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

细胞调控基因操控造血干细胞技术的开发与应用

基本信息

批准号：
11557075
负责人：
OZAWA Keiya
金额：
$ 7.36万
依托单位：
Jichi Medical School
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B).
财政年份：
1999
资助国家：
日本
起止时间：
1999 至 2000
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-11557075/
关键词：
Hematopoietic stem cell in vivo expansion ex vivo expansion Gene transfer differentiation-regulatory gene Selective amplifier gene

项目摘要

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.

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。