Proc. Natl. Acad. Sci. USA Vol. 76, No. 8, pp. 3987-3990, August 1979 Genetics Molecular analysis of chromosome-mediated gene transfer (thymidine kinase of herpes simplex virus/Southern blot hybridization/gene rearrangement/gene amplification) GEORGE A. SCANGOS*, KENNETH M. HUTTNERt, SAUL SILVERSTEIN*, AND FRANK H. RUDDLE*t *Biology Department, Kline Biology Tower, and tDepartment of Human 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 chromosomes isolated from a cell 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+ phenotype. 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 Cell Culture. The mouse 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 parents. 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 hr 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 host 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 population that retained the transferred phe- marker for which a nucleic acid 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. Phosphorylation 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 genome 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 plasmid, termed pTK1, was grown in Escherichia coli 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 enzymes. 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.