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

开发用于高等植物和动物杂交瘤生产的新细胞融合方法

基本信息

批准号：
59890008
负责人：
YAMADA Yasuyuki
金额：
$ 8.26万
依托单位：
Kyoto University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Developmental Scientific Research
财政年份：
1984
资助国家：
日本
起止时间：
1984 至 1985
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-59890008/
关键词：
Cell fusion Electrofusion Hybridoma Protoplast Human T cells Berberine Anthocyanin タバコヒトT細胞

项目摘要

(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.

（1）从烟草和N. langsdorffii中产生的体细胞植物叶肉原生质体被电载（1kv/cm。0.1UF）。从总共9 x <10^4>原生质体（每个4.5 x <10^4>）中，我们在培养10周后获得了96多个绿色杂种菌落（杂交分离率为0.2％）。馏分I蛋白分析证实了这些菌落的杂种性质。经过4个月的培养，所有殖民地都恢复了芽和一些根。经过8个月的培养，有2个殖民地再生完整的植物。 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).我们开发了一个新系统，该系统通过将原生质体混合物填充在平行电极室中，通过微孔拾取异性虫，并用护士细胞将其微培养它们。我们发现在内部杂质杂质者中形成了深红色的froplets。我们使用这些液滴作为异性恋制造商，我们捡起了2830个异性恋并进行了培养。其中三个显示细胞分裂，一个人发展成十至二十个细胞的细胞群。荧光显微镜表明在细胞群体中产生berberine。（3）人类T细胞的电渗渗；我们发现，当用0.1至1.0％的pronase E预处理30mmin并用电脉冲施加0.1至1.0％pronase E时，可以刺激培养的人类细胞的电渗，并在5KV/CM，0.1 UM MM MM MM <Cl_25中进行施加。 0.3m山梨糖醇。通过使用这些条件，我们通过将细胞混合物堆积在平行电极中，电染料的人类T淋巴细胞和培养的CCRF-CEMR细胞。到目前为止尚未获得杂化细胞。