The AID technology for ES cells and mice

• Project/Area Number: 16K15095
• Research Category: Grant-in-Aid for Challenging Exploratory Research
• Allocation Type: Multi-year Fund
• Research Field: Applied molecular and cellular biology
• Research Institution: National Institute of Genetics
• Principal Investigator: Kanemaki Masato 国立遺伝学研究所, 分子遺伝研究系, 教授 (20444507)
• Research Collaborator: HIRATANI Ichiro 理化学研究所, 多細胞システム形成研究センター, グループリーダー (40583753)
• Project Period (FY): 2016-04-01 – 2018-03-31
• Project Status: Completed (Fiscal Year 2017)
• Budget Amount: ¥3,640,000 (Direct Cost: ¥2,800,000、Indirect Cost: ¥840,000); Fiscal Year 2017: ¥1,820,000 (Direct Cost: ¥1,400,000、Indirect Cost: ¥420,000); Fiscal Year 2016: ¥1,820,000 (Direct Cost: ¥1,400,000、Indirect Cost: ¥420,000)
• Keywords: 発現制御 / デグロン / 植物ホルモン / 細胞工学 / ES細胞 / 発現抑制 / オーキシン / バイオテクノロジー

Outline of Final Research Achievements

In order to use auxin-inducible degron in mouse ES cells, it is required to introduce the ubiquitin ligase subunit, OsTIR1. We constructed a vector for introducing the OsTIR1 gene at the ROSA26 locus. We are currently making OsTIR1 expressing cells in E14TG2a background. In other ES lines, we successfully tagged genes with the degron to make conditional mutants.
To use in mice, we synthesized auxin analogs that might have low toxicity. We identified couples of analogs that showed a similar degradation-inducing activity to the natural auxin, IAA.

