Aust. J. BioI. Sci., 1978, 31, 527-43

Fusion and Hybridization of 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 , 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 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 ( 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 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 and Banding Cells were harvested, washed in medium without serum, incubated in hypotonic medium (1: 5, v!v, medium-water or 0·05-0·75 M KCI) for 15 min at 37°C, then fixed in 3: 1 (vjv) methanol­ acetic acid. Air-dried preparations were made by dropping the cell suspension onto clean slides, and cells were stained with Giemsa (Gurr's R66). Chromosomes were C-banded by pretreatment of slides with 0·2 M HCI at room temperature for 1 h, followed by 30 s-10 min in 5% (wjv) Ba(OH)2 at 50°C, thorough rinsing in distilled water, and 1 h in 2 X SSC at 60°C, before staining for 12 min in Giemsa (Sumner 1972). G-banded preparations were obtained by pretreating slides with 0·1 % trypsin (NBC of Difco) at O°Cfor 1-15 min, followed by staining with Giemsa (Seabright 1971).

Biochemical Techniques (i) Cell extracts Cell extracts were made from cultured cells as described previously (Hope and Graves 1978). Extracts were also prepared from freshly collected red blood cells by washing cells in cold phosphate- 530 R. M. Hope and J. A. M. Graves

buffered saline, lysing with an equal volume of lysis buffer (Hope and Graves 1978), adding 1/4 of the total volume of toluene, then shaking vigorously for 10 min at 4°C, and centrifuging at 2000 g for 20 min. Lipid material was removed, and the tubes were centrifuged at 17000 g for 30 min, then the supernatant was removed and used fresh for enzyme studies.

(ii) Enzyme studies Hypoxanthine phosphoribosyltransferase isozymes were separated and identified by starch-gel electrophoresis (Watson et ale 1972). The marsupial form of HPRT was easily distinguishable from

Table 1. Cell types used in the present study

Name and Description Obtained Reference type of from? cells

(a) Marsupial established lines PtKITG HPRT- derivative of PtKI (, .Potorous tridaetylus (1, aneuploid)" (4) PtK2 P. tridaetylus Sf?, aneuploid (1) PtK2BNl TK- derivative of PtK2B (4) PtK2TNI HPRT- derivative of PtK2 (4) Sc9/01 Sminthopsis erassieaudata Sf?, pseudodiploid (1) Ford et ale (1978) Sc3F S. erassieaudata Sf?, pseudodiploid (1) Sc-a5 HPRT- derivative of Sc3F (4) (b) Marsupial diploid lines Pt12 P. tridaetylus, ear fibroblast (3) SG60 Eastern grey (Maeropus Professor D. W. Cooper giganteus) Sf?, ear fibroblast Macquarie University (D.W.C.) SG9 Eastern grey kangaroo Sf?, ear fibroblast (1) K2 Red kangaroo (Maeropus rufus) Sf?, ear fibroblast (2) K7 Red kangaroo (1, ear fibroblast (4) ewe32 Wallaroo x euro hybrid Sf? (M. robustus robustus x M. r. erubeseens), ear fibroblast (1) OKI Wallaroo x red kangaroo hybrid Sf?, ear fibroblast (1) (e) Marsupial lymphocytes P. tridaetylus Eastern grey kangaroo Sf? G54 D.W.C. Eastern grey kangaroo (1 G65 D.W.C. Red kangaroo Dr D. L. Hayman, University of Adelaide Whiptail wallaby (M. parryi) Kangaroo Island wallaby (M. eugenii) Sf? E228 D.W.C. (M. bieolor) (1 B2 D.W.C. Bennett's wallaby (M. rufogriseus) (1 Wallaroo x euro hybrid Sf? ewe32 D.W.C. Wallaroo x red kangaroo hybrid Sf? OKI D.W.C. (Triehosurus vulpeeula) Ringtail possum (Pseudoeheirus peregrinus) (1 Anteehinus rosamondae Dr P. A. Woolley, La Trobe University Growth of Marsupial and Eutherian Cells. VI 531

