Development and application of the technologies for manipulationg hematopoietic stem cells using cell-regulatory genes

1)对于体外扩增的造血干细胞，在体外培养过程中需要一过性的分化阻断，我们利用了显性阴性形式的维甲酸受体α(REARE)。将含有稀有基因的逆转录病毒载体转导至32D细胞后，即使在G-CSF存在的情况下，细胞仍保持未成熟状态，继续增殖，未显示分化。当表达该稀有基因的32D细胞进一步转导Cre重组酶基因时，在G-CSF存在下，细胞分化为粒细胞，表明分化障碍被取消。利用Cre/loxP系统对稀有基因的可逆整合可能适用于HSC的体外扩增。2)为了克服基因转移效率低的缺点，我们开发了一种选择性扩增转导造血细胞的新策略。这个系统涉及“选择性放大基因(SAGs)”，它编码细胞因子受体和类固醇受体激素结合域(HBD)之间的融合蛋白。原型SAG编码了粒细胞集落刺激因子受体(GCR)和雌激素结合域(ER)之间的融合分子(GCRER)。我们使用原代小鼠骨髓细胞在体内对SAG系统进行了评估。将转导SAG基因的骨髓细胞移植到致死性照射的小鼠体内，对重组的小鼠进行TM攻击。结果提示，SAG对体内转导的造血细胞具有激素反应性生长能力，选择性扩增这些细胞是可行的。

1) For ex vivo hematopoietic stem cell (HSC) expansion, the transient block of differentiation would be required during the ex vivo culture and we utilized a dominant negative form of the retinoic acid receptor α (RARE). The 32D cells transduced with a retroviral vector containing the RARE gene flanked by the loxP sites at the both ends remained immature and continued to proliferate without showing differentiation even in the presence of G-CSF.When the 32D cells expressing the RARE gene were further transduced with the Cre recombinase gene, the cells differentiated into granulocytes in the presence of G-CSF, indicating that the differentiation block was abolished. The reversible integration of the RARE gene using the Cre/loxP system may be applicable to ex vivo HSC expansion.2) To overcome the low efficiency of gene transfer into HSCs, we have developed a novel strategy for selective expansion of transduced hematopoietic cells. This system involves "selective amplifier genes (SAGs)" which encode fusion proteins between cytokine receptors and the steroid receptor hormone-binding domains (HBDs). The prototype SAG encoded a fusion molecule (GCRER) between the granulocyte colony-stimulating factor receptor (GCR) and the estrogen-binding domain (ER). We have evaluated the SAG system using primary murine bone marrow cells in vivo. The SAG-transduced bone marrow cells were transplanted into lethally irradiated mice, and the reconstituted animals were challenged with Tm. The results suggested that SAG conferred a steroid-responsive growth ability on the transduced hematopoietic cells in vivo, and that selective expansion of these cells is feasible.