| Project/Area Number |
15310090
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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 |
Nanomaterials/Nanobioscience
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| Research Institution | Kyoto University |
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Principal Investigator |
KATO Koichi Kyoto University, Institute for Frontier Medical Sciences, Associate Professor, 再生医科学研究所, 助教授 (50283875)
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| Co-Investigator(Kenkyū-buntansha) |
IWATA Hiroo Kyoto University, Institute for Frontier Medical Sciences, Professor, 再生医科学研究所, 教授 (30160120)
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| Project Period (FY) |
2003 – 2005
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| Project Status |
Completed (Fiscal Year 2005)
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| Budget Amount *help |
¥15,900,000 (Direct Cost: ¥15,900,000)
Fiscal Year 2005: ¥2,900,000 (Direct Cost: ¥2,900,000)
Fiscal Year 2004: ¥2,900,000 (Direct Cost: ¥2,900,000)
Fiscal Year 2003: ¥10,100,000 (Direct Cost: ¥10,100,000)
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| Keywords | RNA interference / gene function / high-throughput analysis / micropatterned surface / cell microarray / transfectional array / self-assembled monolayer / electroporation / 遺伝子機能解析 / 微細加工 / 細胞マイクロアレイ |
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| Research Abstract |
The aim of this study was to develop microarrays that allow the parallel transfection of multiple siRNAs for the cell-based high-throughput analysis of gene functions. Because transfection efficiency is one of the most crucial factors that determine the usefulness of such microarrays, we were mostly involved in the preparation of siRNA arrays with improved transfection efficiency.
First we made an attempt to prepare siRNA microarrays in the way that complexes consisting of siRNA and a cationic lipid (transfection enhancer) were loaded in an array format taking advantage of micropatterned, self-assembled monolayers formed on a gold-evaporated glass plate. The feasibility of the method was demonstrated by directly seeding HEK293 cells to the microarray on which EGFP-coding plasmid and EGFP-targeting siRNA were simultaneously loaded. The result of this study was submitted for publication in the international journal.
In addition, the method could also be applied to the transfection of siRNA that was prepared by in vitro transcription of cDNA into long double-stranded RNA followed by digestion with endonuclease, dicer. Because dicer-processed RNA contains a variety of oligonucleotides, it is not necessary to select one of the most effective sequences targeted by siRNA. This feature makes the siRNA array more useful.
In order to enhance much more transfection efficiency, we studied another system in which an electric pulse was applied to detach the loaded siRNA from the array to introduce into cells (electroporation). By optimizing conditions for array preparation and electric pulsing, the highly-efficient transfer of siRNA to the directly-cultured cells could be achieved.
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