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Efficient retrovirus-mediated gene transduction into murine hematopoietic
stem cells and long-lasting expression using a Transwell coculture system
WT Germeraad, N Asami, S Fujimoto, O Mazda and Y Katsura
Department of Immunology, Kyoto University, Japan.
The neomycin phosphotransferase (neo) gene was transduced into murine
hematopoietic stem cells by culturing a recombinant retrovirus- producing
cell line in a Transwell (Costar, Cambridge, MA) (bottomed with a porous
membrane) hung into a Dexter-type long-term bone marrow (BM) culture. Gene
transduction into stem cells retaining long-term reconstitution ability was
successfully performed by using protocols of total 15 to 18 days of culture
including establishment of the Dexter culture, transduction, and G418
selection. In the irradiated recipients of these cells, a large majority of
the BM, thymus, and spleen cells as well as peripheral blood (PB)
leukocytes were of donor origin and the neo gene was present in these
organs up to 21 weeks after cell transfer. One third to two thirds of the
in vitro colony-forming cells in the BM of the recipient mice were
resistant to cultivation with G418. It was further found that the
hematopoietic system of secondary recipients given BM cells from a primary
recipient mouse was predominated by original donor-type cells. The
transduced neo gene was detected in the PB, BM, thymus, and spleen cells of
these secondary recipients. These results indicate that our procedure of
retroviral vector-mediated gene transfer is highly effective in safely
introducing a gene into pluripotent hematopoietic stem cells.
Volume 84,
Issue 3,
pp. 780-788,
08/01/1994
Copyright © 1994 by The American Society of Hematology
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