Protection of uninfected human bone marrow cells in long-term culture from G418 toxicity after retroviral-mediated transfer of the NEOr gene.
The long-term effect of retroviral-mediated gene transfer into human hematopoietic cells in vitro was studied in bone marrow culture. Two retroviral vectors (pN2 or pZIP NEO) were used to transfer the gene coding for neomycin phosphotransferase, which confers neomycin resistance, as a dominant selectable marker. Following infection, bone marrow cells of multiple hematopoietic lineages displayed resistance for the duration of the cultures (greater than 80 days) to normally cytotoxic doses of the neomycin analog G418. However, upon DNA analysis of cells surviving in G418, the NEOr (neomycin resistance) gene was not detected under conditions where single copy genes could readily be seen, despite the presence of NEOr RNA sequences. In order to investigate this observation further, infected and uninfected cells were separated by a filter, and cultured in the same medium containing G418. The uninfected cells continued to survive in the presence of normally toxic concentrations of G418. Medium alone from infected cells was able to protect uninfected cells the same way. Efficiency of transfer of this and perhaps other selectable marker genes to cells in the long-term culture system may consequently be overestimated if survival of cells alone is quantitated. These results indicate that long-term cultures are a useful in vitro model for the study of retroviral-mediated gene transfer to human hematopoietic cells.
Bayever
E
E
Division of Allergy-Immunology-Bone Marrow Transplantation, Children's Hospital of Philadelphia, Pennsylvania 19104.
Haines
K
K
Duprey
S
S
Rappaport
E
E
Douglas
S D
SD
Surrey
S
S
D002478:Cells, Cultured; D004279:DNA, Viral / Q000032:analysis; D004352:Drug Resistance, Microbial; D005814:Genes, Viral; D006412:Hematopoietic Stem Cells / Q000166:cytology / Q000187:drug effects*; D006801:Humans; D019868:Kanamycin Kinase; D009355:Neomycin / Q000494:pharmacology; D010770:Phosphotransferases / Q000235:genetics*; D012190:Retroviridae / Q000235:genetics*; D014162:Transfection
D004279:DNA, Viral; D010770:Phosphotransferases; D019868:Kanamycin Kinase; D009355:Neomycin