| 研究課題/領域番号 |
19F19386
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| 研究機関 | 国立研究開発法人理化学研究所 |
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研究代表者 |
SHIN JAE・WOO 国立研究開発法人理化学研究所, 生命医科学研究センター, チームリーダー (60553849)
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| 研究分担者 |
PRABHU ANIKA 国立研究開発法人理化学研究所, 生命医科学研究センター, 外国人特別研究員
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| 研究期間 (年度) |
2019-11-08 – 2022-03-31
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| キーワード | iPSC to neuron / cholesterol modulation / neuron-astrocyte |
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| 研究実績の概要 |
The aim of this project is to investigate the functionality of non-coding RNAs, with a focus on long non-coding RNAs, in neuronal cholesterol metabolism. Thus far, we have established the model system to be utilized, a highly-scalable iPSC-derived neurons that can be co-cultured with human astrocytes and/or astrocyte-conditioned media. The iPSC differentiation process has been optimized in-house, and we have identified that cholesterol levels increase approximately 10-fold during this process. Cellular cholesterol levels at various stages of this differentiation are ready to be determined, which will allow us to identify the optimal time-points at which to analyze the transcriptomic changes that occur during the dramatic increase in cholesterol synthesis in neurons.
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| 現在までの達成度 (区分) |
現在までの達成度 (区分)
2: おおむね順調に進展している
理由
We plan to use targeted qPCR to finalize the timepoints at which neurons will be analyzed by bulk CAGE sequencing, which will measure total RNA expression and map transcriptional start sites. In addition, small-scale tests with sterol modulators will be used to optimize treatment conditions, prior to sequencing. Genes involved in sterol regulation exhibit a particular transcriptomic pattern in response to sterol modulation. Analysis of the sequencing results will confirm this pattern in known sterol regulatory elements, and help identify novel candidates for further interrogation.
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| 今後の研究の推進方策 |
In FY2020, we will finalize CAGE library production and sequencing. Due to COVID19, the sequencing will be significantly delayed. We ask for kind understanding. Once the data is generated, the top candidates identified will be validated and characterized using CRISPRi/sgRNAs against the lncRNAs of interest and systematically test the effect of lncRNA knockdown on cholesterol metabolism in neurons. This involves performing a longitudinal survival assay to determine the role of the lncRNAs on neuronal health, and measuring the effects of lncRNA knockdown on cholesterol synthesis, uptake, and trafficking. Focusing on a few relevant lncRNAs, we will identify the interactions formed by these lncRNAs through ChIRP-seq which allows the simultaneous profiling of binding proteins and chromatin interacting regions.
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