Improved Retroviral Transfer of Genes Into Canine Hematopoietic Progenitor Cells Kept in Long-Term Marrow Culture

Amphotropic helper-free retroviral vectors containing either the bacterial neomycin phosphotransferase gene (NEO) or a mutant dihydrofolate reductase gene (DHFR *) were used to infect canine hematopoietic progenitor cells. In previous experiments, successful transfer and expression of both genes in canine CFU-GM were achieved after 24-hour cocultivation with virus-producing cells. The average rate of gene expression was 10% (6% to 16%) as measured by the number of CFU-GM resistant to either the aminoglycoside G418 or methotrexate. In an attempt to increase the efficiency of gene transfer, marrow was cocultured for 24 hours with either NEO or DHFR * virus-producing packaging cells and then kept in long-term marrow culture fed three times with virus-containing supernatant (2 to 5 × 106 CFU/mL). After six days, cells were harvested and cultured in CFU-GM assay with and without a selective agent. The average rate of gene expression in CFU-GM in five independent experiments was 46% and ranged from 19% to 87%. In conclusion, the efficiency of gene transfer into canine hematopoietic progenitor cells has been increased fourfold by combining cocultivation with long-term marrow culture as compared with results obtained with cocultivation only.