Pellicer, A., Wigler, M., Axel, R. & Silverstein, S. (1978) Cell 14, copy of the TK fragment. A contemporary version maintained 133-141. in selective medium for 18 months appears to have an increased 12. Barile, M. F., Bodey, G. P., Snyder, J., Riggs, D. B. & Grabowski, number of copies. Multiple copies of the TK fragment in LH7 M. W. (1966) J. Natl. Inst. 36, 155-168. 13. W. Science could result from tandem duplication of a region including the Littlefield, J. (1964) 145,709-710. 14. Smiley, J. R., Steege, D. A., Juricek, D. K., Summers, W. P. & fragment, or .from an increase in the copy number of the Ruddle, F. H. (1978) Cell 15,455-468. chromosome into which the fragment integrated, or both. If 15. Greene, P. J., Heyneker, H. L., Bolivar, F., Rodriguez, R. L., the amplification were due to tandem duplication, it is possible Betlach, M. C., Covarrubias, A. A., Backman, K., Russell, A. J., that we transferred multiple copies to the recipient lines. Toit, R. & Boyer, H. L. (1978) Nucleic Acids Res. 5, 2373- However, the rearrangement in line LHM23 argues against the 2380. transfer of multiple copies. It is difficult to explain how multiple 16. Southern, E. M. (1975) J. Mol. Biol. 98,503-517. copies could have been transferred and subsequently rear- 17. Maniatis, T., Jeffrey, A. & Kleid, D. G. (1975) Proc. NatI. Acad. ranged in precisely the same manner. It seems more likely that Sci. USA 72, 1184-1188. a single copy was rearranged and subsequently amplified. 18. Wagner, M. M. & Summers, W. C. (1978) J. Virol. 27, 374- To distinguish between the two mechanisms of amplification 387. in LH7 we performed microcell experiments in which one or 19. Tabak, H. F. & Flavell, R. A. (1978) Nucleic Acids Res. 7, only a few donor chromosomes were transferred. In these ex- 2321-2332. L. M. P. & Van de periments, we LH7 20. Enquist, W., Madden, J., Schiop-Stansly, fused microcells derived from with the F. Science TK-deficient RJK. We two Woude, G. (1979) 203,541-544. Chinese hamster line have analyzed 21. Guerry, P., Leblanc, D. J. & Falkow, S. (1973) J. Bacteriol. 116, microcell hybrids obtained in this manner by Southern blot 1064-1066. hybridization. Both lines have TK gene-containing 22. Jeffreys, D. T. & Flavell, R. A. (1977) Cell 12,429-439. bands of the same mobility as the donor LH7 but of lessened 23. McBride, 0. W., Burch, J. W. & Ruddle, F. H. (1978) Proc. Natl. intensity. These data suggest that only one or at most a few of Acad. Sci. USA 75,914-918. the TK genes present in LH7 were transferred and that the 24. Summers, W. C. & Summers, W. P. (1977) J. Virol. 24, 314- chromosome into which the TK fragment has integrated exists 318. in multiple copies. 25. Degnen, G. E., Miller, I. L., Adelberg, E. A. & Eisenstadt, J. M. The rearrangement of the transgenome in line LHM23 has (1976) Proc. NatI. Acad. Sci. USA 73,2838-2842. Downloaded by guest on September 24, 2021