Development of a versatile vector system for single-cell labeling and gene function studies in vivo

• Project/Area Number: 16K14559
• Research Category: Grant-in-Aid for Challenging Exploratory Research
• Allocation Type: Multi-year Fund
• Research Field: Neurophysiology / General neuroscience
• Research Institution: National Institute of Genetics
• Principal Investigator: IWASATO Takuji 国立遺伝学研究所, 遺伝形質研究系, 教授 (00311332)
• Project Period (FY): 2016-04-01 – 2019-03-31
• Project Status: Completed (Fiscal Year 2018)
• 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: 神経科学 / 神経回路 / 単一細胞解析 / 細胞標識 / 遺伝子ノックアウト / 発達 / マウス / 遺伝学 / 脳 / 新規手法 / 遺伝子 / ノックアウト / 蛍光標識 / in vivo / 哺乳類

Outline of Final Research Achievements

We developed “Supernova” that is a versatile vector system for single-cell labeling and gene function studies in vivo. Supernova system enables single-cell labeling and labeled cell-specific gene manipulation, when introduced by in utero electroporation (IUE) or adeno-associated virus (AAV).

Academic Significance and Societal Importance of the Research Achievements

遺伝子を操作するためにTALEN, CRISPR/Cas9などの最新のゲノム編集技術を取り入れることで、高性能で汎用性のあるSupernovaシリーズを構築した。その結果、遺伝子組換え動物ではない多くのモデル生物においても単一細胞遺伝子ノックアウトできるようになった。また、ベクター導入に、これまでの子宮内電気穿孔法に加えてアデノ随伴ウイルス（AAV）が利用できるようになり、幅広い組織への適用が可能になった。