Table 1 (Continued)

Name and Description Obtained Reference type of from? cells

(d) Eutherian established lines EUE Human, HPRT-, aneuploid (1) De Carli et al. (1964) D98-AH Human, HPRT-, aneuploid (3) BU-25 Human, TK-, aneuploid, HeLa derivative Dr M. Nabholz, Oxford LNSV Human, HPRT-, SV40-transformed Dr R. Kennett fibroblast Univ. of Pennsylvania Croce et al. (1973a) T5-1 Human, HPRT-, lymphoblastoid Dr R. Kennett, Oxford Sato et al. (1972) FU5-AH Rat, HPRT-, hepatoma Dr M. C. Weiss, Schneider and Weiss C.N.R.S., Gif-sur­ (1971), Croce Yvette et al. (1973b) A9 Mouse, HPRT-, L cell derivative (1) PG19 Mouse, HPRT- (3) Na Mouse, HPRT- (2) 1R Mouse, HPRT-, L cell derivative (1) CI-1D Mouse, TK-, L cell derivative Dr I. Craig, Oxford Weiss and Green (1967) 3T3TK - Mouse, TK- (1) B1 4Iso Chinese hamster ~, TK- '.pseudodiploid (3) BIo Ouabain-resistant derivative of B1 4Iso (4) (e) Eutherian diploid lines MAR Human C!, diploid fibroblast Professor W. F. Miggiano et al. Bodmer, Oxford (1969) (I) Eutherian lymphocytes Human c!

A Additional details of cells are given by (1) Graves and Hope (1977a), (2) Graves and Hope (1977b), (3) Graves et al. (1977), and (4) Hope and Graves (1978). B TK-, deficient in thymidine kinase. HPRT-, deficient in hypoxanthine phosphoribosyltransferase. the rodent or human form (Hope, Donald, MacGregor and Chew, unpublished data) for every combination of marsupial and eutherian cells fused. It was also possible, in some combinations, to distinguish the marsupial forms of phosphoglycerate kinase (PGK) (EC 2.7.2.3), purine-nucleoside phosphorylase (NP) (EC 2.4.2.1), adenine phosphoribosyltransferase (APRT) (EC2.4.2.7) and lactate dehydrogenase (LDH) (EC 1.1.1.27). Starch-gel electrophoresis for PGK isozyme identification was carried out using the method described by Cooper et al. (1971). NP, APRT and LDH were typed at the MRC Human Biochemical Genetics Unit, The Galton Laboratory, University College, London.

Results Many combinations of marsupial and eutherian cells were fused. The hybridi­ zations and their outcomes are listed in Table 2, and are described more fully below.

Early Events in Hybrid Formation The formation of a hybrid requires that the chromosomes of the nuclei in a heterokaryon become enclosed in a single nuclear membrane (probably at mitosis), forming a synkaryon, which may then divide normally, to yield a hybrid clone. It was difficult to follow the formation and early divisions of synkaryons because of the large numbers of dying parental cells. However, several attempts were made to observe events shortly after fusion, in cultures fixed in situ, or in suspension. It was 532 R. M. Hope and J. A. M. Graves

found that during the first week after fusion mitotic abnormalities including anaphase bridges, fragments and micronuclei, were very frequent. However, it was clear that synkaryons could be formed, even though rarely. Fig. 1 shows a mitotic cell from a fusion of marsupial (potoroo PtK2BNl) and eutherian (mouse lR) cells. Several potoroo chromosomes are easily distinguishable from mouse chromosomes. That these .synkaryons may undergo massive chromosome damage is suggested by the presence in fused cultures of many cells with fragmented chromosomes (Fig. 2).

