Development of new cell fusion method for production of hybridomas in higher plants and animals

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(1) Production of somatic hybrid plant Mesophyll protoplasts from Nicotiana glauca and N. langsdorffii were electrofused (1kV/cm. 0.1uF). From a total of 9 x <10^4> protoplasts (4.5 x <10^4> each), we obtained more than 96 green hybrid colonies after 10 weeks of culture (the hybrid isolation yield was about 0.2%). Fraction I protein analysis confirmed the hybrid nature of these colonies. After 4 months of culture, all colonies regenerated shoots and some roots. After 8 months of culture, 2 colonies regenerted complete plants. The morphology of these plants was very similar to that of the sexually obtained hybrid plants.(2) Production of plant hybridomas Protoplasts from high berberine-producing Coptis japonica (Polycarpiidae) and high anthocyanin-producing Euphorbia millii (Sapindaifloriidae) suspension cultures were electrofused (2kV/cm, 0.1uF). We developed a new system that uses a large scale electrofusion by packing protoplast mixture in a parallel electrode chamber, picking up heterokaryons by a micropipette and microculture them with nurse cells. We found that dark red froplets are formed in the inter-infraclass heterokaryons. Using these droplets as a heterokaryon maker, we picked up 2830 heterokaryons and cultured. Three of them showed cell division and one developed into a cell colony of ten to twenty cells. Fluorescence microscopy indicated production of berberine in the cell colony.(3) Electrofusion of human T cells to obtain T cell hybridoma We found that electrofusion of cultured human cells could be stimulated when they had been pretreated with 0.1 to 1.0% Pronase E for more than 30min and applied with an electric pulse (e.g. 5kV/cm, 0.1uF) in the presence of 1.25 to 2.5mM Mg <Cl_2> and 0.3M sorbitol. By use of these conditions and we electrofused human T lymphocytes and cultured CCRF-CEMR cells by packing the cell mixture in a parallel electrode. So far no hybrid cells were obtained.