Research Products

• [Journal Article] In Vivo Two-photon Imaging of Cortical Neurons in Neonatal Mice (2018)
  • Author(s): Mizuno H*, Nakazawa S, Iwasato T (* correspondence)
  • Journal Title: Journal of Visualized Experiments Volume: 140 Issue: 140
  • DOI: 10.3791/58340
  • Peer Reviewed / Open Access
• [Journal Article] Differential dynamics of cortical neuron dendritic trees revealed by long-term in vivo imaging in neonates (2018)
  • Author(s): Nakazawa Shingo、Mizuno Hidenobu、Iwasato Takuji
  • Journal Title: Nature Communications Volume: 9 Issue: 1 Pages: 3106-3106
  • DOI: 10.1038/s41467-018-05563-0
  • Peer Reviewed / Open Access
• [Journal Article] Development of tactile sensory circuits in the CNS (2018)
  • Author(s): Iwasato Takuji、Erzurumlu Reha S.
  • Journal Title: Current Opinion in Neurobiology Volume: 53 Pages: 66-75
  • DOI: 10.1016/j.conb.2018.06.001
  • Peer Reviewed / Int'l Joint Research
• [Journal Article] Patchwork-type spontaneous activity in neonatal barrel cortex layer 4 transmitted via thalamocortical projections. (2018)
  • Author(s): Mizuno, H., Ikezoe, K., Nakazawa, S., Sato, T., Kitamura, K., Iwasato, T.
  • Journal Title: Cell Rep. Volume: 22 Issue: 1 Pages: 123-135
  • DOI: 10.1016/j.celrep.2017.12.012
  • Peer Reviewed / Open Access
• [Journal Article] Protocadherin-αC2 is required for diffuse projections of serotonergic axons (2017)
  • Author(s): Katori Shota、Noguchi-Katori Yukiko、Okayama Atsushi、Kawamura Yoshimi、Luo Wenshu、Sakimura Kenji、Hirabayashi Takahiro、Iwasato Takuji、Yagi Takeshi
  • Journal Title: Scientific Reports Volume: 7 Issue: 1 Pages: 15908-15908
  • DOI: 10.1038/s41598-017-16120-y
  • Peer Reviewed / Open Access
• [Journal Article] Spinal RacGAP α-Chimaerin Is Required to Establish the Midline Barrier for Proper Corticospinal Axon Guidance (2017)
  • Author(s): Katori Shota、Noguchi-Katori Yukiko、Itohara Shigeyoshi、Iwasato Takuji
  • Journal Title: The Journal of Neuroscience Volume: 37 Issue: 32 Pages: 7682-7699
  • DOI: 10.1523/jneurosci.3123-16.2017
  • Peer Reviewed
• [Journal Article] Prenatal thalamic waves regulate cortical area size prior to sensory processing. (2017)
  • Author(s): Moreno-Juan, V., Filipchuk, A., Antoｎ-Bolarios, N., Mezzera, C., Gezelius, H., Andes, B., Rodriguez-Malmierca, L., Susin, R., Schaad, O., Iwasato, T., Schuele, R., Rutlin, M., Nelson, S., Ducret, S., Valdeolmillos, M., Rijli, F., Lopez-Bendito, G.
  • Journal Title: Nature Commun. Volume: 8 Issue: 1 Pages: 14172-14172
  • DOI: 10.1038/ncomms14172
  • Peer Reviewed / Open Access / Int'l Joint Research
• [Journal Article] Supernova: A Versatile Vector System for Single-Cell Labeling and Gene Function Studies in vivo. (2016)
  • Author(s): Luo, W., Mizuno, H., Iwata, R., Nakazawa, S., Yasuda, K., Itohara, S., Iwasato, T.
  • Journal Title: Sci Rep. Volume: 6 Issue: 1 Pages: 35747-35747
  • DOI: 10.1038/srep35747
  • Peer Reviewed / Open Access
• [Journal Article] CAPS1 stabilizes the state of readily releasable synaptic vesicles to fusion competence at CA3-CA1 synapses in adult hippocampus (2016)
  • Author(s): 6.Shinoda Y, Ishii C, Fukazawa Y, Sadakata T, Ishii Y, Sano Y, Iwasato T, Itohara S, Furuichi T
  • Journal Title: Scientific Reports Volume: 6 Issue: 1 Pages: 31540-31540
  • DOI: 10.1038/srep31540
  • Peer Reviewed / Open Access / Int'l Joint Research
• [Journal Article] In vitro Assay for Dendritic Spine Retraction of Hippocampal Neurons with Sparse Labeling (2016)
  • Author(s): Iwata, R., Iwasato, T.
  • Journal Title: Bio-Protocol Volume: 6
  • Peer Reviewed / Open Access / Acknowledgement Compliant
