ウィルス導入法を用いた誘導調節可能なタンパク機能阻害法の確立

病毒导入法建立诱导可控蛋白功能抑制方法

• 批准号: 23930009
• 负责人: 岡村 理子
• 金额: $ 0.38万
• 依托单位: The University of Tokyo
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Encouragement of Scientists
• 财政年份: 2011
• 资助国家: 日本
• 起止时间: 2011 至 无数据
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-23930009/
• 关键词: RNA干渉; テトラサイクリン

これまで、Creリコンビナーゼやテトラサイクリン応答システムを用い、誘導的RNA干渉法を行うベクターを構築してきた。本研究では、誘導効率をこれまでのものより増大できる次世代テトラサイクリンシステムを利用し、生体内(脳)でのタンパク機能阻害を特異的に調節する系の開発のため、ウィルスベクターの適用を行った。まず新世代テトラサイクリン応答性のRNA干渉実現のため、TRE(tetracycline response element)の変異配列の下流で、Dox(ドキシサイクリン:テトラサイクリン誘導体)作用後に、RNA干渉を起こすshRNA配列が組み込まれたmiRNA配列と、蛍光蛋白質(TagRFP)が同一ベクター上で発現するdual cassetteベクターを構築した。また、独立した別のベクターとして、rtTA-Adの改変体が構成的もしくは神経特異的に発現するベクターを作成した。続いて、培養細胞(HEK293T)において、上記の2種ベクター(Tet依存的miR誘導ベクターとrtTA発現ベクター)と目的遺伝子cDNAを含むベクターを発現させ、Doxを作用させたところ、Dox依存的に目的遺伝子の発現抑制と遺伝子導入マーカーであるTagRFPの発現増加を確認できた。また、shRNA抵抗変異型cDNAベクターを作出し、発現抑制から回復できることも確認した。さらに、これらベクターのウィルス化を進め、Dox依存的に生体内で機能する条件検討を進めた。

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