Selection of Hybrids from Matings of Fibroblasts in vitro and Their Presumed Recombinants
14 Aug 1964-Science (American Association for the Advancement of Science)-Vol. 145, Iss: 3633, pp 709-710
TL;DR: Evidence can be obtained which suggests that mating may be followed by segregation in mice, and when two clonal lines of mouse fibroblasts are grown together for 4 days, hybrid cells can be detected by selective conditions.
Abstract: When two clonal lines of mouse fibroblasts, each containing a drug-resistant marker, are grown together for 4 days, hybrid cells can be detected by selective conditions. These hybrid cells are presumed to be the result of mating. By the same method evidence can be obtained which suggests that mating may be followed by segregation.
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TL;DR: The derivation of a number of tissue culture cell lines which secrete anti-sheep red blood cell (SRBC) antibodies is described here, made by fusion of a mouse myeloma and mouse spleen cells from an immunised donor.
Abstract: THE manufacture of predefined specific antibodies by means of permanent tissue culture cell lines is of general interest. There are at present a considerable number of permanent cultures of myeloma cells1,2 and screening procedures have been used to reveal antibody activity in some of them. This, however, is not a satisfactory source of monoclonal antibodies of predefined specificity. We describe here the derivation of a number of tissue culture cell lines which secrete anti-sheep red blood cell (SRBC) antibodies. The cell lines are made by fusion of a mouse myeloma and mouse spleen cells from an immunised donor. To understand the expression and interactions of the Ig chains from the parental lines, fusion experiments between two known mouse myeloma lines were carried out.
19,053 citations
TL;DR: Cell fusion techniques have been used to produce hybrids between myeloma cells and antibody‐producing cells that are permanently adapted to grow in tissue culture and are capable of inducing antibody-producing tumors in mice.
Abstract: Cell fusion techniques have been used to produce hybrids between myeloma cells and antibody-producing cells. The hybrid lines derived are permanently adapted to grow in tissue culture and are capable of inducing antibody-producing tumors in mice. Spleens from mice immunized against sheep red blood cells (SRBC) were fused to an 8-azaguanine-resistant clone (X63-Ag8) of MOPC 21 myeloma. Over 50% of the derived hybrid lines produce and secrete immunoglobulins different from the MOPC 21 myeloma. About 10% of the hybrid lines exhibit anti-SRBC activity. The high proportion of antibody-producing hybrids suggests that the fusion involves a restricted fraction of the spleen cell population, probably cells committed to antibody production. In order to avoid the presence of the MOPC 21 heavy chain in the specific hybrids, another myeloma cell line (NSI/1-Ag4-1) has been used. This is a nonsecreting variant of the MOPC 21 myeloma which does not express heavy chains. Three anti-SRBC (probably of the mu, gamma2b and gamma1 classes, respectively) and two anti-2,4,6-trinitrophenyl (of the mu class) antibody-producing hybrids have been repeatedly cloned. By random selection and by selection of specific clones according to their lytic activity (clone plaque selection), a number of different lines have been constructed. Such lines express different combinations of the four possible chains of each hybrid line: the myeloma gamma and K chains and the specific antibody heavy and light chains. In three cases (Sp1, Sp2 and Sp7) it is shown that only the specific H and L combination has activity and that the myeloma chains are unable to substitute for them. In most cases lines have been derived which no longer express the MOPC 21 chains but only the specific antibody chains.
2,170 citations
TL;DR: The experiments established the usefulness of the bybrid myeloma technique in preparing monospecific antibodies against human cell surface antigens and highlights the possibilities not only of obtaining reagents for somatic cell genetics, but also of obtaining mouse antibodies detecting human antigenic polymorphisms.
1,892 citations
Cites methods from "Selection of Hybrids from Matings o..."
...half the medium was replaced with HAT medium (Littlefield, 1964); this was repeated every day for the first week...
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TL;DR: FUSION between myeloma cells and spleen cells from immunised donors has been shown to be a successful method of deriving homogeneous anti-SRBC (anti-sheep red blood cell) and anti-TNP antibodies.
Abstract: FUSION between myeloma cells and spleen cells from immunised donors has been shown to be a successful method of deriving homogeneous anti-SRBC (anti-sheep red blood cell) and anti-TNP antibodies1,2. One of the most powerful features of this approach is that, by cloning, one may easily derive cell lines synthesising monoclonal antibodies despite using non-purified immunogens. The multiple components of a heterogeneous population of hybrid cells are resolved by cloning techniques. This feature makes the system a very powerful tool in the study of complex antigenic structures. The established cell lines offer the further advantage of unlimited permanent supply of material, and the possibility of worldwide standardisation.
1,749 citations
TL;DR: A permanent human cell line, EA .