Fig. 1. Synkaryon from a fusion of marsupial (PtK2BN1, Potorous tridactylus) and eutherian (1R, mouse) cells. The chromosome preparation was made 5 days after fusion. At least eight potoroo chromosomes (arrowed), including the X, are clearly visible amongst 1R chromosomes.

Colonies Obtainedfrom Hybridizations For some combinations of marsupial and eutherian cells one parental cell type could not be completely eliminated (Table 2). However, many hybridizations yielded discrete colonies which could be further studied. These could be classified into three groups. (i) Aberrant colonies These were small colonies composed oflarge, vacuolated cells having a morphology unlike that of either parent. These cells showed little or no increase in number over periods ofseveral weeks, or months in culture. Although many mitoses were observed, Growth of Marsupial and Eutherian Cells. VI S33

daughter cells frequently failed to reattach. A few colonies were ultimately isolated from hybridizations of ringtail possum lymphocytes with PG 19. These cells initially grew extremely slowly, and sparse cultures failed to survive. However, after several months in culture the growth rate and cloning efficiency increased to the level of PG 19. These cells were propagated, characterized and shown to be hybrids. How­ ever, the great majority of aberrant colonies became progressively smaller and eventu­ ally died, or they grew slowly but failed to survive isolation.

Fig. 2. Cell photographed 1 week after fusion of grey kangaroo (SG9) fibroblasts with mouse (PG 19) cells, showing extensive chromosome fragmentation.

(ii) Eutherian-like colonies These colonies initially grew slowly and consisted of large, vacuolated cells. However, unlike the aberrant colonies their growth rate increased strikingly about a month after fusion, and their morphology, growth rate and cloning efficiency pro­ gressively came to resemble that of the eutherian parent. They were much more frequent in fused cultures than in mixed or single control cultures, and their fre­ quencies were higher when marsupial parental cells were used in excess (Table 3). Careful cytological examination, using C- and G-banding, failed to reveal the presence of any marsupial chromosomes in any of these colonies, except in those derived from hybridizations involving red kangaroo lymphocytes. 534 R. M. Hope and J. A. M. Graves

Table 2. Results of hybridization experiments

Parental cell type Selection Outcome Marsupial Eutherian system?

(a) Established lines PtK1TG BU25 HAT No colonies, incomplete elimination of PtK1TG PtK2 A9, Na, 3T3TK-, D98AH Growth, HAT No overgrowth BIo, Na, 3T3TK-, A9 Ouabain, HAT Aberrant colonies", eutherian-like colonies" PtK2TNl MAR (senescent) HAT, growth No colonies Human lymphocytes HAT, attachment No colonies PtK2BN1 1R HAT No colonies, but synkaryons observed (Fig. 1) MAR HAT, growth No colonies T5-1 HAT, attachment No colonies A9, Na HAT Aberrant colonies, eutherian-like colonies, incomplete elimination of PtK2BN1 PG19 HAT Hybrid colonies BIo, Na Ouabain, HAT Eutherian-like colonies, incomplete elimination of PtK2BN1 Sc9j01 EUE, D98AH, 3T3TK-, 1R Growth, HAT Sc9 colonies, many synkaryons

Sc3F Na, B1 4Iso Growth, HAT Aberrant colonies BIo Ouabain, HAT Eutherian-like colonies

Sea5 B1 4Iso HAT Incomplete elimination of Sca5, eutherian-like colonies (b) Diploid lines Pt12 CI-1D Growth, HAT Eutherian-like colonies, incomplete elimination of Pt12 D98AH, LNSV Growth, HAT Eutherian-like colonies PG19, FU5-AH Growth, HAT No colonies SG60 (senescent) A9, Na Growth, HAT Incomplete elimination of SG60 SG9 (senescent) Na, PG19 Growth, HAT Hybrid colonies