• [Presentation] 赤ん坊の脳の中での神経回路の発達 ―マウス遺伝学と二光子イメージングを用いた研究 (2019)
  • Author(s): 岩里琢治
  • Organizer: 第18回BIRD脳科学セミナー
  • Invited
• [Presentation] In vivo imaging of barrel cortex layer 4 during thalamocortical reorganization (2018)
  • Author(s): Takuji Iwasato
  • Organizer: 遺伝研研究会2018 “Circuit construction in the mammalian brain”
  • Invited
• [Presentation] In vivo imaging of barrel cortex circuit refinement in neonates (2018)
  • Author(s): Takuji Iwasato
  • Organizer: Spontaneous Activity in Brain Development (SPONT 2018)
  • Int'l Joint Research / Invited
• [Presentation] In vivo functional and morphological imaging of neural circuit refinement in the neonatal barrel cortex (2018)
  • Author(s): 岩里琢治
  • Organizer: CSHA(Cold Spring Harbor Asia) on Neuroscience 2018 「Latest Advances in Development and Function of Neuronal Circuits」サテライトシンポジウム
  • Int'l Joint Research / Invited
• [Presentation] 新生仔マウスのバレル皮質神経回路発達のin vivoイメージング (In vivo imaging of barrel cortex circuit refinement in neonates) (2018)
  • Author(s): 岩里琢治
  • Organizer: 第41回日本神経科学大会
  • Invited
• [Presentation] マウスで迫る「赤ん坊の脳の発達のしくみ」 (2018)
  • Author(s): 岩里琢治
  • Organizer: JT生命誌研究館・国立遺伝学研究所共同企画シンポジウム「遺伝学の最先端研究から見る生命誌」
  • Invited
• [Presentation] マウス遺伝学を用いた体性感覚系神経回路発達の解析 (2017)
  • Author(s): 岩里琢治
  • Organizer: 新潟大学脳研究所共同研究合同セミナー
  • Invited
• [Presentation] Unveiling the mechanisms of neuronal circuit refinement in the neonatal mouse barrel Cortex (2017)
  • Author(s): 岩里琢治
  • Organizer: Academia Sinica Symposium
  • Int'l Joint Research / Invited
• [Presentation] マウス大脳皮質の生後発達期神経回路リモデリング (2017)
  • Author(s): 岩里琢治
  • Organizer: 第12回「認識と形成」研究会
  • Invited
• [Presentation] 生後発達期の大脳皮質リモデリング (2017)
  • Author(s): 岩里琢治
  • Organizer: 生理学研究所研究会「大脳皮質回路の機能原理を探る」
  • Invited
• [Presentation] マウス大脳皮質の生後発達期神経回路リモデリング (2017)
  • Author(s): 岩里琢治
  • Organizer: 第5回「幸福脳」研究会（名古屋大学・慶応大学合同ミーティング）
  • Invited
• [Presentation] 哺乳類中枢神経系における神経回路形成の遺伝学的解析 (2017)
  • Author(s): 岩里琢治
  • Organizer: 新潟大学脳研究所共同研究合同セミナー
  • Invited
• [Presentation] In vivo imaging of neonatal mouse barrel cortex (2016)
  • Author(s): T. Iwasato
  • Organizer: Dart Neuroscience Seminar Series at TSRI
  • Place of Presentation: SD, ＵＳＡ
  • Year and Date: 2016-11-18
  • Int'l Joint Research / Invited
• [Presentation] Supernova: Extensible vector systems that enable high intensity single-cell labeling and labeled cell-specific gene knockout and editing In vivo (2016)
  • Author(s): S. Nakazawa., W. Luo., R. Iwata., H. Mizuno., T. Iwasato.
  • Organizer: Society for Neuroscience 2016
  • Place of Presentation: SD, USA
  • Year and Date: 2016-11-12
  • Int'l Joint Research
• [Presentation] 独自の二光子イメージング技術を用いた、新生仔マウス大脳皮質における神経回路発達の解析 (2016)
  • Author(s): 岩里琢治
  • Organizer: 第159回日本獣医学会学術集会
  • Place of Presentation: 藤沢
  • Year and Date: 2016-09-07
  • Invited
• [Presentation] In vivo imaging of neonatal mouse barrel cortex (2016)
  • Author(s): T. Iwasato.
  • Organizer: Circuit Construction in the Mammalian Brain
  • Place of Presentation: 大阪
  • Year and Date: 2016-08-08
  • Int'l Joint Research / Invited
• [Presentation] 単一細胞蛍光標識と標識細胞特異的遺伝子操作を可能とするベクターシステム (2016)
  • Author(s): 岩里琢治
  • Organizer: 第13回アカデミックフォーラム
  • Place of Presentation: 東京
  • Year and Date: 2016-05-13
  • Invited
• [Patent(Industrial Property Rights)] ベクターシステム、遺伝子発現方法、標的遺伝子ノックアウト方法、標的遺伝子ノックダウン方法、標的遺伝子編集方法、及び遺伝子発現キット (2016)
  • Inventor(s): 岩里琢治、水野秀信、羅ブンジュウ、岩田亮平
  • Industrial Property Rights Holder: 情報・システム研究機構
  • Industrial Property Rights Type: 特許
  • Industrial Property Number: 2016-094814
  • Filing Date: 2016-05-10