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Development and characterization of ouabain-resistant human fusion partners
R N Ramachandra1, I Berczi, E S Rector
1Department of Immunology, Faculty of Medicine, University of Manitoba, Winnipeg, Canada.
Abstract:
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.
Insights
Ouabain-resistant mutant cell lines HOA-1 and HOA-20 were developed for hybrid generation. These cell lines, along with their parent, share characteristics like EBNA positivity and cytokine secretion, making them suitable for fusion experiments.
Area of Science:
- Cell Biology
- Immunology
- Genetics
Background:
- Development of drug-resistant cell lines is crucial for genetic manipulation and cell fusion studies.
- Lymphoblastoid cell lines offer a valuable model for investigating B-cell characteristics and Epstein-Barr virus interactions.
Purpose of the Study:
- To develop and characterize ouabain-resistant mutant cell lines (HOA-1, HOA-20) from a parent cell line (WI-L2-729-HF2).
- To assess the suitability of these mutant cell lines for generating hybrid cells through fusion with Epstein-Barr virus-transformed human B-lymphocytes.
Main Methods:
- Development of mutant cell lines through cloning with increasing ouabain concentrations.
- Assessment of resistance to base analogues (6-thioguanine, 8-azaguanine) and sensitivity to HAT-selective medium.
- Characterization of cell surface antigens (CD20, CD71, CD38), immunoglobulin secretion (IgG kappa, IgM kappa), Epstein-Barr virus nuclear antigen (EBNA) expression, and karyotype analysis.
Main Results:
- HOA-1 and HOA-20 exhibited significant resistance to ouabain compared to the parent cell line.
- All cell lines were resistant to 6-thioguanine and 8-azaguanine, and sensitive to HAT medium.
- The cell lines expressed common EBNA, secreted TNF-beta, and had similar HLA profiles, with HOA-20 additionally expressing CD38.
- HOA-1 and HOA-20 were confirmed suitable for hybrid generation via fusion.
Conclusions:
- The developed ouabain-resistant mutant cell lines (HOA-1, HOA-20) are valuable tools for cell fusion experiments.
- These cell lines retain key characteristics of lymphoblastoid cells, including EBNA expression and cytokine secretion.
- The genetic stability and antigen expression profiles support their use in generating hybrid cells for further research.