Viable Hybrids between Adherent Cells: Generation, Yield Improvement, and Analysis
Somatic cell hybridization was discovered and introduced by Barski et al. (1960) and Sorieul and Ephrussi (1961). This technique allows one to examine the result of introducing various genomes in different functional states and from different species into the same cell. Hybrid cells have been widely used in various fields (genetics, cell biology, tumour biology, virology) and the most famous hybrids are hybridomas.
One important application of somatic cell hybridization is chromosomal gene assignment. Breeding analysis, which is effective for this purpose in lower animals and plants, is too slow in mammals (even in mice the generation time is about 3 months) and is impossible in humans. Gene mapping techniques based on somatic cell genetics have been central to the study of human genetics. In 1968, only two genes had been mapped to specific autosomes, and a decade later this number had risen to 300, mostly using human-rodent somatic cell hybrids. Such hybrids present the advantage to retain only a few human chromosomes and they are now currently used as donor cells in irradiation and fusion gene transfer (IFGT) experiments for constructing detailed genetic maps (Walter and Goodfellow, 1993).
Cell fusion was also used to analyse how specialized cells acquire and maintain their differentiation. The activities of somatic cells can be divided into two main categories: essential or ubiquitous functions that are indispensable for cell survival and growth and "luxury" or differentiated functions. Essential functions continue to be expressed in hybrids, whereas differentiated functions are subject to different regulations (expression, extinction, activation) depending on the histogenetic nature of the parental cells that have been fused (see examples in Cassio and Weiss, 1979; Hamon-Benais et al., 1994; Killary and Fournier, 1984; Mevel-Ninio and Weiss, 1981). Cell determination is, however, not modified in hybrids, as extinction or activation requires retention of the chromosomes coding for the appropriate regulatory factors.
Somatic cell hybridization has not only shown that tissue-specific genes are regulated by trans-acting factors, but has provided strong evidence for the existence of tumour suppressor genes (Anderson and Stanbridge, 1993). Cell fusion experiments have also demonstrated that cellular senescence is a dominant active process and that several genes or genes pathways are implicated in the senescence program (Goletz et al., 1994).
Spontaneous fusion of cells in culture occurs at a very low frequency. To obtain hybrid cells, inactivated Sendai virus or, more commonly, polyethylene glycol (PEG), which was introduced by Pontecorvo (1976), is used as the fusogen. Cell hybridization has also been performed by electrofusion on filters (Ramos et al., 2002). The inital products of fusion contain within a common cytoplasm two or more distinct nuclei from one single parent (homokaryons) or from both (heterokaryons). Only a very small proportion of these polykaryons will progress to nuclear fusion and then through mitosis. Moreover, the first divisions of the heterokaryons and of their daughter cells often fail because of abnormalities of the mitotic spindle and abnormal chromosome movements. The formation of viable hybrids from heterokaryons is thus a rare event, and the use of selective methods that favour the survival of the hybrids at the expense of the parental cells is often a requisite. These selective methods are also necessary because hybrid cells often grow more slowly than parental cells and are rapidly overgrown by parental cells.
A. Selective Methods
The best known of such methods is the application of hypoxanthine + aminopterin + thymidine (HAT) selection (Littlefield, 1964) for the fusion of cells deficient in hypoxanthine guanosine phosphoryl transferase (HGPRT-) with cells deficient in thymidine kinase (TK-), but different combinations of selectable markers can be used (Hooper, 1985), provided that the two selective systems do not interfere. If the lines that are fused have no selective markers, a good strategy is to select sequentially for HGPRT deficiency (thioguanine resistance) and ouabain resistance in one parental cell line. Then this marked cell line may be fused with any unmarked cell line and hybrids selected in HAT + ouabain (Jha and Ozer, 1976). Other couples of selective markers, such as TK deficiency (5-bromo- 2'deoxyuridine resistance) and neomycine resistance, can also be used. For producing primate-rodent hybrids, the selection of an HGPRT- rodent parent is sufficient because rodent cells are more resistant to ouabain than primate cells. Moreover, hybrid cells can also be isolated on the basis of their size, morphology, growth parameters, and DNA content.
B. Yield of Viable Hybrids
Whatever the method used to isolate hybrids, the most important is to optimize the fusion conditions in order to obtain a number of viable hybrids as high as possible. In the best cases the fusion of several millions of parental cells leads to the formation of only a few hundred hybrids and often the yield of viable hybrids is much lower, as illustrated in Table I for hepatomaderived hybrids. The protocol described here has been used routinely to produce large amounts of hybrid clones between differentiated rat hepatoma cells and various cells of different histogenetic origin and of different species, particularly mouse and human fibroblasts (Mevel-Ninio and Weiss, 1981; Sellem et al., 1981). Moreover, some of the hybrids obtained have been used themselves as partners of fusion and new hybrids were generated successfully using exactly the same method (Bender et al., 1999; Hamon-Benais et al., 1994). The most important parameters in fusion experiments are the yield of viable and growing hybrids and their stability. Thus it is recommended to define optimal fusion conditions and to vary different parameters, particularly the ratio of parental cells, for improving the yield. It is also recommended to analyze regularly hybrid clones for their phenotype and chromosomal content.
PEG 1000 ultrapure (Merck, Cat. No. 9729)
Trypsin (pig pancreas; United States Bioch. Corp., Cleveland, OH, Cat. No. 22715)
Complete growth medium (available from local suppliers)
Serum-free growth medium (available from local suppliers)
Selective complete growth medium (available from local suppliers)
35- and 50-mm tissue culture dishes (Falcon, Cat. No. 3001, 3002)
15-ml tube (Falcon, Cat. No. 352099)
22 × 22-mm sterile glass coverslips
A. Before Fusion
Grow parental cells in nonselective medium for a short period.
