Improved retroviral transfer of genes into canine hematopoietic progenitor cells kept in long-term marrow culture |
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Authors: | Schuening, FG Storb, R Stead, RB Goehle, S Nash, R Miller, AD |
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Affiliation: | Division of Clinical Research, Fred Hutchinson Cancer Research Center, Seattle, WA 98104. |
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Abstract: | 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 x 10(6) 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. |
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