A9, B 1 4Iso Growth, HAT Eutherian-like colonies" A9, Na, BIo Ouabain, HAT Eutherian-like colonies" K2 (senescent) Na Growth, HAT Eutherian-like colonies, aberrant colonies K7 Na Growth, HAT Eutherian-like colonies, incomplete elimination of K7 ewe32 Na, B1 4Iso, 3T3TK- Growth, HAT Aberrant colonies, eutherian-like colonies BIo, Na Ouabain, HAT Eutherian-like colonies"

OK1 A9, Na, B1 4Iso, PG19 Growth, HAT Eutherian-like colonies, incomplete elimination of OK1 BIo, Na Ouabain, HAT Aberrant colonies, eutherian-like colonies" (c) Lymphocytes Antechinus rosamondaePG19, BIo Attachment, HAT Aberrant colonies Growth of Marsupial and Eutherian Cells. VI 535

Table 2 (Continued)

Parental cell type Selection Outcome Marsupial Eutherian systemA

Potoroo D98AH Attachment, HAT Eutherian-Iike colonies PG19 Attachment, HAT Hybrid colonies Bennett's wallaby D98AH Attachment, HAT Eutherian-Iike colonies EUE, BU25, 3T3TK-, lR Attachment, HAT No colonies Whiptail wallaby PG19 Attachment, HAT No colonies Brushtail possum PG19 Attachment, HAT No colonies Ringtail possum D98AH Attachment, HAT No colonies LNSV Attachment, HAT Eutherian-Iike colonies PG19 Attachment, HAT Hybrid colonies Red kangaroo lR,PG19 Attachment, HAT Hybrid colonies Eastern grey kangaroo Na, PG19 Attachment, HAT Hybrid colonies BIo, A9 Attachment, HAT Eutherian-Iike coloniesc Kangaroo Island wallaby Na, PGI9, A9, BIo Attachment, HAT Eutherian-Iike coloniesc Swamp wallaby Na, PG19 Attachment, HAT Hybrid colonies A9 Attachment, HAT No colonies Euro x wallaroo Na, PG19 Attachment, HAT Hybrid colonies BIo, A9 Attachment, HAT Eutherian-Iike coloniesc WallarooxWallaroo x red kangaroo Na, PG19 Attachment, HAT Hybrid colonies BIo Attachment, HAT Eutherian-IikeEutherian-like coloniescolonies?c

A For details of selective systems, see Materials and Methods. Where two selective systems were used, these are presented in the same order as are the parent celIscells selected against. B Descriptions of these colony types are given in the text. C These colonies are yet to be fully characterized.

Some eutherian-like colonies were probably HAT-resistant eutherian variants. For example, colonies derived from hybridizations with D98AH were clearly of this type since they lacked any detectable HPRT, and possessed no marsupial PGK, APRT, NP, or LDH. Many eutherian-like colonies derived from early experiments

Table 3. Frequency of recovery of eutherian-Iike clones (number of clones per 10 6 eutherian cells) after hybridization of eutherian with marsupial cells

Eutherian Control Marsupial cell type cell type Macropodid Macropodid fibroblasts lymphocytes

A9 0 4 2 Na 1 6 11 PG19 0 12 20 B14B1 4IIso/BIoso/BIo <1 5 3 CI-lDCI-1D 0 8 5 were discarded without characterization of their HPRT since they, too, were assumed to be HAT-resistant variants of the eutherian parent. More recently, however, many such colonies have been shown to possess marsupial HPRT, and are therefore marsupial xX eutherian hybrids (Table 2). 536 R. M. Hope and J. A. M. Graves

Fig. 3. Phase contrast photomicrograph of cells in culture showing morphological differences between (a) marsupial x eutherian hybrid cells (ringtail possum lymphocytes x PG19) and (b) eutherian parental cells (PG19). Growth of Marsupial and Eutherian Cells. VI 537

(iii) Hybrids The first marsupial X eutherian hybrids to be confirmed were the products of fusion between ringtail possum lymphocytes and PG 19. Cells in hybrid colonies were morphologically distinct from PG 19 (Fig. 3). Chromosome preparations obtained from cells washed from the surface of colonies before isolation contained PG 19 and ringtail possum chromosomes, which were readily distinguishable by their C-banding patterns (Fig. 4). After 10 weeks colonies were isolated, propagated and subsequently

