| Project/Area Number |
11557075
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| Research Category |
Grant-in-Aid for Scientific Research (B).
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| Allocation Type | Single-year Grants |
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| Section | 展開研究 |
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| Research Field |
Hematology
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| Research Institution | Jichi Medical School |
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Principal Investigator |
OZAWA Keiya Jichi Medical School, Faculty of Medicine, Professor, 医学部, 教授 (30137707)
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| Co-Investigator(Kenkyū-buntansha) |
HASEGAWA Mamoru DNAVEC Research Inc., Director, 所長
KUME Akihiro Jichi Med.Sch., Faculty of Medicine, Associate Professor, 医学部, 助教授 (10264293)
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| Project Period (FY) |
1999 – 2000
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| Project Status |
Completed (Fiscal Year 2000)
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| Budget Amount *help |
¥11,500,000 (Direct Cost: ¥11,500,000)
Fiscal Year 2000: ¥5,700,000 (Direct Cost: ¥5,700,000)
Fiscal Year 1999: ¥5,800,000 (Direct Cost: ¥5,800,000)
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| Keywords | Hematopoietic stem cell / in vivo expansion / ex vivo expansion / Gene transfer / differentiation-regulatory gene / Selective amplifier gene |
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| Research Abstract |
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.
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