Our previous studies have demonstrated that retro-virus-mediated gene transduction of either the human erythropoietin receptor (EpoR) or H-ras cDNA into single purified hematopoietic progenitor (HPC), CD343+, cells from cord blood (CB) resulted in increased numbers and sizes of erythroid cell containing colonies. We therefore evaluated if there were further effects when H-ras and EpoR genes were co-transduced into the same progenitor cells. Highly purified single sorted CD343+ CB cells were transduced with retroviral vectors encoding EpoR or H-ras cDNA. At the single cell level, and in response to stimulation by a combination of growth factors, including Epo, the number of colonies formed by BFU-E and CFU-GEMM was significantly increased in cells transduced with either single H-ras or EpoR cDNA compared to mock virus-transduced cells as previously described. Increased numbers of BFU-E, but not CFU-GEMM, colonies were produced from cells simultaneously co-transduced with both EpoR and H-ras genes. Little or no growth was seen in transduced cells without exogenously added cytokines. The size of all types of colonies including CFU-GM was increased in cells transduced with H-ras and/or EpoR cDNAs, and the greatest increase was noticed in cells co-transduced with both genes. Integration and expression of either gene in individual colonies as assessed by PCR and RT-PCR analysis were 45-62% and 48-58%, respectively, with approximately 31% of the cells containing and expressing both genes. These results add to information suggesting an enhancing interacting role of H-ras and EpoR in erythroid proliferation/differentiation.
- Retroviral vector
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