2016 Fiscal Year Final Research Report
Development of an efficient method for generation of genetically modified insulin secreting cells and its application for studies on insulin secretion
| 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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| Keywords | インスリン分泌機構 / 膵β細胞 / 遺伝子発現修飾 / recombinase |
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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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| Free Research Field |
代謝学
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