Research Products

• [Int'l Joint Research] NIH, National Cancer Institute/Baylar College of Medicine(米国)
• [Int'l Joint Research] University of Birmingham/University of Aberdeen/University of Edinburgh(英国)
• [Int'l Joint Research] Heidelberg University(Germany)
• [Int'l Joint Research] University of Copenhagen(Denmark)
• [Int'l Joint Research] University of Edinburgh(英国)
• [Journal Article] Endosomal Rab cycles regulate Parkin-mediated mitophagy. (2018)
  • Author(s): Yamano K, Wang C, Sarraf SA, Münch C, Kikuchi R, Noda NN, Hizukuri Y, Kanemaki MT, Harper W, Tanaka K, Matsuda N, Youle RJ.
  • Journal Title: eLife Volume: 7
  • DOI: 10.7554/elife.31326
  • NAID: 120006457172
  • Peer Reviewed / Open Access / Int'l Joint Research
• [Journal Article] Ki-67 and condensins support the integrity of mitotic chromosomes through distinct mechanisms (2018)
  • Author(s): Takagi Masatoshi、Ono Takao、Natsume Toyoaki、Sakamoto Chiyomi、Nakao Mitsuyoshi、Saitoh Noriko、Kanemaki Masato T.、Hirano Tatsuya、Imamoto Naoko
  • Journal Title: Journal of Cell Science Volume: 131 Pages: 212092-212092
  • DOI: 10.1242/jcs.212092
  • Peer Reviewed / Open Access
• [Journal Article] Xrn2 accelerates termination by RNA polymerase II, which is underpinned by CPSF73 activity (2018)
  • Author(s): Eaton Joshua D.、Davidson Lee、Bauer David L.V.、Natsume Toyoaki、Kanemaki Masato T.、West Steven
  • Journal Title: Genes & Development Volume: 32 Issue: 2 Pages: 127-139
  • DOI: 10.1101/gad.308528.117
  • Peer Reviewed / Int'l Joint Research
• [Journal Article] A pathway for mitotic chromosome formation (2018)
  • Author(s): Gibcus Johan H.、Samejima Kumiko、Goloborodko Anton、Samejima Itaru、Naumova Natalia、Nuebler Johannes、Kanemaki Masato T.、Xie Linfeng、Paulson James R.、Earnshaw William C.、Mirny Leonid A.、Dekker Job
  • Journal Title: Science Volume: 359 Issue: 6376
  • DOI: 10.1126/science.aao6135
  • Peer Reviewed / Int'l Joint Research
• [Journal Article] Replication stress induces accumulation of FANCD2 at central region of large fragile genes. (2018)
  • Author(s): Okamoto Y, Iwasaki WM, Kugou K, Takahashi KK, Oda A, Sato K, Kobayashi W, Kawai H, Sakasai R, Takaori-Kondo A, Yamamoto T, Kanemaki MT, Taoka M, Isobe T, Kurumizaka H, Innan H, Ohta K, Ishiai M, Takata M.
  • Journal Title: Nucleic Acids Res. Volume: 46(6) Issue: 6 Pages: 2932-2944
  • DOI: 10.1093/nar/gky058
  • NAID: 120006488604
  • Peer Reviewed / Open Access
• [Journal Article] Functional analysis after rapid degradation of condensins and 3D-EM reveals chromatin volume is uncoupled from chromosome architecture in mitosis (2018)
  • Author(s): Samejima Kumiko、Booth Daniel G.、Ogawa Hiromi、Paulson James R.、Xie Linfeng、Watson Cara A.、Platani Melpomeni、Kanemaki Masato T.、Earnshaw William C.
  • Journal Title: Journal of Cell Science Volume: 131 Issue: 4
  • DOI: 10.1242/jcs.210187
  • Peer Reviewed / Open Access / Int'l Joint Research
• [Journal Article] TAGing for destruction (2018)
  • Author(s): Yesbolatova Aisha、Kanemaki Masato T.
  • Journal Title: Nature Chemical Biology Volume: 14 Issue: 5 Pages: 414-415
  • DOI: 10.1038/s41589-018-0024-5
  • Open Access
• [Journal Article] Exosomes maintain cellular homeostasis by excreting harmful DNA from cells (2017)
  • Author(s): Takahashi, A., Okada, R., Nagao, K., Kawamata, Y., Hanyu, A., Yoshimoto, S., Takasugi, M., Watanabe, S., Kanemaki, M.T., Obuse, C. & Hara, E.
  • Journal Title: Nature Communications Volume: - Issue: 1 Pages: 15287-15287
  • DOI: 10.1038/ncomms15287
  • Peer Reviewed / Open Access / Acknowledgement Compliant
• [Journal Article] Dynamic organization of chromatin domains revealed by super-resolution live-cell imaging. (2017)
  • Author(s): Nozaki, T., Imai, R., Tanbo, M., Nagashima, R., Tamura, S., Tani, T., Joti, Y., Tomita, M., Hibino, K., Wendt, K.S., Okada, Y., Nagai, T., Maeshima, K.
  • Journal Title: Molecular Cell. Volume: 67 Issue: 2 Pages: 282-293
  • DOI: 10.1016/j.molcel.2017.06.018
  • Peer Reviewed / Open Access / Int'l Joint Research
• [Journal Article] Conditional Degrons for Controlling Protein Expression at the Protein Level (2017)
  • Author(s): Natsume Toyoaki、Kanemaki Masato T.
  • Journal Title: Annual Review of Genetics Volume: 51 Issue: 1 Pages: 83-102
  • DOI: 10.1146/annurev-genet-120116-024656
  • Peer Reviewed
• [Journal Article] Knockout-Rescue Embryonic Stem Cell-Derived Mouse Reveals Circadian-Period Control by Quality and Quantity of CRY1. (2017)
  • Author(s): Ode KL, Ukai H, Susaki EA, Narumi R, Matsumoto K, Hara J, Koide N, Abe T, Kanemaki MT, Kiyonari H, Ueda HR.
  • Journal Title: Mol. Cell Volume: 65 Issue: 1 Pages: 176-190
  • DOI: 10.1016/j.molcel.2016.11.022
  • Peer Reviewed / Acknowledgement Compliant
