| Project/Area Number |
15K15351
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| Research Category |
Grant-in-Aid for Challenging Exploratory Research
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| Allocation Type | Multi-year Fund |
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| Research Field |
Metabolomics
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| Research Institution | Nihon University |
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Principal Investigator |
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| Co-Investigator(Renkei-kenkyūsha) |
OTSUKA Yuichiro 日本大学, 医学部, 助手 (40748399)
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| Project Period (FY) |
2015-04-01 – 2017-03-31
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| Project Status |
Completed (Fiscal Year 2016)
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| Budget Amount *help |
¥3,640,000 (Direct Cost: ¥2,800,000、Indirect Cost: ¥840,000)
Fiscal Year 2016: ¥1,820,000 (Direct Cost: ¥1,400,000、Indirect Cost: ¥420,000)
Fiscal Year 2015: ¥1,820,000 (Direct Cost: ¥1,400,000、Indirect Cost: ¥420,000)
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| Keywords | インスリン分泌機構 / 膵β細胞 / 遺伝子発現修飾 / recombinase / インスリン分泌 / リコンビナーゼ / β細胞 / 遺伝子操作 |
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| Outline of Final Research Achievements |
Genetic modyfication of insulin secreting cells is a powerful strategy to study mechanisms of insulin secretion and of cell death. However, one of the disadvantages encountered in using these cells is the low transfection efficiency of nucleotides. To circumvent the difficulties, we have established a very efficient system based on the recombinase-cassette exchange for generation of a insulin secreting MIN6-derived master cell line.
A recipient platform was generated that has the zeocin-resistant gene flanked with a set of yeast flippase recognition target sites and placed on the genome of MIN6 cells. Overexpression and suppression of gene-of-interest are achieved by delivery with an exchange vector of cDNAs and shRNAs, respectively. Using this cell line, we showed that engineered clones were created within 6 weeks. Moreover, we could generate more than 100 transfectant cell lines and identified novel genes involved in regulation of glucose-stimulated insulin secretion.
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