2001 Fiscal Year Final Research Report Summary
Development of mammalian cells capable of growing rapidly in serum-free media
| 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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| Keywords | chimeric receptor / antibody variable region / erythropoietin receptor / hybridoma cells / gp130 / growth control / serum-free medium / antibody production |
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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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[Publications] Ueda, H., Kawahara, M., Aburatani, T., Tsumoto, K., Todokoro, K., Suzuki, E., Nishimura, H., Schueler, P.A., Winter, G., Mahoney, W.C., Kumagai, L, and Nagamune, T.: "Cell-growth control by monomeric antigen; the cell surface expression of lysozyrne-specific Ig V-domains fused to truncated Epo receptor"Immuriol. Methods. 241. 159-170 (2000)
Description
「研究成果報告書概要(欧文)」より
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[Publications] Kawahara, M., Ueda, H., Tsumoto, K., Kumagai, I., Mahoney, W. and Nagamune, T.: "A growth signal with an artificially induced erythropoietin receptor-gp130 cytoplasmic domain heterodimer"J. Biochem.. 130. 305-312 (2001)
Description
「研究成果報告書概要(欧文)」より
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[Publications] Kawahara, M., Natsume, A., Terada, S., Kato, K., Tsumoto, K., Kumagai, I., Miki, M., Mahoney, W., Ueda, H., and Nagamune, T.: "Replacing factor-dependency with that for lysozyme: Affordable culture of IL-6-dependent hybridoma by transfecting artificial cell surface receptor"Biotechnol. Bioeng.. 74. 416-423 (2001)
Description
「研究成果報告書概要(欧文)」より
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[Publications] Kawahara, M, Ueda, H., Tsumoto, K., Kumagai, I., Mahoney, W., and Nagamune, T.: "Selection of highly productive mammalian cells based on an inducible growth advantage using an antibody/receptor chimera"J. Biosci. Bioeng.. 93. 399-404 (2002)
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「研究成果報告書概要(欧文)」より
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[Publications] Kawahara, M., Ueda, H., Tsumoto, K., Todokoro, K., Mahoney, W., Kumagai, I. and Nagamune, T.: "Monovalent antigen activates antibody/cytokine receptor chimera and controls hematopoietic cell growth. Animal Cell Technology: Challenges for the 21st Century(JAACT/ESACT) (Sasaki, R., Ed.)"Kluwer Academic Publishers, Dordrecht. 255-259 (1999)
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「研究成果報告書概要(欧文)」より
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[Publications] Kawahara, M., Natsume, A., Terada, S., Kato, K., Tsumoto, K., Kumagai, L, Miki, M., Mahoney, W., Ueda, H. and Nagamune, T.: "Lysozyme-dependent proliferation switch: Hematopoietic and hybridoma cell growth control with the use of antibody/gp130 chimera. Animal cell technology: basic & applied aspects (Shirahata, S. et al. Eds.)"Kluwer Academic Publishers, Dordrecht. 337-341 (2002)
Description
「研究成果報告書概要(欧文)」より
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[Publications] Kawahara, M., Ueda, H., Tsumoto, K., Kumagai, I., Mahoney, W. and Nagamune, T.: "Antigen-mediated genetically modified cell amplification. Advances in Biochemical Production Technologies (Nagamune, T., Marten, M., Park, T. H., Eds.)"Oxford University Press (in press).
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「研究成果報告書概要(欧文)」より
