Development of tagged protein imaging during translation and folding steps by using genome editing

2018 Fiscal Year Final Research Report

• Project/Area Number: 16K01931
• Research Category: Grant-in-Aid for Scientific Research (C)
• Allocation Type: Multi-year Fund
• Section: 一般
• Research Field: Chemical biology
• Research Institution: Tokyo Medical and Dental University
• Principal Investigator: Nomura Wataru 東京医科歯科大学, 生体材料工学研究所, 准教授 (80463909)
• Project Period (FY): 2016-04-01 – 2019-03-31
• Keywords: ゲノム編集 / 蛍光イメージング / ペプチド / タンパク質 / タグ－プローブ

Outline of Final Research Achievements

In this project, a method to observe processes of protein translation, folding, and translocation was developed using our tag-probe system. Membrane receptor and histone protein were chosen as targets and their fusion with fluorescent protein were expressed in cells. Expression and localization of target proteins were observed by fluorescent microscopy. To introduce tag gene after starting codon, guide RNA was designed and genome editing by CRISPR-Cas9 was performed. We have established a method to select cells with drug-resistant marker to increase editing efficiency. The method can widely applied to introduce various gene to genome.

Free Research Field

生物化学

Academic Significance and Societal Importance of the Research Achievements

細胞内に存在するタンパク質はその機能自体の解明も重要であるが，細胞の中にどれくらいの量が存在し，どこに存在するのか、存在場所は機能とともに変化するか，などを顕微鏡で観察することが必要になる。それを解決する手段として、細胞のゲノムに存在するタンパク質を定義している遺伝子に目印となるタグを付けることで細胞内のタンパク質の絶対量や動きを正確に観察することができる。本研究では独自のタグをゲノムに組み入れる方法を確立することができた。