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

建立了一种新的小鼠基质细胞系Hess-M28，该细胞系支持人CD34 CD38-细胞在人IL-3和SCF存在下体外扩增300倍以上。利用这些细胞，我们试图评估逆转录病毒载体在人原始造血细胞中的顺式作用元件。脐带血细胞在Hess-M28基质细胞系和逆转录病毒载体产生细胞的混合细胞层上培养。FMEV型载体SF/Lyt含有脾病灶形成病毒U3和MESV引物结合位点(PBS)，而MO3/Lyt含有来自MoMLV的U3区和PBS。经FMEV型和基于MoMLV的载体转导后，标记基因小鼠CD8(MCD8)在CD34、CD34和CD34 CD38-细胞中的表达被检测。在CD34和CD34 CD38-细胞中，FMEV型载体SF/Lyt的mCD8表达高于基于MoMLV的载体MO3/Lyt，而在CD34-细胞中mCD8的表达水平与之相当。标记基因在长期培养启动细胞(LTC-ICs)和SCID再填充细胞(SRCs)中的表达也得到证实。

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).