| Project/Area Number |
20K21763
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| Research Category |
Grant-in-Aid for Challenging Research (Exploratory)
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| Allocation Type | Multi-year Fund |
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| Review Section |
Medium-sized Section 59:Sports sciences, physical education, health sciences, and related fields
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| Research Institution | Kumamoto University |
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Principal Investigator |
ONO YUSUKE 熊本大学, 発生医学研究所, 独立准教授 (60601119)
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| Project Period (FY) |
2020-07-30 – 2022-03-31
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| Project Status |
Completed (Fiscal Year 2021)
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| Budget Amount *help |
¥6,500,000 (Direct Cost: ¥5,000,000、Indirect Cost: ¥1,500,000)
Fiscal Year 2021: ¥2,600,000 (Direct Cost: ¥2,000,000、Indirect Cost: ¥600,000)
Fiscal Year 2020: ¥3,900,000 (Direct Cost: ¥3,000,000、Indirect Cost: ¥900,000)
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| Keywords | 骨格筋 / ゲノム編集 / 筋幹細胞 / 筋再生 / 筋可塑性 / サテライト細胞 |
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| Outline of Research at the Start |
筋可塑性や筋再生を制御する分子機構については不明な点が未だ多く残されており，研究の加速が望まれる。本研究計画では，筋可塑性・再生研究を加速させるために骨格筋を標的にした新たなゲノム編集技術の開発を行う。Cre依存性にCRISPR/Cas9システムを誘導できるR26-LSL-Cas9ノックインマウスを用いて、in vivoおよびin vitroにおけるgRNA導入方法の確立とゲノム編集の効率を評価する。
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| Outline of Final Research Achievements |
The purpose of this study was to establish genome editing techniques for muscle cells using Cas9 mice. To induce satellite cell-specific Cas9 expression, Pax7-IRES-CreER/+ mice were crossed with R26-LSL-Cas9 mice to create Pax7-IRES-CreER/+;Rosa26-LSL-Cas9 mice. We also generated MlcCre/+;R26-LSL-Cas9 mice that express Cas9 specifically in myofibers. Using primary cultured cells, we investigated transfection efficiency of gRNA by electroporation or lipofection. We observed that the transfection of gRNA was more efficient with electroporation compared to the lipofection method. Genome editing efficiency for target genes was determined by qPCR. We also confirmed the genome editing method is applicable for multiple genes. The knockout efficiency varied with the length of the target genome.
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| Academic Significance and Societal Importance of the Research Achievements |
骨格筋の可塑性や再生を制御する分子機構については不明な点が多く，研究分野を加速する技術開発が望まれている。今回検討したゲノム編集技術を，今後さらに効率を改善することで，単一遺伝子のみならず複数遺伝子の同時欠損や遺伝子発現調節領域の編集も可能となり，当該分野を加速する強力なツールになる。
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