• [Journal Article] Acute Inactivation of the Replicative Helicase in Human Cells Triggers MCM8-9-dependent DNA Synthesis (2017)
  • Author(s): Natsume T, Nishimura K, Minocherhomji S, Bhwmick R, Hickson ID, Kanemaki MT
  • Journal Title: Genes & Development Volume: advanced online Issue: 8 Pages: 816-829
  • DOI: 10.1101/gad.297663.117
  • Peer Reviewed / Int'l Joint Research / Acknowledgement Compliant
• [Journal Article] Rapid Protein Depletion in Human Cells by Auxin-Inducible Degron Tagging with Short Homology Donors (2016)
  • Author(s): Natsume T, Kiyomitsu T, Saga Y, Kanemaki MT
  • Journal Title: Cell Reports Volume: 15 Issue: 1 Pages: 210-218
  • DOI: 10.1016/j.celrep.2016.03.001
  • Peer Reviewed / Open Access / Acknowledgement Compliant
• [Journal Article] Conditional Budding Yeast Mutants with Temperature-Sensitive and Auxin-Inducible Degrons for Screening of Suppressor Genes (2016)
  • Author(s): Devrekanli A, Kanemaki MT
  • Journal Title: Methods in Molecular Biology Volume: 1369 Pages: 257-278
  • DOI: 10.1007/978-1-4939-3145-3_18
  • ISBN: 9781493931446, 9781493931453
  • Peer Reviewed / Int'l Joint Research
• [Journal Article] Chromatin folding and DNA replication inhibition mediated by a highly antitumor-active tetrazolato-bridged dinuclear platinum(II) complex. (2016)
  • Author(s): Imai R, Komeda S, Shimura M, Tamura S, Matsuyama S, Nishimura K, Rogge R, Matsunaga A, Hiratani I, Takata H, Uemura M, Iida Y, Yoshikawa Y, Hansen JC, Yamauchi K, Kanemaki MT, Maeshima K.
  • Journal Title: Scientific Reports Volume: 6 Issue: 1 Pages: 24712-24725
  • DOI: 10.1038/srep24712
  • Peer Reviewed / Open Access / Int'l Joint Research
• [Journal Article] Prichromosomal protein Ki67 supports mitotic chromosome architecture. (2016)
  • Author(s): Takagi M, Natsume T, Kanemaki MT, Imamoto N.
  • Journal Title: Genes to Cells Volume: 21(10) Issue: 10 Pages: 1113-1124
  • DOI: 10.1111/gtc.12420
  • Peer Reviewed / Open Access / Acknowledgement Compliant
• [Presentation] オーキシンデグロン法によるヒト細胞内タンパク質の特異的除去と機能解析 (2017)
  • Author(s): 鐘巻将人
  • Organizer: 日本呼吸器学会
  • Place of Presentation: 東京
  • Year and Date: 2017-04-22
  • Invited
• [Presentation] Acute inactivation of the replicative helicases induces MCM8-9-dependent DNA synthesis (2017)
  • Author(s): Masato Kanemaki
  • Organizer: US-Japan DNA repair meeting
  • Int'l Joint Research / Invited
• [Presentation] Acute inactivation of the replicative helicases induces MCM8-9-dependent DNA synthesis (2017)
  • Author(s): 鐘巻将人
  • Organizer: 日本細胞生物学会年会
• [Presentation] Rapid protein depletion in human cells by the auxin-inducible degron technology (2017)
  • Author(s): Masato Kanemaki
  • Organizer: 日本ゲノム編集学会サテライトミーティング
  • Int'l Joint Research / Invited
• [Presentation] MCM-BP is an MCM chaperon protecting the MCM sub-complexes from degradation (2017)
  • Author(s): Masato Kanemaki
  • Organizer: CSH meeting on DNA replication
• [Presentation] Use of the auxin-inducible degron to study a backup mechanism against failure of DNA replication (2017)
  • Author(s): 鐘巻将人
  • Organizer: ConBio2017 Symposium
• [Presentation] Artificial destruction of active DNA replication forks reveals the HR-dependent fork recovery involving MCM8-9 (2016)
  • Author(s): 鐘巻将人
  • Organizer: The 10th 3R Symposium
  • Place of Presentation: 松江
  • Year and Date: 2016-11-17
  • Int'l Joint Research
• [Presentation] MCM8-9は切断誘導DNA複製（BIR）に関与する (2016)
  • Author(s): 鐘巻将人
  • Organizer: 第75回日本癌学会学術総会
  • Place of Presentation: 横浜
  • Year and Date: 2016-10-08
• [Presentation] MCM8-9複合体は切断誘導DNA複製（BIR）に関与する (2016)
  • Author(s): 鐘巻将人
  • Organizer: 日本生化学会年会
  • Place of Presentation: 仙台
  • Year and Date: 2016-09-26
• [Presentation] 短鎖ホモロジードナーを利用したオーキシンデグロンタグ付加による迅速なヒト細胞中のタンパク質除去 (2016)
  • Author(s): 鐘巻将人
  • Organizer: 第一回日本ゲノム編集学会
  • Place of Presentation: 広島
  • Year and Date: 2016-09-07
• [Presentation] ヒト細胞における染色体機能を理解するためのオーキシンデグロン（AID）法 (2016)
  • Author(s): 鐘巻将人
  • Organizer: 日本細胞生物学会
  • Place of Presentation: 京都
  • Year and Date: 2016-06-15
• [Book] 実験医学 2017年7月号 News & Hot Paper Digest (2017)
  • Author(s): 鐘巻将人
  • Total Pages: 3
  • Publisher: 羊土社
  • ISBN: 9784758101653
• [Book] 実験医学 2017年11月号 News & Hot Paper Digest (2017)
  • Author(s): 鐘巻将人
  • Total Pages: 2
  • Publisher: 羊土社
  • ISBN: 9784758125017
• [Remarks] タンパク質レベルで発現調節を可能とするデグロン技術
  • URL: https://www.nig.ac.jp/nig/ja/2017/11/research-highlights_ja/20171122.html
• [Patent(Industrial Property Rights)] オーキシンデグロンシステムにおけるタンパク質分解阻害剤及びその使用 (2017)
  • Inventor(s): 鐘巻将人、夏目豊彰、林謙一郎
  • Industrial Property Rights Holder: 大学共同利用機関法人情報・システム研究機構、学校法人加計学園
  • Industrial Property Rights Type: 特許
  • Industrial Property Number: 2017-228310
  • Filing Date: 2017