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Gene transduction into murine primitive hematopoietic cells with 2‐gene retroviral vectors using a Transwell coculture system
Author(s) -
Asami N.,
Germeraad W. T. V.,
Fujimoto S.,
Nagai S.,
Izumi T.,
Katsura Y.
Publication year - 1996
Publication title -
european journal of haematology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.904
H-Index - 84
eISSN - 1600-0609
pISSN - 0902-4441
DOI - 10.1111/j.1600-0609.1996.tb01378.x
Subject(s) - reporter gene , haematopoiesis , transduction (biophysics) , biology , microbiology and biotechnology , transfection , selectable marker , viral vector , cell culture , gene , progenitor cell , gene expression , transgene , stem cell , recombinant dna , genetics , biochemistry
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 α 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 promoterneo‐3′ LTR. In experiments investigating the expression of a reporter gene in hematopoietic cells, we used the Escherichia coli β‐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.