Notes: Steps 4 to 11 must be done dish per dish. Some control dishes must be included. They will be treated as the others except that the PEG solution will be replaced by serum-free medium.
C. After Fusion
The production of hybrid clones in large amounts depends greatly on the parental cells (cell type and growth capacity) and on the fusion conditions. These two points are illustrated in Tables I and II for rat hepatoma-derived hybrids. The frequency of occurrence of hybrids between rat hepatoma and normal fibroblast was particularly low compared to other hepatoma-derived hybrids (Table I). Consequently, various fusion conditions were tested. The use of unbalanced ratios of parental cells is one of the most important parameters to improve the hybrid yield (Table II). Therefore, to save time and to obtain the highest number of hybrid cells, it is recommended to fuse parental cells in different ratios.
Mixed parental cell populations that have not been treated by PEG can give rise to colonies that grow in selective medium at low frequency. These colonies could be either spontaneous hybrids or revertants from parental cells. The isolation of revertants is one of the most common difficulties that may arise in selecting hybrids. Therefore the hybrid nature of the cells selected has to be verified by checking their chromosomal content.
I thank M. C. Weiss for training in cell culture and C. H. Sellem and C. Hamon-Benais for the illustrations.
Anderson, M. J., and Stanbridge, E. J. (1993). Tumor suppressor genes studied by cell hybridization and chromosome transfer. FASEB J. 7, 826-833.
Barski, G., Sorieul, S., and Cornefert, E (1960). Production dans des cultures in vitro de deux souches cellulaires en association, de cellules de caractére "hybride". C. R. Acad. Sci. Paris 251, 1825-1827.
Bender, V., Bravo, P., Decaens, C., and Cassio, D. (1999). The structural and functional polarity of the hepatic human-rat hybrid WIF-B is a stable and dominant trait. Hepatology 30, 1002-1010.
Cassio, D., and Weiss, M. C. (1979). Expression of fetal and neonatal hepatic functions by mouse hepatoma-rat hepatoma hybrids. Som. Cell Genet. 5, 719-738.
Goletz, T. J., Smith, J. R., and Pereira-Smith, O. M. (1994). Molecular genetic approaches to the study of cellular senescence. Cold Spring Harb. Symp. Quant. Biol. LIX, 59-66.
Hamon-Benais, C., Delagebeaudeuf, C., Jeremiah, S., Lecoq, O., and Cassio, D. (1994). Efficiency of a specific albumin extinguisher locus in monochromosomal hepatoma hybrids. Exp. Cell Res. 213, 295-304.
Hooper, M. (1985). In "Mammalian Cell Genetics" (E. Bittar, ed.), pp. 77-81. Wiley-InterScience, New York.
Jha, K. K., and Ozer, H. L. (1976). Expression of transformation in cell hybrids. I. Isolation and application of density-inhibited Balb/3T3 cells deficient in hypoxanthine phosphoribosyl transferase and resistant to ouabain. Som. Cell Genet. 2, 215-233.
Killary, A. M., and Fournier, R. E. K. (1984). A genetic analysis of extinction: Trans-dominant loci regulate expression of liverspecific traits in hepatoma-hybrid cells. Cell 38, 523-534.
Littlefield, J. W. (1964). Selection of hybrid from matings of fibroblasts in vitro and their presumed recombinants. Science 145, 709-710.
Mevel-Ninio, M., and Weiss, M. C. (1981). Immunofluorescence analysis of the time-course of extinction, reexpression and activation of albumin production in rat hepatoma-mouse fibroblast heterokaryons and hybrids. J. Cell Biol. 90, 339-350.
Polokoff, M. A., and Everson, G. T. (1986). Hepatocyte-hepatoma cell hybrids: Characterization and demonstration of bile acid synthesis. J. Biol. Chem. 261, 4085--4089.
Pontecorvo, G. (1976). Production of indefinitely multiplying mammalian somatic cell hybrids by polyethylene glycol (PEG) treatment. Somat. Cell Genet. 1, 397--400.
Ramos, C., Bonenfant, D., and Teissie, J. (2002). Cell hybridization by electrofusion on filters. Anal. Biochem. 302, 213-219.
Sellem, C. H., Cassio, D., and Weiss, M. C. (1981). No extinction of tyrosine aminotransferase inducibility in rat hepatoma-human fibroblast hybrids containing the human x chromosome. Cytogenet. Cell Genet. 30, 47-49.
Sorieul, S., and Ephrussi, B. (1961). Karyological demonstration of hybridization of mammalian cells in vitro. Nature (Lond.) 190, 653-654.
Walter, M. A., and Goodfellow, P. N. (1993). Radiation hybrids: Irradiation and fusion gene transfer. Trends Genet. 9, 352-356.
Worton, R. G., and Duff, C. (1979). Karyotyping. Methods Enzymol. 58, 322-344.
Harris, H. (1995). "The Cells of the Body: A History of Somatic Cell Genetics." Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.
Ringertz, N. R., and Savage, R. E., (1976). "Cell Hybrids." Academic Press, London.
Zallen, D. T., and Burian, R. M. (1992). On the beginnings of somatic cell hybridization: Boris EPHRUSSI and chromosome transplantation. Genetics 132, 1-8.
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