Development of a method for efficient transfer and expression of genes in hematopoietic cells.

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A novel murine stromal cell line ,HESS-M28, was established, which supports the expansion of human CD34+CD38-cells more than 300-fold in vitro in the presence of human IL-3 and SCF. Utilizing this cells, Attempt was made to evaluate cis-acting elements of retroviral vectors in human primitive hematopoietic cells.Cord blood cells were cultured on top of the mixed cell layers of the stromal cell line, HESS-M28, and retroviral vector producing cells. The FMEV-type vector ,SF/Lyt, contained the spleen focus-forming virus U3 and the MESV primer binding site (PBS), while MO3/Lyt contained the U3 region and PBS from MoMLV. Following transduction by the FMEV-type and the MoMLV-based vectors, expression of the marker gene, murine CD8(mCD8), was examined in CD34-, CD34+ and CD34+CD38-cells. In CD34+and CD34+CD38-cells, expression of mCD8 was higher with the FMEV-type vector, SF/Lyt, compared to the cells transduced by the MoMLV-based vector,MO3/Lyt,although the expression was comparable in CD34-cells. Expression of marker genes was also confirmedimed in long-term culture-initiating cells(LTC-ICs)and SCID-repopulating cells(SRCs).