| Project/Area Number |
14350434
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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 | 一般 |
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| Research Field |
生物・生体工学
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| Research Institution | Nagoya University |
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Principal Investigator |
KAMIHIRA Masamichi Nagoya University, Graduate School of Engineering, Associate Professor, 工学研究科, 助教授 (40202022)
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| Co-Investigator(Kenkyū-buntansha) |
IIJIMA Shinji Nagoya University, Graduate School of Engineering, Professor, 工学研究科, 教授 (00168056)
NISHIJIMA Ken-ichi Nagoya University, Graduate School of Engineering, Assistant Professor, 工学研究科, 助手 (10262891)
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| Project Period (FY) |
2002 – 2004
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| Project Status |
Completed (Fiscal Year 2004)
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| Budget Amount *help |
¥16,600,000 (Direct Cost: ¥16,600,000)
Fiscal Year 2004: ¥4,000,000 (Direct Cost: ¥4,000,000)
Fiscal Year 2003: ¥5,800,000 (Direct Cost: ¥5,800,000)
Fiscal Year 2002: ¥6,800,000 (Direct Cost: ¥6,800,000)
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| Keywords | transgenic avian / monoclonal antibody / retroviral vector / scFv-Fc / animal cell / chimeric antibody / chicken / quail |
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| Research Abstract |
Transgenic chickens have been proposed as a transgenic bioreactor for the production of recombinant proteins. To date, although various procedures for generating transgenic avians have been reported, the expression of a transgene at a commercially feasible level has not been attained. We used a replication-defective pantropic retroviral vector based on Moloney murine leukemia virus(MoMLV) pseudotyped with vesicular stomatitis virus G protein(VSV-G) for gene introduction to avian embryos. For the generation of transgenic chickens producing monoclonal antibodies, we firstly constructed a retroviral vector plasmid for the expression of a single-chain antibody fragment(scFv), derived from an anti-prion protein monoclonal antibody, fused with the Fc region of human IgG1, as a model antibody. We steadily generated transgenic chickens (GO) by injecting a concentrated retroviral vector into the heart of developing embryos after 55 h incubation and followed by embryo culture to hatch. The GO ch
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ickens expressed the transgene product in their serum and eggs at high levels (~5.6 mg/ml) throughout the breeding period. The expression level in egg white corresponds to almost one tenth that of ovalbumin, a major protein in egg white, and more than equivalent to that of lysozyme. Furthermore, we could obtain G1 and G2 transgenic progeny derived from a GO chicken. The transgenic progeny also expressed the transgene product in their serum and eggs. Next, we constructed a retroviral vector for the production of a chimeric anti-CD2 antibody, in which the bicistronic expression of H-and L-chain genes of the antibody was mediated by an internal ribosomal entry site(IRES) sequence derived from encephalomyocarditis virus(EMCV). The viral vector was injected to chicken embryos after 55 h incubation to generate GO transgenic chickens. The chickens hatched expressed anti-CD2 antibody in the serum and eggs. This system demonstrates the reality of a transgenic chicken bioreactor for the commercial production of monoclonal antibodies. Less
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