Development and characterization of ouabain-resistant human fusion partners

The ouabain-resistant mutant cell, lines, HOA-1 and HOA-2 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-thiaguanine (6-TG) and 8-azaguanine (8-AG) to the level of 20 μg/ml in the culture medium. The parent cell line WI-L2-729-HF2 was highly senstive to ouabain, whereas HOA-1 and HOA-20 were resistant to ouabain to the level of 1 μM and 20 μM, 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(κ), which was also present in their cytoplasm and had IgM(κ) as surface bound immunoglobulin. They also expessed 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.