Fig. 4. C-banded chromosome preparation of marsupial x eutherian somatic cell hybrid (ringtail possum lymphocytes x PG19). The possum chromosomes are characterized by their extensive area of centromeric C-banding material. examined cytologically and biochemically. The presence of mouse and marsupial chromosomes was confimed by G-banding, and it was apparent that marsupial chromosomes had been preferentially lost. Enzyme studies showed that the cells contained the ringtail possum form of HPRT, as would be expected, since PG19 is HPRT deficient and hybrids were selected in HAT. Both mouse and ringtail possum forms of PGK were present. These results clearly establish the hybrid nature of these cells. 538 R. M. Hope and J. A. M. Graves

Preliminary characterization of hybrids obtained from fusions of red kangaroo lymphocytes with eutherian cells indicated that these contained the red kangaroo X chromosome and red kangaroo HPRT (Donald and Hope, unpublished data). Hybrids obtained from fusions involving other macropodids apparently contained no marsupial chromosomes, but could be identified as hybrids by the presence of mar­ supial HPRT (Hope, Graves and Chew, unpublished data). The results of our detailed studies on these and other marsupial X eutherian cell hybrids will be presented in forthcoming publications.

Discussion We describe here the derivation of several marsupial x eutherian cell hybrids. We believe that these are the first such hybrids to have been positively identified. There has been only one previous report of marsupial X eutherian hybrids. Potoroo X Chinese hamster hybrids were described by Jakob and Ruiz (1970) but, in the absence of chromosome banding and isozyme studies, the authors relied entirely on gross chromosome morphology for hybrid identification. Examination of their published photographs casts doubt on the hybrid nature of these cells; the chromosomes claimed to be of potoroo origin do not correspond in morphology to normal potoroo chromosomes, and the cells depicted contain tclocentrics, dicentrics and fragments not present in either parental cell type. The chromosomes claimed to be of potoroo origin may well be aberrant Chinese hamster chromosomes. It is quite common to observe polyploidy and chromosome rearrangement in DON, or in other Chinese hamster cells subjected to non-permissive selective culture conditions (Terzi 1974; Graves, unpublished data). Because of our initial difficulties in obtaining marsupial x eutherian hybrids, we haveinvestigated the steps which must be completed for successful hybrid formation -fusion of parental cells to form functional heterokaryons, synkaryon formation, and growth and selection of hybrid clones. We have shown previously that marsupial and eutherian cells may be fused with frequencies comparable to those of interspecific eutherian cell combinations (Graves and Hope 1977b; Graves et ale 1977), forming heterokaryons which are synthetically active and whose nuclei are able to enter mitosis synchronously (Graves and Hope 1978). We have developed selective systems for the isolation of marsupial X eutherian hybrids (Hope and Graves 1978), and have shown that marsupial and eutherian cells may be cocultivated with little or no inhibition of growth of either cell type (Graves and Hope 1977a). These studies have revealed no special difficulties in completion of any of these steps. During the present study, however, two sources of problems have become apparent. The first involves technical difficulties with selection systems; the second derives from the nature of the hybrids. In practice, several of the selective techniques we have used in these hybridizations failed to give complete elimination of one parental cell type. The drug-resistant HAT-sensitive derivatives of marsupial cell lines (Hope and Graves 1978) were found to be sensitive to HAT medium only at low cell densities, and at the higher densities necessary for plating fused cultures they remained attached in growing patches which were often difficult to distinguish from hybrids (e.g. in the fusion between PtK1TG and BU25). Also, selection on the basis of differences in growth rate or cloning Growth of Marsupial and EutherianCells. VI 539

