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. Addition of granulocyte-macrophage colony stimulating factor (GM-CSF) to the culture medium is required for long-term growth of the cells. Erythropoietin (Epo) can also be used to sustain TF-1 cells but for only limited periods (approximately a week). Low levels of both growth factors can act synergistically to maintain proliferation for a longer period of time than Epo alone. To eliminate the requirement of exogenous Epo for growth, TF-1 cells were co-cultured with a retroviral secreting cell line containing the human erythropoietin (hEpo) gene and a neomycin (neo) selectable marker. TF-1 cells which exhibited neo resistance (indicating infection by the retrovirus) were then grown in low concentrations of GM-CSF without the addition of Epo. Under these conditions growth of normal TF-1 cells was not sustained. The neo-resistant cells survived for more than 14 days indicating synergy between GM-CSF and the Epo synthesised by the co-cultured TF-1 cells. Radioimmunoassays performed on growth media detected concentrations up to 1 mU/ml of Epo, implying that stable integration of the retroviral vector and expression of the hEpo gene have been achieved.
D002455:Cell Division / Q000187:drug effects; D002460:Cell Line; D003001:Cloning, Molecular; D004262:DNA Restriction Enzymes; D018076:DNA, Complementary; D004921:Erythropoietin / Q000032:analysis / Q000096:biosynthesis* / Q000235:genetics*; D018014:Gene Transfer Techniques; D005819:Genetic Markers; D016178:Granulocyte-Macrophage Colony-Stimulating Factor / Q000494:pharmacology; D006801:Humans; D019868:Kanamycin Kinase; D004915:Leukemia, Erythroblastic, Acute; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000235:genetics; D011863:Radioimmunoassay; D014407:Tumor Cells, Cultured
D018076:DNA, Complementary; D005819:Genetic Markers; D004921:Erythropoietin; D016178:Granulocyte-Macrophage Colony-Stimulating Factor; D017853:Phosphotransferases (Alcohol Group Acceptor); D019868:Kanamycin Kinase; D004262:DNA Restriction Enzymes