Nakano Risako, Ihara Naoki, Morikawa Shota, Nakashima Ai, Kanemaki Masato T, Ikegaya Yuji, Takeuchi Haruki
Laboratory of Chemical Pharmacology.
Molecular Cell Engineering Laboratory, National Institute of Genetics, Research Organization of Information and Systems (ROIS).
Neuroreport. 2019 Sep 4;30(13):908-913. doi: 10.1097/WNR.0000000000001299.
Genetic manipulation of protein levels is a promising approach to identify the function of a specific protein in living organisms. Previous studies demonstrated that the auxin-inducible degron strategy provides rapid and reversible degradation of various proteins in fungi and mammalian mitotic cells. In this study, we employed this technology to postmitotic neurons to address whether the auxin-inducible degron system could be applied to the nervous system. Using adeno-associated viruses, we simultaneously introduced enhanced green fluorescent protein (EGFP) fused with an auxin-inducible degron tag and an F-box family protein, TIR1 from Oryza sativa (OsTIR1), into hippocampal neurons from mice. In dissociated hippocampal neurons, EGFP enhanced green fluorescent protein fluorescence signals rapidly decreased when adding a plant hormone, auxin. Furthermore, auxin-induced enhanced green fluorescent protein degradation was also observed in hippocampal acute slices. Taken together, these results open the door for neuroscientists to manipulate protein expression levels by the auxin-inducible degron system in a temporally controlled manner.
对蛋白质水平进行基因操作是一种很有前景的方法，可用于确定特定蛋白质在活生物体中的功能。先前的研究表明，生长素诱导降解子策略能使真菌和哺乳动物有丝分裂细胞中的各种蛋白质快速且可逆地降解。在本研究中，我们将该技术应用于有丝分裂后的神经元，以探讨生长素诱导降解子系统是否可应用于神经系统。利用腺相关病毒，我们将与生长素诱导降解子标签融合的增强型绿色荧光蛋白（EGFP）和来自水稻的F-box家族蛋白TIR1（OsTIR1）同时导入小鼠海马神经元。在解离的海马神经元中，添加植物激素生长素后，EGFP增强型绿色荧光蛋白荧光信号迅速减弱。此外，在海马急性脑片中也观察到了生长素诱导的增强型绿色荧光蛋白降解。综上所述，这些结果为神经科学家通过生长素诱导降解子系统以时间可控的方式操纵蛋白质表达水平打开了大门。