efficiency between marsupial and eutherian cells proved .to be less effective than expected. For instance, after fusion of Sc9 with 1R, colonies were obtained which were presumed to be of hybrid origin since Sc9 formed no colonies when plated alone in HAT at similar cell densities. However, enzyme and chromosome analysis revealed these colonies to be of Sc9 origin. It is now clear that in combination with a feeder layer of HAT-sensitive eutherian cells Sc9 cloning efficiency is increased 1ODD-fold (Graves and Hope 1977a; Graves 1978). Similarly, senescent fibroblasts (e.g. SG60), which in single cultures showed no increase in cell number and a mitotic index of less than 0 · 1%, were stimulated to divide in combination with HAT-sensitive eutherian cells, and were difficult to eliminate from fused cultures. Reversion of HAT-sensitive eutherian lines to HAT resistance was also a problem. For instance, many eutherian-like colonies isolated from fusions of marsupial cells with the HAT-sensitive HPRT- human line D98AH, selected in HAT, were shown to possess no marsupial chromosomes or enzymes, and lacked any detectable HPRT. We assumed, therefore, that these colonies represented aminopterin-resistant D98AH variants. Many aminopterin- and amethopterin-resistant cell lines have been described previously (see review by Siminovitch 1976). Some of the other HAT-sensitive eutherian lines used also revertedto HAT resistance with appreciable frequencies. For instance, colonies were obtained at a frequency of about 10- 6 when the TK­ line BIo was grown in HAT, and it was apparent that 95 %of these revertants lacked TK, so were probably also aminopterin-resistant (Graves, Kellow and Norton, unpublished data). It now appears that many of our early difficultiesin obtaining marsupial X eutherian hybrids were due to problems in recognition, rather than production, ofthe hybrids. For example, a number of combinations of marsupial and eutherian cells (e.g. grey kangaroo and mouse) appear to yield hybrids which contain no identifiable marsupial chromosomes, but which do possess the marsupial form of the selected marker (HPRT), so that cytological screening of colonies, the method relied on in earlier experiments, failed to reveal their hybrid nature. Those combinations of marsupial and eutherian cells listed in Table 2 as yielding no hybrids should therefore not necessarily be regarded as being incompatible. It is likely that some of our earlier experiments did in fact yield hybrids but that these could either not be satisfactorily selected or could not be recognized by the techniques employed. The most striking feature of the marsupial X eutherian cell hybrids is the extensive or complete loss of marsupial chromosomes. In the extent of this loss these hybrids appear to be intermediate between interspecific eutherian hybrids and hybrids between eutherian cells and cells from more distantly related classes. Chromosome loss in interspecies eutherian hybrids varies from very slight progressive loss [e.g. in rodent X rodent crosses such as mouse X Chinese hamster (Marin and Pugliatti-Crippa 1972)] through slow but ultimately complete loss of human chromosomes from rodent X human hybrids (Weiss and Green 1967) to rapid elimination of all non-selected chromosomes [e.g. retention of only the X chromosome in mouse X Microtus agrestis cell hybrids (Cook 1975)]. Rapid and complete loss of chick chromosomes from mouse fibroblast X chick erythrocyte hybrids, identified by the presence of chick HPRT, has been described (Schwartz et ale 1971),although a single chick chromosome is stably retained in other mouse X chick hybrids (Kao 1973). Generally, therefore, it appears that the more distantly related are the cell parents, the more extensive is the chromosome loss from one parental complement. 540 R. M. Hope and J. A. M. Graves

