Protein knockown system using IMiDs in mice.

2023 Fiscal Year Final Research Report

• Project/Area Number: 21K19188
• Research Category: Grant-in-Aid for Challenging Research (Exploratory)
• Allocation Type: Multi-year Fund
• Review Section: Medium-sized Section 42:Veterinary medical science, animal science, and related fields
• Research Institution: Kyoto University
• Principal Investigator: Naruse Chie 京都大学, 医学研究科, 准教授 (30372486)
• Co-Investigator(Kenkyū-buntansha): 浅野 雅秀 京都大学, 医学研究科, 教授 (50251450)
• Project Period (FY): 2021-07-09 – 2024-03-31
• Keywords: マウス / デグロン / プロテインノックダウン

Outline of Final Research Achievements

We are currently aiming to develop a simple protein knockdown method using thalidomide analogs. Since thalidomide analogs have been considered to have no neoligand degrading activity in mouse cells, we generated CRBN humanized mice by genome editing and evaluated the protein knockdown efficiency by measuring the fluorescence intensity of EGFP-degglutination tag derivatives. CRBN humanized mice developed normally and had no reproductive problems. The fluorescence intensity of the EGFP-degglutag fusion was measured in cells harvested from living animals. We also found that protein knockdown of the neoligand occurred even in wild-type mouse cells. Experiments using several cultured cell lines suggested that the activity was higher in undifferentiated cell lines. We also applied this protein knockdown system to endogenous PD-1 in vivo in mice. We succeeded in suppressing the expression of PD-1 in T cells and the growth of cancer cell lines transplanted into mice.

Free Research Field

実験動物学

Academic Significance and Societal Importance of the Research Achievements

これまでの神経研究のための実験動物は遺伝子ノックアウトによるものが多く，タンパク質の欠損は不可逆的であった。また，一過性に遺伝子発現を制御するTetシステムは，Doxなどの薬剤が脳血液関門を通過しないため脳の一部領域に直接投与する以外には使用できなかった。そこで，薬剤を腹腔内投与することによって，脳神経系組織全体において標的タンパク質を簡便にかつ迅速にノックダウンできるシステムを開発すれば，脳神経研究に役立てられる。さらに，この方法を拡張して，組織・細胞特異的にデグロンシステムを機能させることができれば，脳神経研究だけでなく，様々な分野の研究に応用が可能になると考えられる。