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

(1)将灰烟和朗斯多夫烟草的体细胞杂交植株叶肉原生质体进行电融合(1kV/cm)。0.1uF)。从9个原生质体(每个4.5个)中，经过10周的培养，我们获得了96个以上的绿色杂交菌落(杂种分离率约为0.2%)。组分I蛋白分析证实了这些菌落的杂交性质。经过4个月的培养，所有的菌落都再生了不定芽和部分根。经过8个月的培养，2个菌落再生了完整的植株。这些植株的形态与有性获得的杂交植株非常相似。(2)将黄连(Polycarpiidae)和大戟(Sapindaifloriidae)悬浮培养产生的植物杂交瘤原生质体进行电融合(2KV/cm，0.1uF)。我们开发了一种新的系统，它使用大规模的电融合，将原生质体混合物包装在平行的电极室中，用微吸管拾取异核体，并用哺育细胞进行微培养。我们发现，在下层异核体中形成了暗红色的小体。利用这些液滴作为异核体的标记物，我们获得了2830个异核体并进行了培养。其中3个细胞分裂，1个细胞形成10-20个细胞的集落。(3)人T细胞的电融合获得T细胞杂交瘤。我们发现，用0.1%~1.0%的Pronase E预处理30min以上，并在1.25~2.5 mM mg&lt；Cl2&gt；0.3M山梨醇中施加电脉冲(如5kV/cm，0.1uF)，可刺激培养的人T细胞的电融合。在此条件下，我们将人T淋巴细胞电融合，将细胞混合物包装在平行电极中培养CCRF-CEMR细胞。到目前为止，还没有获得杂交细胞。

(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.