| 研究課題/領域番号 |
22F22072
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| 配分区分 | 補助金 |
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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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| 受入研究者 |
石川 文彦 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 (30403918)
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| 外国人特別研究員 |
LIANG MINGGAO 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) |
2022-07-27 – 2025-03-31
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| キーワード | transcription / non-coding / electronmicroscopy |
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| 研究実績の概要 |
We developed a novel methodology to directly visualize chromatin and gene regulatory dynamics in situ. Our strategy involves 2 components:
1) the chromEMT protocol, which combines targeted staining of DNA with OsO4, tilt-axis electron microscopy imaging, and tomography to generate ultra-resolution 3D models of chromatin in-situ.
2) immunogold labeling of regulatory genome landmarks (polymerases, histone modifications, and specific RNAs) for visualization by EM.
Combining the two, our approach aims to directly visualize regulatory genome features (enhancers, promoters, transcriptional units) in the context of ultra-resolution chromatin imaging in-situ.
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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 have spent the past 2 years developing a working protocol and overcoming two key challenges:
1) Establishing chromEMT at RIKEN, including sample prep, staining, sectioning, and imaging
2) Developing an in-situ labeling strategy compatible with chromEMT. For the latter, we used immuno-fluoronanogold particles that can easily enter fixed nuclei, bind specific targets, and can be chemically enhanced for visualization by EM.
We have successfully labeled various targets, including lncRNA (XIST), polymerases (POL1, RNAPII), and histone modifications (H3K27Ac) and have optimized our method for sensitivity and specificity. These are supported by preliminary 2D EM imaging and fluorescent imaging data. We are now ready to proceed with 3D tilt-axis imaging and tomography.
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| 今後の研究の推進方策 |
Using the established protocol, we will proceed with tilt-axis imaging of labeled samples for tomography and 3D modeling.
Experiments will be performed in the MCF10A cell line where our assay is currently established and optimized. Labeling will be done for XIST, RNAPII-S5, and histone modifications to cover a variety of regulatory element classes. Imaging will be performed at OIST, where specialized TEM is available for tilt-axis imaging.
Sample preparation and imaging for the final dataset will be completed in 2 months time, and computational analysis will proceed immediately after. We will write a manuscript to report achievements for the program.
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| 備考 |
We were able to make considerable progress over the past 2 years and develop a working protocol. Our findings have been well received at internal symposia and have sparked new collaborations.
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