In vivo imaging of a secreted glycoprotein, Wnt5a

• Project/Area Number: 17K08633
• Research Category: Grant-in-Aid for Scientific Research (C)
• Allocation Type: Multi-year Fund
• Section: 一般
• Research Field: General medical chemistry
• Research Institution: Osaka University
• Principal Investigator: Harada Takeshi 大阪大学, 医学系研究科, 助教 (30362768)
• Project Period (FY): 2017-04-01 – 2021-03-31
• Project Status: Completed (Fiscal Year 2020)
• Budget Amount: ¥4,810,000 (Direct Cost: ¥3,700,000、Indirect Cost: ¥1,110,000); Fiscal Year 2019: ¥1,430,000 (Direct Cost: ¥1,100,000、Indirect Cost: ¥330,000); Fiscal Year 2018: ¥1,430,000 (Direct Cost: ¥1,100,000、Indirect Cost: ¥330,000); Fiscal Year 2017: ¥1,950,000 (Direct Cost: ¥1,500,000、Indirect Cost: ¥450,000)
• Keywords: Wnt5a / ライブイメージング / 線維芽細胞 / シングルセル解析 / CRISPR / 生体分子

Outline of Final Research Achievements

To elucidate the kinetics of Wnt5a protein in vivo, we generated Venus-Wnt5a knock-in mouse (Venus-Wnt5a KI) using CRISPR/Cas system. We confirmed that Venus-Wnt5a was expressed in lung, heart, stomach and kidney same as the endogenous Wnt5a protein. Because the homozygote of Venus-Wnt5a KI was embryonic lethal, we revealed that Venus-Wnt5a did not work in vivo.

Academic Significance and Societal Importance of the Research Achievements

本研究ではCRISPR/Cas システムを用いることにより従来のES細胞を用いる方法と比べて大幅に短期間でVenus-Wnt5a KIを作製することが出来た。Venus-Wnt5aはin vitroではWnt5aとしてシグナルを伝えられたが、生体内において機能しなかった。実際にノックインマウスを作製することにより初めてVenusのような分子量の大きい蛍光タンパク質はWntタンパク質のタグとしては不適切であることが明らかとなった。この事実は今後の
分泌タンパク質の動態研究を行う上で非常に重要な知見である。

Research Products

• [Journal Article] Palmitoylated CKAP4 regulates mitochondrial functions through an interaction with VDAC2 at ER?mitochondria contact sites (2020)
  • Author(s): Harada Takeshi、Sada Ryota、Osugi Yoshito、Matsumoto Shinji、Matsuda Tomoki、Hayashi-Nishino Mitsuko、Nagai Takeharu、Harada Akihiro、Kikuchi Akira
  • Journal Title: Journal of Cell Science Volume: 133 Issue: 21
  • DOI: 10.1242/jcs.249045
  • Peer Reviewed / Open Access
• [Journal Article] ARL4C is associated with initiation and progression of lung adenocarcinoma and represents a therapeutic target (2020)
  • Author(s): Kimura Kenji、Matsumoto Shinji、Harada Takeshi、Morii Eiichi、Nagatomo Izumi、Shintani Yasushi、Kikuchi Akira
  • Journal Title: Cancer Science Volume: 111 Issue: 3 Pages: 951-961
  • DOI: 10.1111/cas.14303
  • Peer Reviewed / Open Access
• [Journal Article] Efficient generation of Knock-in/Knock-out marmoset embryo via CRISPR/Cas9 gene editing (2019)
  • Author(s): Kumita Wakako、Sato Kenya、Suzuki Yasuhiro、Kurotaki Yoko、Harada Takeshi、Zhou Yang、Kishi Noriyuki、Sato Kengo、Aiba Atsu、Sakakibara Yasubumi、Feng Guoping、Okano Hideyuki、Sasaki Erika
  • Journal Title: Scientific Reports Volume: 9 Issue: 1 Pages: 12719-12719
  • DOI: 10.1038/s41598-019-49110-3
  • Peer Reviewed / Open Access / Int'l Joint Research
• [Journal Article] Chemically Modified Antisense Oligonucleotide Against ARL4C Inhibits Primary and Metastatic Liver Tumor Growth (2019)
  • Author(s): Harada Takeshi、Matsumoto Shinji、Hirota Suguru、Kimura Hirokazu、Fujii Shinsuke、Kasahara Yuuya、Gon Hidetoshi、Yoshida Toshihiko、Itoh Tomoo、Haraguchi Naotsugu、Mizushima Tsunekazu、Noda Takehiro、Eguchi Hidetoshi、Nojima Satoshi、Morii Eiichi、Fukumoto Takumi、Obika Satoshi、Kikuchi Akira
  • Journal Title: Molecular Cancer Therapeutics Volume: 18 Issue: 3 Pages: 602-612
  • DOI: 10.1158/1535-7163.mct-18-0824
  • Peer Reviewed
• [Journal Article] CKAP4, a DKK1 Receptor, Is a Biomarker in Exosomes Derived from Pancreatic Cancer and a Molecular Target for Therapy (2019)
  • Author(s): Kimura Hirokazu、Yamamoto Hideki、Harada Takeshi、Fumoto Katsumi、Osugi Yoshihito、Sada Ryota、Maehara Natsumi、Hikita Hayato、Mori Soichiro、Eguchi Hidetoshi、Ikawa Masahito、Takehara Tetsuo、Kikuchi Akira
  • Journal Title: Clinical Cancer Research Volume: 25 Issue: 6 Pages: 1936-1947
  • DOI: 10.1158/1078-0432.ccr-18-2124
  • Peer Reviewed
• [Presentation] パルミトイル化CKAP4はVDAC2を介してミトコンドリア機能を制御する (2020)
  • Author(s): 原田武志、佐田僚太、大杉祥仁、松本真司、菊池章
  • Organizer: 第93回 日本生化学学会大会
• [Presentation] CKAP4-VDAC2相互作用によるミトコンドリア機能の制御 (2019)
  • Author(s): 原田武志、松本真司、大杉祥仁、菊池章
  • Organizer: 第42回日本分子生物学会年会
• [Presentation] 小胞体―ミトコンドリア連携ゾーンにおけるII型膜タンパク質CKAP4の機能解析 (2018)
  • Author(s): 原田武志、大杉祥仁、松本真司、菊池章
  • Organizer: 第91回日本生化学大会
• [Presentation] Wnt5b含有エソソームは癌細胞の増殖及び運動能を亢進させる (2017)
  • Author(s): 原田武志
  • Organizer: 第40回日本分子生物学会年会