Aust. J. BioI. Sci., 1978, 31, 527-43 Fusion and Hybridization of Marsupial and Eutherian Cells VI. * Hybridization Rory M. Hope A and Jennifer A. Marshall Grapes" A Department of Genetics, University of Adelaide, G.P.O. Box 498, Adelaide, S.A. 5001. B Department of Genetics and Human Variation, La Trobe University, Bundoora, Vic. 3083. Abstract We have fused many different combinations of marsupial and eutherian cells, in order to test their capacity to form proliferating marsupial x eutherian somatic cell hybrids. During the first week after fusion we were able to identify marsupial x eutherian synkaryons, a number of which had undergone extensive chromosome fragmentation. For some combinations of marsupial and eutherian cells one parental cell type could not be completely eliminated from the fused cultures by the various selective systems employed, making it difficult to distinguish possible hybrid cells from parental cells. However, many hybridizations yielded discrete colonies which could be isolated and further studied. The first hybrids to be confirmed were produced from fusions between Pseudocheirus peregrinus (Marsupialia) lymphocytes and cultured mouse cells from the line PG19. These hybrid cells possessed clearly identifiable marsupial and mouse chromosomes and enzyme markers. Certain other combinations of marsupial and eutherian cells yielded 'eutherian-like' colonies. Although initially slow growing, these cells came to resemble the eutherian parental cell type in morphology, growth rate and cloning efficiency. Some of these colonies were shown to be composed of variant eutherian cells. However, others were clearly marsupial x eutherian hybrids as they possessed enzyme markers of each parental cell type even although, in some cases, these cells possessed no identifiable marsupial chromosomes. All the marsupial x eutherian hybrids isolated showed either partial or complete loss of marsupial chromosomes. We discuss the difficulties associated with the derivation of marsupial x eutherian hybrids, the important features of these hybrids, and their potential use for further studies. Introduction Somatic cell hybrids are of great value for studies of the biology and genetics of mammalian cells. By using somatic cell genetic techniques it is possible to assign human genes to particular chromosomes (Ruddle and Creagan 1975), and to establish gene order (Ricciutti and Ruddle 1973; Goss and Harris 1975). The same technique can be applied to gene mapping in other mammals, e.g. mouse (Kozak et al. 1975). Somatic cell hybrids are also useful for analysis of the control of cell processes such as DNA synthesis (Graves 1972, 1975; Lin and Davidson 1975) and X' chromosome inactivation (Migeon 1972; Kahan and De Mars 1975). Somatic cell hybrids have useful application in medical genetics (Hope 1977). Hybrids between marsupial and eutherian cells would be particularly useful. For instance, mapping of human enzyme markers, using electrophoresis to distinguish between parental forms in man X mouse hybrids, is restricted to isozymes which differ in molecular charge. This excludes 10-20 % of enzymes (Ruddle 1970), at least some of which should have distinguishable electrophoretic forms in mammals * Part V, Aust. J. Bioi. Sci., 1978, 31, 293-301. 528 R. M. Hope and J. A. M. Graves as distantly related as marsupials and eutherians. For example, the human and rodent forms of several mitochondrial enzymes are not readily separable by electrophoresis (van Heyningenet al. 1973; Tolley and Craig 1975; Shimizu et al. 1977). However, Bennett's wallaby (Macropus rufogriseus) cells do possess forms of the mitochondrial enzymes citrate (si)-synthase (EC 4.1.3.7) and aspartate aminotransferase (EC 2.6.1.1) which are electrophoretically distinct from the human forms of these enzymes (I. Craig and S. Povey, personal communication). Marsupial genetic studies have been severely restricted by the problems of main­ taining and breeding the animals in captivity (Smith et al. 1978), and by the paucity of suitable markers. Several protein polymorphisms have been described (Hope 1972; Cooper1974),butsince fewspecieshave beenfound to be multiply polymorphic, classical gene linkage studies have been difficult; such studies have been confined to the, assignment of genes to the X chromosome (Cooper et al. 1975). As yet there is no example of linked genes in any marsupial. If, however, marsupial X eutherian cell hybrids could be obtained, many interspecific isozyme differences would be available as genetic markers. The genes for these markers could then be assigned to chromo­ somes, and their syntenic relationships established. Comparison of chromosome maps from different marsupial species would enable theories of the evolution of marsupial karyotypes (Hayman and Martin 1974) to be examined. It would also be possible to compare marsupial with eutherian chromosome maps and, for example, to examine further the hypothesis (Ohno 1969) that the X chromosome has been highly conserved in evolution. Marsupial cells also provide excellent material for cytogenetic investigations because of the low diploid numbers, large size, and distinctive morphology of the chromosomes (Sharman 1973; Hayman 1977). Since the marsupial chromosomes can be so readily identified, hybrids between marsupial and eutherian cells would be ideal for chromo­ some studies; for instance, studies of chromosome loss from hybrid cells, and studies of the regulation of chromosome replication. Of particular interest are cell hybridization studies of the mechanism of X chromosome inactivation. Since marsupials have a paternal rather than a random X inactivation system (Cooper et al. 1975)the inactive X can be distinguished genetically and/ or cytologically in cells from heterozygotes or interspecies hybrid animals. In addition, it now appears that X inactivation in marsupials is incomplete in some tissues (Cooper et al. 1977; VandeBerg et al. 1977). This 'leaky' system may be more amenable to experimental manipulation than is the more rigidly controlled eutherian system, and therefore efforts to discover the factors determining and maintaining inactivation may be more successful. None of our early hybridization experiments appeared to yield viable marsupial X eutherian hybrids for reasons which were not at all evident. Previous papers in this series have described the co-cultivation of marsupial and eutherian cells (Graves and Hope 1977a) and their fusion to form homo- and heterokaryons (Graves and Hope 1977b; Graveset al. 1977),whose nuclei were synthetically active, and underwent synchronous chromosome condensation (Graves and Hope 1978). Satisfactory selective systems have also been developed which now enable us to select against marsupial and eutherian parental cells in a variety of combinations (Hope and Graves 1978). In this paper we describe experiments to test marsupial cells from many different species, and from different tissues, for their ability to form hybrids with a variety of Growth of Marsupial and Eutherian Cells. VI 529 eutherian cells. We report here the results of these studies, and describe the first marsupial X eutherian hybrids isolated. Materials and Methods Parental cells are listed and described in Table 1. Media and culture conditions are described by Graves and Hope (1977a). To isolate and propagate clones, metal or glass rings were placed over well separated colonies, and sealed to the flask or Petri dish by sterile silicone grease. Cells were removed by using 0·1 % trypsin-versene, and were trans­ ferred to separate wells in multi-well tissue culture plates (Lux or Linbro) or to small plastic culture flasks. As these became confluent, they were each transferred to larger culture vessels for further propagation and analysis. Cell Fusion and Selection of Hybrids Cells were dissociated, mixed, and fused in suspension with inactivated Sendai virus as described previously (Graves and Hope 1977b; Graves et ale 1977). Hybrids were selected from fused cultures using four techniques (and combinations of these) described fully by Hope and Graves (1978). (i) HAT selection One or both parental cell types were deficient in hypoxanthine phosphoribosyltransferase (HPRT) (EC 2.4.2.8) or thymidine kinase (TK) (EC 2.7.1.75), and were therefore unable to survive in hypo­ xanthine-aminopterin-thymidine (HAT) selective medium (Littlefield 1964). Hybrids were expected to survive in this medium, and colonies could be isolated and studied. (ii) Ouabain selection Parental cells differed in their sensitivity to the drug ouabain. Hybrids were expected to show intermediate levels of resistance (Giles and Ruddle 1973), so that the sensitive parent could be eliminated by growth in an appropriate concentration of ouabain. Selection against the resistant parent required use of one of the other methods. (iii) Attachment Lymphocytes and lymphoblastoid cell lines could be readily eliminated from fused cultures by changing the medium, because of their failure to attach to the culture vessel. (iv) Growth Some parental cells (diploid fibroblasts, PtKl, PtK2 and their derivatives, and 3T3TK -) showed density-dependent growth inhibition, so that it should be possible to identify and isolate multilayered hybrid clones overgrowing the monolayer. Some of the diploid marsupial lines were in the senescent phase of their growth, so that they could be eliminated by further subculturing. The other parent in these hybridizations was an established line which could be eliminated by HAT selection. Chromosome Preparations