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. These cells were used in an attempt 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 Mo-MuLV. After transduction by the FMEV-type and Mo-MuLV-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 with the cells transduced by the Mo-MuLV-based vector MO3/Lyt, although the expression was comparable in CD34- cells. Expression of marker genes was also confirmed in long-term culture-initiating cells (LTC-ICs) and SCID-repopulating cells (SRCs).

译文

建立了一种新型的鼠基质细胞系HESS-M28,该细胞系在存在人IL-3和SCF的情况下,在体外支持人CD34 CD38细胞扩增300倍以上。这些细胞用于评估人类原始造血细胞中逆转录病毒载体的顺式作用元件。在基质细胞系、HESS-M28和逆转录病毒载体产生细胞的混合细胞层的顶部培养脐带血细胞。FMEV型载体SF/Lyt包含脾脏焦点形成病毒U3和MESV引物结合位点 (PBS),而MO3/Lyt包含来自Mo-MuLV的U3区域和PBS。通过FMEV型和基于Mo-MuLV的载体转导后,在CD34-,CD34和CD34 CD38-细胞中检查了标记基因鼠CD8 (mCD8) 的表达。在CD34和CD34 CD38-细胞中,与基于Mo-MuLV的载体MO3/Lyt转导的细胞相比,FMEV型载体SF/Lyt的mCD8表达更高,尽管其表达在CD34-细胞中相当。标记基因的表达也在长期培养起始细胞 (LTC-ICs) 和SCID再填充细胞 (src) 中得到证实。

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