Our limited range of marsupial X eutherian combinations shows chromosome loss patterns, depending on the marsupial species used, which overlap those ofeutherian x eutherian and eutherian X avian cell hybrids. As many as ten ringtail possum chromo­ somes are retained in possum X mouse cells, whereas only the X chromosome and possibly a single autosome are retained in red kangaroo X mouse hybrids (Donald and Hope, unpublished data), and no identifiable marsupial material at all is present in hybrids between other macropodid and mouse cells, although the marsupial form of the selected HPRT marker is present. In the last case, as in mouse xchick hybrids, presumably a small chromosome fragment bearing the HPRT gene has been translo­ cated to a mouse chromosome where it is replicated and expressed. Because of the great evolutionary differences between genera within the subclass Marsupialia (Metatheria), which are at least as great as that between rodents and humans, it would be hazardous to generalize, from the limited number of combinations we have studied, about the patterns of loss in all marsupial X eutherian cell hybrids. It is interesting, however, that in the ringtail possum all chromosomes have large amounts of centric heterochromatin, and in the red kangaroo (but not in the other Macropus species studied here) the X chromosome has a heavily C-banded arm. It is possible, therefore, that the species differences in the extent of marsupial chromosome loss reflect a tendency of hybrids to retain marsupial constitutive heterochromatin. It has recently become apparent that the direction of chromosome elimination from mouse X human cell hybrids depends not only on the species of origin but also on the tissue of origin of parental cells. Whereas human chromosomes are invariably lost from hybrids with established mouse lines, mouse chromosomes are preferentially eliminated from hybrids between primary diploid mouse cells and established human lines (Minna and Coon 1974). No tissue-related differences in the pattern of chromo­ some loss were observed among the marsupial X eutherian cell hybrids described here. All hybrids derived from the established potoroo line PtK2 as well as from fresh potoroo lymphocytes showed complete elimination of marsupial chromosomes, as did the hybrids derived from grey kangaroo lymphocytes and fibroblasts. Combinations of marsupial established cell lines and eutherian lymphocytes are not yet available for study, but will be particularly interesting. The usefulness of mouse X human somatic cell hybrids for mapping of human genes has depended on the retention of partial human chromosome complements by these hybrids. It is likely that some of the marsupial X eutherian hybrid combinations in which one or a few marsupial chromosomes are retained will prove useful for assigning marsupial genetic markers to chromosomes. Other hybrids in which complete loss of marsupial chromosome occurs cannot be used for chromosome assignment in the same way, but it may be/possible to use these hybrids for determining the order of genes syntenic with the selective markers by comparing the frequencies of cotransfer of markers, in the same manner as described by Goss and Harris (1975). Such mapping studies have been initiated. Other studies which we had hoped to carry out using marsupial X eutherian cell hybrids do not, at present, appear to be feasible. For instance, mapping of the eutherian genes for which there is no isozyme difference known among eutherian species would require marsupial X eutherian cell hybrids which segregate eutherian chromosomes. None of the hybrids so far obtained are appropriate for this type of study. It is possible that combinations of mouse cells with cells from marsupial Growth of Marsupial and Eutherian Cells. VI. 541

genera not yet hybridized or combinations of established marsupial cell lines with eutherian lymphocytes may yield such hybrids. Alternatively, it may be possible to manipulate the direction of chromosome loss by pre-irradiation of the eutherian cell parent (cf. Pontecorvo 1971).

Acknowledgments We thank Dr D. L. Hayman and Professor D. W. Cooper for many helpful dis­ cussions and for their generous gifts of marsupial cells and blood. R.M.H. wishes to thank Professor W. F. Bodmer, Dr. R. Kennett and their colleagues, University of Oxford, for their hospitality, encouragement and advice. We thank Ms Jennifer Donald for permission to quote unpublished data. We wish to acknowledge the ex­ pert technical assistance of Mrs Lorraine Billett, Mrs Iole Barbieri and Mrs Clare Greer. We thank Dr Brian Watson and colleagues and Dr Guat Kin Chew for carrying out some of the enzyme assays; Professor Henry Harris for providing cul­ tures of Sendai virus; and Mrs Ruth Rofe and Mr Roy Goodwins for assistance. To the people who supplied us with marsupial and eutherian cells and cell lines (Table 1) we express our thanks. This project is supported by grants to R.M.H. and J.M.G. from the Australian Research Grants Committee.

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Manuscript received 23 February 1978