| Project/Area Number |
14571127
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| Research Category |
Grant-in-Aid for Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
General surgery
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| Research Institution | The University of Tokyo |
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Principal Investigator |
ANDO Yuichi The University of Tokyo, The Institute of Medical Science, Research Associate, 医科学研究所, 助手 (00262080)
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| Co-Investigator(Kenkyū-buntansha) |
TAHARA Hideaki The University of Tokyo, The Institute of Medical Science, Professor, 医科学研究所, 教授 (70322071)
TAKAYAMA Takuya The University of Tokyo, The Institute of Medical Science, Research Associate, 医科学研究所, 助手 (10332579)
BECK Yoshifumi The University of Tokyo, The Institute of Medical Science, Lecturer, 医科学研究所, 講師 (70199454)
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| Project Period (FY) |
2002 – 2003
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| Project Status |
Completed (Fiscal Year 2003)
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| Budget Amount *help |
¥4,000,000 (Direct Cost: ¥4,000,000)
Fiscal Year 2003: ¥1,100,000 (Direct Cost: ¥1,100,000)
Fiscal Year 2002: ¥2,900,000 (Direct Cost: ¥2,900,000)
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| Keywords | dendritic cells / electroporation / RNA / Flt3L / cancer / immunotherapy / genetherapy / 骨髄非破壊的幹細胞移植 |
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| Research Abstract |
[Background] Dendritic cells are professional antigen presenting cells that can efficiently activate antigen specific T cells. Various strategies have been investigated to load antigen on dendritic cell for efficient CTL induction. Recent reports have shown that strong immune responses can be induced by dendritic cells transfected with antigen-coding or tumor-derived RNA using electroporation. However, the optimal condition of RNA-transfection with electroporation is still controversial. [Aim] We examined various conditions of RNA electroporation for dendritic cells to determine the optimal conditions for expression. [Materials and Methods] We used EGFP RNA transcribed in vitro from pTNT/EGFP and pGEM4Z/EGFP/A64, and lacZ RNA transcribed in vitro from pTNT/lacZ and pGEM4Z/lacZ/A64. In vitro transcribed RNA was transfected into day-7 bone marrow-derived dendritic cells of C57BL/6 mice with an. 5x10e6 cells in 200μl Opti-MEM (Invitrogen) were electroporated with RNA in 0.2-cm gapped cuve
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tte. Multiple conditions of voltage, pulse length, number of pulse and RNA amount were examined ranging between 200-lOOOV, 150-3000μs, 1-5pulses and 0-50μg, respectively. Effect of 0K-432 or LPS on EGFP expression in transfected dendritic cell was also examined.
[Results] Flow cytometry and X-gal staining showed the expression of EGFP and 13 -galactosidase in dendritic cells electroporated with EGFP and lacZ RNA transcribed in vitro, respectively. Capped RNA better expressed EGFP than by RNA added β globin leader sequence or by uncapped RNA. The best efficiency with minor cell damage of electroporation was obtained with a condition at 300V, 500μs and one pulse. EGFP expression reached a plateau in DC transfected with 25μg-capped EGFP RNA. EGFP expression was detected in 12hr after electroporation, and was at its peak at 24hr after electroporation. LPS and OK-432 argued EGFP expression. [Conclusion] We demonstrated that RNA of interest could be efficiently transfected and expressed with electroporation. only with well-examined specific conditions. Less
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