Ramachandra R N, Berczi I, Rector E S, Sehon A H
Department of Immunology, Faculty of Medicine, University of Manitoba, Winnipeg, Canada.
J Immunol Methods. 1991 Aug 28;142(1):21-9. doi: 10.1016/0022-1759(91)90288-q.
The ouabain-resistant mutant cell lines, HOA-1 and HOA-20 were developed from WI-L2-729-HF2 by cloning with increasing concentration of ouabain. Both parent and mutant cell lines were resistant to base analogues, 6-thioguanine (6-TG) and 8-azaguanine (8-AG) to the level of 20 micrograms/ml in the culture medium. The parent cell line WI-L2-729-HF2 was highly sensitive to ouabain, whereas HOA-1 and HOA-20 were resistant to ouabain to the level of 1 microM and 20 microM, respectively. However, all the cell lines were sensitive to HAT-selective medium which is essential for hybrid selection after fusion. All three lymphoblastoid cell lines were positive for Epstein-Barr virus nuclear antigen (EBNA), secreted TNF-beta (lymphotoxin) without any external stimulation, secreted trace amounts of IgG(kappa), which was also present in their cytoplasm and had IgM(kappa) as surface bound immunoglobulin. They also expressed the CD20, CD71 (transferrin receptor) as surface antigens. In addition to these antigens, HOA-20 also expressed CD38 antigen. The karyotype analysis of these cell lines revealed modal chromosomal numbers ranging from 40 to 47. The HLA-A, -B and -C antigens expressed by WI-L2-729-HF2 and its mutants HOA-1 and HOA-20 were identical. Both the HOA-1 and HOA-20 mutants were found suitable for the generation of hybrids after fusion with EBV-transformed human B-lymphocytes.
哇巴因抗性突变细胞系HOA - 1和HOA - 20是通过用浓度递增的哇巴因进行克隆，从WI - L2 - 729 - HF2细胞系中衍生而来的。亲代细胞系和突变细胞系对碱基类似物6 - 硫鸟嘌呤（6 - TG）和8 - 氮鸟嘌呤（8 - AG）均有抗性，在培养基中的抗性水平可达20微克/毫升。亲代细胞系WI - L2 - 729 - HF2对哇巴因高度敏感，而HOA - 1和HOA - 20对哇巴因的抗性水平分别为1微摩尔/升和20微摩尔/升。然而，所有细胞系对融合后杂交选择所必需的HAT选择性培养基均敏感。所有这三种淋巴母细胞系的EB病毒核抗原（EBNA）均呈阳性，在无任何外部刺激的情况下分泌肿瘤坏死因子 - β（淋巴毒素），分泌微量的IgG（κ），其也存在于细胞质中，并且具有IgM（κ）作为表面结合免疫球蛋白。它们还表达CD20、CD71（转铁蛋白受体）作为表面抗原。除了这些抗原外，HOA - 20还表达CD38抗原。这些细胞系的核型分析显示众数染色体数在40至47之间。WI - L2 - 729 - HF2及其突变体HOA - 1和HOA - 20所表达的HLA - A、 - B和 - C抗原是相同的。发现HOA - 1和HOA - 20这两种突变体都适合与EB病毒转化的人B淋巴细胞融合后产生杂交细胞。
