| 研究課題/領域番号 |
20K06865
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| 研究機関 | 国立遺伝学研究所 |
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研究代表者 |
ZHU YAN 国立遺伝学研究所, 遺伝形質研究系, 助教 (50464235)
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| 研究期間 (年度) |
2020-04-01 – 2023-03-31
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| キーワード | ATP sensor / Bioenergetics / metabolic pathways / Neuronal migration |
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| 研究実績の概要 |
There are two objectives of this research: (1) visualize the energy status of migrating neurons; (2) tease out the metabolic sources that fuel neuronal migration.
For the first objective, I have subcloned ATeam series of ATP sensors to enable efficient neuronal expression. FRET-based imaging of intracellular ATP is currently being established. For the second objective, I have established a 3D whole hindbrain organotypic culture system using which I have started testing a range of pharmacological inhibitors of metabolic pathways. I have also successfully generated a knockout mouse line of a transcription factor known to mediate switching of cellular reliance on different metabolic pathways. Detailed phenotype analysis of thees knockout mice is underway.
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| 現在までの達成度 (区分) |
現在までの達成度 (区分)
3: やや遅れている
理由
The progress of this study is slightly delayed for the following reasons.
I encountered some difficulty in establishing FRET-based imaging using confocal microscopy, because the CFP-YFP FRET pair in ATeam sensors require a specific laser line.
A second reason for the delay is because of a two months’ absence from work due to an emergency oversea trip which is further extended by COVID-related border control and the quarantine policy.
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| 今後の研究の推進方策 |
(1)For real-time imaging of ATP level in migrating neurons, I will introduce the fluorescent ATP sensors into the migrating neurons via in utero electroporation and subsequently set up in vitro explant culture to recapitulate chain neuronal migration. I will then use the DeltaVision imaging platform to track the FRET signals in migrating cells. I am also considering using a single wavelength ATP sensor, iATPSnFRs, as a complementary approach.
(2)I will test aspects of neuronal migration when the metabolic pathways are manipulated by application of a range of pharmacological inhibitors.
(3)I will perform detailed phenotype analyses of the knockout mouse line we have generated.
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| 次年度使用額が生じた理由 |
For the next fiscal year, the incurring amount of the grant will be used partly for employing a technician who will be involved in the generation and maintenance of knockout mouse lines, their phenotype analyses, as well as other aspects of animal husbandry. The rest of the grant will be spent on the tissue and cell culture medium and disposable plasticware, purchasing the pharmacological reagents, antibodies for phenotype analyses, and molecular biology kits and reagents.
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