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Improved retroviral transfer of genes into canine hematopoietic progenitor
cells kept in long-term marrow culture
FG Schuening, R Storb, RB Stead, S Goehle, R Nash and AD Miller
Division of Clinical Research, Fred Hutchinson Cancer Research Center,
Seattle, WA 98104.
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.
Volume 74,
Issue 1,
pp. 152-155,
07/01/1989
Copyright © 1989 by The American Society of Hematology
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