| Project/Area Number |
16K14595
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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 |
Laboratory animal science
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| Research Institution | Nagasaki University (2017)
Osaka University (2016) |
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Principal Investigator |
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| Project Period (FY) |
2016-04-01 – 2018-03-31
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| Project Status |
Completed (Fiscal Year 2017)
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| Budget Amount *help |
¥3,770,000 (Direct Cost: ¥2,900,000、Indirect Cost: ¥870,000)
Fiscal Year 2017: ¥2,080,000 (Direct Cost: ¥1,600,000、Indirect Cost: ¥480,000)
Fiscal Year 2016: ¥1,690,000 (Direct Cost: ¥1,300,000、Indirect Cost: ¥390,000)
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| Keywords | オートファジー / 測定系 / Haloタグ / タンデムタグ / in vivo測定 / モデル動物 / tandem-fluoresent tag |
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| Outline of Final Research Achievements |
In this study, we aimed to establish a novel strategy to monitor autophagic activity which could be effective even at in vivo situation. To this end, we developed Halotag-LC3 based method using different kinds of Halo tag ligands. We established cell line stably expressing Halotag-LC3 and applied the cells for transient pulse labeling of LC3 with biotin, which allow us to monitor degradation kinetics of LC3. In contrast to conventional autophagy flux assay, this method can avoid the side-effect of lysosomal dysfunction which could potentially cause change in autophagy. Moreover, using features of TMR and Oregon green for different sensitivity to lysosomal low pH condition, we developed pulse labeling of autophagosome and autolysosome.
In summary, we showed that Halotag-LC3 system is highly flexible and efficient way to monitor autophagic activity.
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