| Project/Area Number |
11555216
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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 |
生物・生体工学
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| Research Institution | The University of Tokyo |
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Principal Investigator |
NAGAMUNE Teruyuki Graduate School of Engineering, The University of Tokyo, Professor, 大学院・工学系研究科, 教授 (20124373)
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| Co-Investigator(Kenkyū-buntansha) |
TERADA Satoshi Fukui Univ., School of Engineering, Research Associate, 工学部, 助手 (60311685)
KITAYAMA Atsushi Graduate School of Engineering, The University of Tokyo, Research Associate, 大学院・工学系研究科, 助手 (70270882)
UEDA Hiroshi Graduate School of Engineering, The University of Tokyo, Lecturer, 大学院・工学系研究科, 講師 (60232758)
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| Project Period (FY) |
1999 – 2000
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| Project Status |
Completed (Fiscal Year 2001)
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| Budget Amount *help |
¥8,900,000 (Direct Cost: ¥8,900,000)
Fiscal Year 2000: ¥3,400,000 (Direct Cost: ¥3,400,000)
Fiscal Year 1999: ¥5,500,000 (Direct Cost: ¥5,500,000)
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| Keywords | chimeric receptor / antibody variable region / erythropoietin receptor / hybridoma cells / gp130 / growth control / serum-free medium / antibody production / c-Kit |
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| Research Abstract |
Cytokines and growth factors are indispensable for the propagation and maintenance of factor-dependent mammalian cells. However, cytokines are often so expensive that the use of factor-dependent cells for the industrial applications such as protein production is not practical. Here we try to establish a method to maintain cell growth with an inexpensive ligand quite different from cytokines. Based on the fact that interaction between V_H and V_L regions of anti-hen egg lysozyme (HEL) antibody HyHEL-10 becomes strong only in the presence of HEL, part of the extracellular region of erythropoietin receptor (EpoR) was replaced with either V_H or V_L region of HyHEL-10 to create Vn-EpoR or V_L-EpoR, respectively. When V_H-EpoR and V_L-EpoR were cotransfected into an interleukin-3 (IL-3) dependent pro-B cell line Ba/F3, the transfectant proliferated in a HEL dose-dependent manner without IL-3. To substitute IL-6 dependency of hybridoma cells, the intracellular domain of V_H-EpoR and V_L-EpoR was replaced with that of gp130 to create V_H-gp130 and V_L-gp130, respectively. When V_H-gp130 and V_L-gp130 genes were co-transfected to Ba/F3 cells, the transfectant proliferated in a HEL dose-dependent manner without IL-3. When V_H-gp130 and V_L-gp130 genes were co-transfected to an IL-6 dependent hybridoma cell line 7TD1, the transfectant showed clear HEL dose-dependent cell growth and monoclonal antibody production both in serum-containing and serum-free media without IL-6, resulting in significant medium cost reduction. The results further suggest the possibility of applying this technology for the control of many other cell functions by exchanging the receptor intracellular domain.
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