Abstract: A permanent human cell line, EA . hy 926, has been established that expresses at least one highly differentiated function of vascular endothelium, factor VIII-related antigen. This line was derived by fusing human umbilical vein endothelial cells with the permanent human cell line A549. Hybrid cells that survived in selective medium had more chromosomes than either progenitor cell type and included a marker chromosome from the A549 line. Factor VIII-related antigen can be identified intracellularly in the hybrids by immunofluorescence and accumulates in the culture fluid. Expression of factor VIII-related antigen by these hybrid cells has been maintained for more than 100 cumulative population doublings, including more than 50 passages and three cloning steps. This is evidence that EA . hy 926 represents a permanent line.
1,487 citations
References
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TL;DR: Mouse fibroblasts cultivated in the presence of bromodeoxyuridine (BUDr) incorporated the thymidine (TDr) analogue into DNA but a resistant subline has been selected which failed to incorporate BUDr into DNA, as shown by CsCl density gradient centrifugation experiments.
540 citations
TL;DR: This chapter illustrates that formal genetic analysis by means of mitotic recombination is perfectly feasible and leads to the identification of linkage groups, of the order of genes, and of their distances, though in units, it is different from those of meiotic analysis.
Abstract: Publisher Summary This chapter illustrates that formal genetic analysis by means of mitotic recombination is perfectly feasible. It leads to the identification of linkage groups, of the order of genes, and of their distances, though in units, it is different from those of meiotic analysis. In aspergillus nidulans, which has a sexual cycle and in which the results of genetic analysis based on meiosis are available, mitotic mapping has been compared with, and helped by, meiotic mapping. But in other similar organisms, such as aspergillus niger, in which a sexual cycle is unknown, mitotic recombination is the only means available so far toward formal genetic analysis and breeding for scientific or applied purposes. The possibility of applying mitotic analysis to somatic tissues of higher organisms, better if in tissue cultures, is clearly at hand. Even if somatic crossing-over were not of widespread occurrence, the analysis could at least identify linkage groups making use of breakdowns of mitosis, similar to the haploidization described in the chapter, which are known to occur as rare accidents in higher organisms. The principles of mitotic analysis expounded in the chapter are clearly adaptable, with minor modifications, to a wide variety of special cases.
253 citations
TL;DR: The analyses of pentachlorophenol were based on its absorption maximum at 320 m/t in alkali, and the results confirmed its importance as a raw material for biochemistry.
Abstract: 3 Weinbach, E. C., J. Biol. Chem., 221, 609 (1956). 4Lehninger, A. L., J. Biol. Chem., 234, 2187 (1959). 6 Lehninger, A. L., B. L. Ray, and M. Schneider, J. Biophys. Biochem. Cytol., 5, 97 (1959). 6 The analyses of pentachlorophenol were based on its absorption maximum at 320 m/t in alkali. 7 Lehninger, A. L., and L. E. Remmert, J. Biol. Chem., 234, 2459 (1959). 8 Weinbach, E. C., J. Biol. Chem., 234, 1580 (1959). 9 Rall, J. E., R. Michel, J. Roche, 0. Michel, and S. Varrone, J. Biol. Chem., 238, 1848 (1963). 0 Lehninger, A. L., Physiol. Revs., 42, 467 (1962). 11 Weinbach, E. C., in Abstracts, 144th Meeting, American Chemical Society, March 31, 1963, p. 20L. 12 Weinbach, E. C., and W. J. Bowen, Biochim. et Biophys. Acta, 30, 476 (1958). 13 Palade, G. E., in Enzymes: Units of Biological Structure and Function, ed. 0. H. Gaebler (New York: Academic Press, 1956), p. 185. 14 Racker, E., Advances in Enzymol., 23, 323 (1961). 15 Goodwin, T. W., and 0. Lindberg, ed., Biological Structure and Function (New York: Academic Press, 1961), vol. 2, pp. 3-268.
140 citations
TL;DR: A confirmation of the results of Ephrussi and Sorieul1 with data concerning a new somatic hybrid between a polyoma-induced and a spontaneous mouse tumour, each originating in a different inbred mouse strain.
Abstract: Ephrussi and Sorieul1 recently reported a somatic hybrid between a spontaneous mouse tumour derived from a C3H mouse, and a polyoma-induced mouse tumour originating from a non-inbred Swiss mouse. These findings extended their own and other reports of somatic cell hybridization2–4, in showing that cells with different genotypes originating in different strains of mice can form a somatic hybrid when cultured together in vitro. In this communication we describe a confirmation of the results of Ephrussi and Sorieul1 with data concerning a new somatic hybrid between a polyoma-induced and a spontaneous mouse tumour, each originating in a different inbred mouse strain. The origin of each parental cell in a different inbred strain has made it possible to undertake transplantation investigations of the antigenic properties of the somatic hybrid.
39 citations