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

(1)灰绿烟草与烟草体细胞杂种叶肉原生质体的制备。电融合（1 kV/cm. 0.1uF）。从总共9 × 10^4>个原生质体（每个4.5 × 10^4>）中，我们在培养10周后获得了超过96个绿色杂交菌落（杂交分离产率约为0.2%）。组分I蛋白质分析证实了这些菌落的杂交性质。培养4个月后，所有菌落都再生出芽和一些根。培养8个月后，2个菌落再生出完整植株。这些植株的形态与有性杂交植株的形态非常相似。(2)植物杂交瘤的产生将来自高产小檗碱的日本黄连（Coptis japonica，Polycarpiidae）和高产花色素苷的小米大戟（Euphorbia millii，无患子科（Sapindaifloriidae））悬浮培养物的原生质体电融合（2kV/cm，0.1uF）。我们开发了一个新的系统，使用大规模的电融合，通过包装原生质体混合物在一个平行的电极室，拿起异核体的微量吸管和微量培养他们与护士细胞。我们发现在类内异核体中有暗红色的小团形成。利用这些液滴作为异核体标记物，我们挑选了2830个异核体并进行了培养。其中三个显示出细胞分裂，一个发展成10到20个细胞的细胞集落。荧光显微镜检查表明细胞集落中产生小檗碱。(3)电融合人T细胞以获得T细胞杂交瘤我们发现，在1.25 - 2.5mM Mg和0.3M山梨醇存在下，用0.1 - 1.0%链霉蛋白酶E预处理30分钟以上，并施加电脉冲（例如5 kV/cm，0.1 μ F），可以刺激培养的人细胞的电融合<Cl_2>。利用这些条件，我们电融合人T淋巴细胞，并通过将细胞混合物填充在平行电极中培养CCRF-CEMR细胞。到目前为止，没有获得杂交细胞。

(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.