Improved Retroviral Transfer of Genes into Canine Hematopoietic Progenitor Cells
Canine hematopoietic progenitor cells were infected with amphotropic helper-free retroviral vectors containing either the neomycin phosphotransferase gene (NEO) or a mutant dihydrofolate reductase gene (DHFR*). Successful transfer and expression of both genes in hematopoietic progenitor cells was demonstrated by the ability of the viruses to confer resistance to either the aminoglycoside G418 or methotrexate (MTX). It was shown that the incidence of resistant granulocyte- macrophage colony-forming units (CFU-GM) after cocultivation for 24 hours with helper-free virus-producing cells was 10% (6% to 16%). Autologous marrow cocultivated for 24 hours with virus-producing packaging cells was then transplanted into six dogs after lethal total body irradiation. All dogs showed engraftment within two weeks and four dogs survived 5–7 months without adverse effects. One dog that had been given marrow infected with a DHFR* virus and that had received MTX as in vivo selection after marrow transplantation and survived, showed 0.1% and 0.03% MTX-resistant CFU-GM at weeks 3 and 5, respectively. In an attempt to increase the efficiency of gene transfer, which could improve our yield of cells with a transformed phenotype posttransplant, marrow was cocultivated for 24 hours with virus-producing packaging cells and then kept in long-term marrow culture which was fed with virus-containing supernatant. Cells were harvested after six days and cultured in CFU-GM assay with and without selective agent. The average rate of gene expression in CFU-GM was 41% (19% to 86%). Successful gene transfer into canine hematopoietic progenitor cells can be achieved using retroviral vectors and the efficiency of gene transfer is increased by combining cocultivation with long-term marrow culture. It is uncertain at this point whether canine pluripotent hematopoietic stem cells can be infected.
KeywordsPackaging Cell Roller Bottle Neomycin Phosphotransferase Gene Autologous Marrow Dihydrofolate Reductase Gene
Unable to display preview. Download preview PDF.
- 6.Kantoff PW, Gillio AP, McLachlin JR, Bordignon C, Eglitis MA, Kernan NA, Moen RC, Kohn DB, Yu S, Karson E, Karlsson S, Zwiebel JA, Gilboa E, Blaese RM, Nienhuis A, O’Reilly RJ, French Anderson W (1987) Expression of human adenosine deaminase in nonhuman primates after retrovirus-mediated gene transfer. J Exp Med 166: 219–234PubMedCrossRefGoogle Scholar
- 14.Schuening F, Emde C, Schaefer UW (1983) Improved culture conditions for granulocyte-macrophage progenitor cells (abstr). Exp Hematol 11 (Suppl 14): 205Google Scholar
- 16.Toksoez D, Dexter TM, Lord BI, Wright EG, Lajtha LG (1980) The regulation of hemopoiesis in long-term bone marrow cultures. II. Stimulation and inhibition of stem cell proliferation. Blood 55: 931–936Google Scholar