RETROVIRAL-MEDIATED GENE-TRANSFER OF THE HUMAN ERYTHROPOIETIN GENE INTO A FACTOR-DEPENDENT CELL-LINE, TF-1

The factor-dependent cell line, TF-1, established from a patient with erythroleukaemia, shows characteristics of immature erythroblasts. Add ition of granulocyte-macrophage colony stimulating factor (GM-CSF) to the culture medium is required for long-term growth of the cells. Eryt hropoietin (Epo) can also be used to sustain TF-l cells but for only l imited periods (approximately a week). Low levels of both growth facto rs can act synergistically to maintain proliferation for a longer peri od of time than Epo alone. To eliminate the requirement of exogenous E po for growth, TF-I cells were cocultured with a retroviral secreting cell line containing the human erythropoietin (hEpo) gene and a neomyc in (neo) selectable marker. TF-1 cells which exhibited neo resistance (indicating infection by the retrovirus) were then grown in low concen trations of GM-CSF without the addition of Epo. Under these conditions growth of normal TF-1 cells was not sustained. The neo-resistant cell s survived for more than 14 days indicating synergy between GM-CSF and the Epo synthesised by the cc-cultured TF-l cells. Radioimmunoassays performed on growth media detected concentrations up to 1 mU/ml of Epo , implying that stable integration of the retroviral vector and expres sion of the hEpo gene have been achieved.