| 研究課題/領域番号 |
22KF0405
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| 配分区分 | 基金 |
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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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研究代表者 |
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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| 研究分担者 |
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) |
2023-03-08 – 2025-03-31
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| キーワード | transcription / electron microscopy / epigenomics |
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| 研究実績の概要 |
We successfully established the chromEMT protocol at RIKEN for cultured adherent cells (MCF10A), including EM-fixation, photo-oxidation and staining of DNA with DRAQ5 + OsO4, resin embedding, and ultrathin sectioning. We next developed a modified chromEMT protocol to incorporate immunolabeling of specific genomic landmarks with fluorescent nanogold probes for visualization by light microscopy and EM.
We have acquired TEM images of labeled samples that demonstrate successful labeling of single targets, including lncRNA (XIST), histone modifications (H3K27Ac), and RNA polymerases (RNAPII-S2 and POL1). In addition, we have preliminary data (fluorescent microscopy) supporting on-target labeling of additional targets (H3K27me3, H3K4me3, RNAPII-S5).
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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 developed a modified chromEMT protocol to label genomic landmarks for EM imaging. Preliminary data from conventional TEM imaging confirm quality, specificity, and good signal-to-noise of labeling. We are now preparing samples with labeling for regulatory genome landmarks (H3K27me3, H3K27Ac, and RNAPII-S2) for tilt-axis EM imaging and to construct 3D tomograms. These will form the primary dataset for our study, after which we will be ready for analysis and manuscript writing.
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| 今後の研究の推進方策 |
We have revised the original goal of imaging of patient-specific regulatory genome mutations due to time constraints. We will instead aim to complete the project with comprehensive labeling of different classes (active, repressed, and transcribed) of regulatory elements in the MCF10A system where our assay is established and working. Final sample preparation will be completed by the end of May. Tilt-axis imaging will be done in early June at OIST. 3D tomography, chromatin structure modeling, and manuscript writing will be performed thereafter.
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| 次年度使用額が生じた理由 |
ChromEMT is a new and complicated methodology. Modifying the existing protocol to accommodate targeted labeling required optimization at multiple steps including testing different labeling, permeabilization, gold enhancement, and photo oxidation.
In FY2023, we took a careful, steady approach to solving these challenges. Although our timelines for experiments and spending were delayed, we successfully developed a working protocol optimized for signal-to-noise. We are now ready for full scale sample preparation and data acquisition. The carryover budget is needed to cover associated costs.
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