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 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.
Schuening
F G
FG
Division of Clinical Research, Fred Hutchinson Cancer Research Center, Seattle, WA 98104.
Storb
R
R
Stead
R B
RB
Goehle
S
S
Nash
R
R
Miller
A D
AD
D000818:Animals; D001854:Bone Marrow Cells; D002478:Cells, Cultured; D004285:Dogs; D005818:Genetic Engineering / Q000379:methods; D005822:Genetic Vectors; D006412:Hematopoietic Stem Cells / Q000502:physiology*; D012190:Retroviridae / Q000235:genetics; D013997:Time Factors; D014162:Transfection