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Gene Transfer by Means of Cell Fusion

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Gene Transfer by Means of Cell Fusion J. Cell Sci. 25, 17-37 (i977) 17 Printed in Great Britain GENE TRANSFER BY MEANS OF CELL FUSION I. STATISTICAL MAPPING OF THE HUMAN X-CHROMOSOME BY ANALYSIS OF RADIATION- INDUCED GENE SEGREGATION S. J. GOSS AND H. HARRIS The Sir William Dunn School of Pathology, University of Oxford, South Parks Road, Oxford OXi 3RE, England SUMMARY Hybrid cells were obtained by virus-induced fusion of hamster cells with irradiated human cells. The analysis of such hybrids permits a study of the effects of lethal doses of radiation on human cells and provides a method of sub-chromosomal genetic mapping that is independent of karyological analysis. Radiation-induced chromosome exchanges are shown to be extremely localized, and a map of 4 X-linked genes is presented. INTRODUCTION Methods have recently been developed that have led to rapid progress in the assign- ment of genes to specific human chromosomes. Cell fusion induced by Sendai virus is used to make hybrids between human and rodent cells (Harris & Watkins, 1965); from such hybrids human chromosomes are preferentially eliminated (Weiss & Green, 1967). Since specific human chromosomes can be recognized by their banding patterns after appropriate staining procedures (Caspersson, Lomakka & Zech, 1971), it is possible to search for a correlation between the expression of a chosen human gene in the hybrid cell and the retention of a particular human chromosome. By means of such cor- relations approximately 100 genes have been assigned to specific human chromosomes (McKusick, Klinger, Bootsma & Ruddle, 1976). Regional assignment of genes within a chromosome can subsequently be achieved by examining a second set of hybrids, in which the chromosome of interest is translocated or otherwise rearranged in some identifiable way. The expression of the human gene can then be correlated with the presence of a section of the chromosome in question. Progress in regional mapping is being made by this approach (Burgerhout & Jongsma, 1976), but it is limited, to some extent by the availability of suitable translocations, and more seriously by the diffi- culty of identifying the exact nature of the chromosome rearrangements observed. Thus, despite extensive studies, there are still inconsistent regional assignments for genes on the X-chromosome, most probably because of incorrect determination of the chromosome break-points (Brown et al. 1976). Karyological mapping is, moreover, limited in resolution, in that it can only localize genes to a segment of chromosome large enough to be identified by light microscopy. Much smaller chromosomal frag- ments may, however, be retained in hybrid cells (Schwartz, Cook & Harris, 1971; Boyd & Harris, 1973; McBride & Ozer, 1973); so that the full potential of gene 18 S.J.Goss and H.Harris mapping by the techniques of somatic cell genetics has clearly not yet been rea- lized. The present paper describes a new approach to the problem, which relies on a study of the retention of human genes in hybrids made by fusing hamster fibroblasts with human lymphocytes given different doses of ionizing radiation. By using a hamster cell line, Wg3-h, that lacks the enzyme hypoxanthine phosphoribosyl transferase (HPRT), and isolating the hybrids in 'HAT' medium (Littlefield, 1964), we could ensure the selective retention, in all hybrids, of the human X-linked gene for HPRT. It is known from the cytological studies of Westerveld, Visser, Meera Khan & Bootsma (1971) that human chromosomes not selected for are rapidly lost from Wg3-h/ human lymphocyte hybrids. In the first part of this paper, we show that unselected X-linked genes are, in general, retained in the hybrids only if their linkage to HPRT has not been disrupted by radiation damage. We describe such genes as having been 'co-transferred' with HPRT. Autosomal genes, and those X-linked genes whose link- age with HPRT has been disrupted by the radiation, are rapidly eliminated. By analysing the co-transfer of X-linked genes, we can derive a map of the human X- chromosome. Preliminary reports of this work have appeared elsewhere (Goss & Harris, 1975; Goss, 1976). MATERIALS AND METHODS Cell culture The Chinese hamster cell lines, Wg3-h and a-23, were kindly supplied by Dr A. Westerveld (Westerveld et al. 1971), Wg3-h is deficient in the enzyme hypoxanthine phosphoribosyl transferase (HPRT, E.C. No. 2.4.2.8.) and a-23 exhibits a greatly reduced level of thymidine kinase (TK, E.C. No. 2.7.1.75). All cells were grown in monolayers in medium based on Eagle's minimal essential medium (MEM), supplemented with 10 % foetal calf serum. To keep Wg3-h stocks free from HPRT+ revertants, 6-thioguanine was added to the MEM to a final concentration of 6 /tg/ml. Medium containing 6-thioguanine was also