| Project/Area Number |
19F19386
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| Research Category |
Grant-in-Aid for JSPS Fellows
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| Allocation Type | Single-year Grants |
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| Section | 外国 |
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| Review Section |
Basic Section 43050:Genome biology-related
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| Research Institution | Institute of Physical and Chemical Research |
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Principal Investigator |
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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| Co-Investigator(Kenkyū-buntansha) |
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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| Project Period (FY) |
2019-11-08 – 2022-03-31
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| Project Status |
Completed (Fiscal Year 2021)
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| Budget Amount *help |
¥2,000,000 (Direct Cost: ¥2,000,000)
Fiscal Year 2021: ¥500,000 (Direct Cost: ¥500,000)
Fiscal Year 2020: ¥900,000 (Direct Cost: ¥900,000)
Fiscal Year 2019: ¥600,000 (Direct Cost: ¥600,000)
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| Keywords | CRISPR / single-cell / transcriptomics / iPS-derived neurons / iPSC differentiation / Non-coding RNA / Genomics / Neurodegeneration / iPSC to neuron / cholesterol modulation / neuron-astrocyte |
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| Outline of Research at the Start |
This study will investigate how brain cholesterol levels are regulated by long non-coding RNAs (lncRNAs). Cellular cholesterol levels are controlled by a careful balance of synthesis, uptake and efflux. Notably, 40% of long ncRNAs are expressed in the brain. Therefore, this project will be the first to investigate lncRNA functionality on cholesterol metabolism in neurons.
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| Outline of Annual Research Achievements |
Through this collaboration we first established iPS cell lines conducible for CRISPR screening. Cell lines were engineered to express GFP in specific loci responsible for sodium channel. Using this newly established cell line, we generated single cell RNA gene expression profiles from over 5000 neurons and identified 1000 long non-coding RNAs involved in neuron differentiation. We next designed sgRNA libraries targeting these long non-coding RNAs compatible for large-scale CRISPR screening. The experimental design also led to the development of novel assays to evaluate the effectiveness of the screening, which involved optimization of cytometry analysis and cell sorting, immunohistochemistry, cell culture protocols, where we have successfully developed a method to selectively isolate population of neurons that can distinguish partial to full responsiveness to genetic perturbations using fluorescently tagged antibodies. Overall, the development of cell lines, single cell data, CRISPR screening assay have been successful and will result in discovering novel regulators of neuronal differentiation and sodium channel regulation.
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| Research Progress Status |
令和3年度が最終年度であるため、記入しない。
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| Strategy for Future Research Activity |
令和3年度が最終年度であるため、記入しない。
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