Somatic cell hybridization between buffalo and mouse cells

Rank, D.N.; Joshi, C.G.; Kanakaraj, P.; Brahmkshtri, B.P.; Kharadi, V.B.; Nainar, A.M. and Krishnan, A.R. (1998) Somatic cell hybridization between buffalo and mouse cells. Indian Journal of Animal Sciences, 68 (9). pp. 991-992.

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Abstract

Breeding of livestock for heightened disease resistance and better productivity is the ultimate goal of livestock genome mapping. Interspecific somatic cell hybridization is one of the several approaches for gene mapping. Mouse and hamster cell lines have been used, as fusion partner to somatic cells from various livestock species (Saidi - Mehtar et al. 1981, Womack and Moll 1986). Research on buffalo gene mapping using this technique is of recent origin and only a few reports are available (El-Nahas et al. 1993). Present study aims at standardization of this methodology forthe production of hybrid cells between river buffalo cells and mouse LM (TK-) cell line. Hybridization study involved buffalo embryo kidney fibroblasts (BEKF) and mouse connective tissue cell line, LM (TK). Parent cells were cultured in Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 10% fetal bovine serum and incubated under 5% CO2 tension at 37°C. Hybridization was carried out by monolayer fusion technique as described by Davidson and Gerald (1976) with modifications using polyethylene glycol (PEG), average mol. wt. 3350 as fusogen. Hybrid cells were grown in HAT - ouabain selective medium containing hypoxanthine (2.5 x 10-4M), thymidine (1.6 x l0-5M), aminopterin (l.0 x 10-7M) and ouabain (5.0x 10-7M). Initially 50% PEG solution was tried for fusion. Later multinucleated cells (heterokaryone) were observed 1-2 hr. Many of them surviving up to 20-25 days but failed to multiply. Hence, dimethyl sulfoxide (DMSO) was added in fusogen solutions as it widens the range over which PEG is acting (Norwood et al. 1976). More number of hybrid cell colonies were obtained when PEG was used at 45% concentration (6.5 and 22) than at 50% (0 and 1) for fusion of cells at 2 different concentrations, high (BEKF) 5.0 x 105 ; LM (TK) 5.0 x 105and low (BEKF) 2.5 x 105;

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