Gene transduction into murine primitive hematopoietic cells with 2-gene retroviral vectors using a Transwell coculture system.
The present study aims at expressing a reporter gene in hematopoietic cells in vivo by introducing it into primitive hematopoietic cells with a 2-gene retroviral vector. Various constructs of retroviral vectors containing the human IL-2 receptor alpha chain gene (TAC) as the reporter and the neomycin phosphotransferase gene (neo) as a selectable marker were engineered, and the effectiveness of these vectors for expression of the reporter gene was evaluated after transfection into the packaging cell line GP + E86. It was found that the highest levels of reporter gene expression were attained with constructs ordered 5' long terminal repeat (LTR)-TAC-internal promoter-neo-3' LTR. In experiments investigating the expression of a reporter gene in hematopoietic cells, we used the Escherichia coli beta-galactosidase gene (lacZ) instead of TAC, because a very sensitive detection method was available for lacZ. For transduction of hematopoietic progenitors, packaging cell lines producing recombinant viruses were cultured in a Transwell hung into a Dexter-type bone marrow (BM) culture. The BM cells were selected with G418, and transferred into irradiated recipient mice. LacZ enzyme activity was detectable in the peripheral blood lymphocytes (PBL) of recipients taken 8 wk after reconstitution.
Asami
N
N
Department of Immunology, Kyoto University, Japan. asami@chest.chest.kyoto-u.ac.jp
Germeraad
W T
WT
Fujimoto
S
S
Nagai
S
S
Izumi
T
T
Katsura
Y
Y
D000818:Animals; D001854:Bone Marrow Cells; D002478:Cells, Cultured; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D018014:Gene Transfer Techniques; D005798:Genes, Bacterial; D017930:Genes, Reporter; D005822:Genetic Vectors; D006412:Hematopoietic Stem Cells; D051379:Mice; D008810:Mice, Inbred C57BL; D011994:Recombinant Proteins; D012190:Retroviridae / Q000235:genetics; D013997:Time Factors; D014018:Tissue Distribution; D014161:Transduction, Genetic; D001616:beta-Galactosidase / Q000235:genetics
D011994:Recombinant Proteins; D001616:beta-Galactosidase