used for the back-selec- tion of hybrids. HAT medium (Littlefield, 1964) was used for the isolation and propagation of the man-hamster hybrid cells. Our formulation of HAT is MEM containing hypoxanthine (6 x 10" 5M), thymidine (1 x IO~5M), aminopterin (4-5 x IO"'M) and glycine (4 x IO"4M). Prior to back-selection, cells were withdrawn from HAT by 2 passages in HAT without amino- pterin. This avoids the massive cell death due to the persistent effects of aminopterin. Human peripheral blood lymphocytes were prepared essentially according to the method of Boyum (1968). The same male donor (SJG) was used throughout, in order to reduce as much as possible any variation between batches of cells. Cells prepared in this way showed no DNA synthesis, as detected by the incorporation of radioactivity into acid-insoluble material, after culture of the cells for 1 h in MEM containing tritiated thymidine. We therefore assumed that most of these cells were non-dividing, and that each contained one complete copy of the human X-chromosome. No abnormalities could be detected in the G-banded X-chromosome of these cells (E. P. Evans and M. Burtenshaw, personal communication). Irradiation of the lymphocytes The cells were irradiated in a Gammacell 40 (Atomic Energy of Canada Ltd, Ottawa.). This equipment contained two 137Cs sources and delivered gamma-rays at a fixed rate of 1-2 J kg"1 min"1. The lymphocytes were suspended in ice-cold MEM without serum for the period of irradiation. Cold medium was used to minimize repair of the radiation damage (Kleijer, Hoek- sema, Sluyter & Bootsma, 1973), since repair would be expected to diminish the total effect of the irradiation (Dewey, Miller & Leeper, 1971). Statistical mapping of the human X-chromosome 19 Cell fusion Three million hamster cells were fused with approximately io7 lymphocytes by the tech- nique of Harris & Watkins (1965). To facilitate fusion, the lymphocytes were first trypsinized lightly by the method of Sun, Chang & Chu (1974), and then suspended together with the hamster cells. Each fusion was done in a total volume of 05 ml, 5000 haemagglutinating units of ultraviolet-inactivated Sendai virus being added. Cytological examination of the cell suspen- sion immediately after cell fusion by the method of Obara, Chai, Weinfeld & Sandberg (1974) showed that the majority of heterokaryons contained a single lymphocyte nucleus. The fusion was thus likely to produce hybrid cells containing, at least initially, a single human X-chromo- some. The fused cells were plated in 20 srrall.plastic tissue culture flasks (Falcon Plastics) and cul- tured for 1 day in MEM. The medium was then changed to HAT, which was renewed after 1 week. A week after that, the hybrid clones were isolated and grown up. Only one clone from each flask was analysed, so we can be certain of the independent origin of all the clones studied. Chromosome spreads The karyotypes of parental cells and hybrids were studied in chromosome spreads prepared according to Miller et al. (19716). The chromosomes were sometimes banded by the method of Kao (i973)- Identification of human enzymes in the hybrids Human enzymes were detected in lysates of hybrid cells by Cellogel electrophoresis. The enzymes studied are listed in Table 1. The basic procedure is that of Meera Khan (1971). All electrophoresis was done in a Shandon U77 tank in the cold room at 4 °C. Buffer I of Meera Khan (1971) was used for G6PD, PGK and IPO, and his buffer V for HPRT and aGAL. Table 2 shows the running conditions for the electrophoresis. The lysates (approx. 2 fd of each sample) were applied 1 cm from the cathodal bridge in all cases. Table 1. Human enzymes analysed in the hybrid cells Gene Chromo- catalogue some as- Enzyme (trivial name) Abbreviation E.C. no. no.* signmentf Hypoxanthine phospho- ribosyl transfers se HPRT 2.4. 2.8 30800 X Glucose-6-phosphate dehydrogenase G6PD 1.1. 1.49 30590 X 3-phosphoglycerate kinase PGK 2.7. 2-3 31180 X a-galactosidase A aGAL 3.2. I .22 30150 X Indophenol oxidase A IPO-A 1. iS;. 1.1 14745 21 Indophenol oxidase B IPO-B 1.15;. 1.1 14746 6 * McKusick (1975). f Chromosome assignments are taken from McKusick, Klinger, Bootsma & Ruddle (1976). The method for developing bands of enzyme activity on the gels after electrophoresis is des- cribed for HPRT, G6PD, PGK and IPO, by Meera Khan (1971), and for aGAL by Grzeschik (Grzeschik et al. 1972). The HPRT gels were incubated in the appropriate reaction mixture and then soaked for 15 min in a buffered lanthanum chloride solution (Nichols & Ruddle, 1974) to precipitate radiolabelled inosinic acid on the gel. Unincorporated radioactive hypoxanthine was 20 S. J. Goss and H. Harris removed from the gel by washing with fast-running tap water for 15 min. Each track on the gel was sliced into 5-mm strips and the radioactivity of the strips measured by scintillation counting.
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