The University of Tokyo

15 articles published in JoVE

• Medicine

  
  Modified Heterotopic Abdominal Heart Transplantation and a Novel Aortic Regurgitation Model in Rats Shigeto Tsuji¹, Shogo Shimada¹, Minoru Ono^{1 1}Department of Cardiac Surgery, The University of Tokyo This study demonstrates a reproducible heterotopic abdominal heart transplantation technique in rats that beginners can learn and perform. Additionally, a novel aortic regurgitation model in rats is generated by performing heterotopic abdominal heart transplantation and damaging the donor's aortic valve using a guidewire after harvesting.

• Biology

  
  Semi-Automated Isolation of the Stromal Vascular Fraction from Murine White Adipose Tissue Using a Tissue Dissociator Kaede Saito^1,2, Yuta Hiraike^1,2,3, Misato Oguchi², Toshimasa Yamauchi^{2 1}Division for Health Service Promotion, The University of Tokyo, ²Department of Diabetes and Metabolic Diseases, Graduate School of Medicine, The University of Tokyo, ³The University of Tokyo Excellent Young Researcher Program, The University of Tokyo This protocol describes the semi-automated isolation of the stromal vascular fraction (SVF) from murine adipose tissue to obtain preadipocytes and achieve adipocyte differentiation in vitro. Using a tissue dissociator for collagenase digestion reduces experimental variation and increases reproducibility.

• Neuroscience

  
  Easy and Reproducible Low-Density Primary Culture using Frozen Stock of Embryonic Hippocampal Neurons Noriko Koganezawa¹, Reiko T. Roppongi², Yuko Sekino^3,4, Izuo Tsutsui³, Ayaka Higa⁵, Tomoaki Shirao^{1,5 1}Department of Pharmacology, Graduate School of Medicine, Gunma University, ²Gunma University Initiative for Advanced Research, Gunma University, ³Department of Veterinary Pathophysiology and Animal Health, Graduate school of Agricultural and Life Sciences, The University of Tokyo, ⁴Institute for Drug Discovery Innovation, ⁵AlzMed, Inc. A ready-to-use frozen stock of neurons is a powerful tool for evaluating synaptic functions. Here, we introduce an easy low-density primary culture from frozen stock using a 96-well plate.

• Developmental Biology

  
  CRISPR/Cas9-Mediated Highly Efficient Gene Targeting in Embryonic Stem Cells for Developing Gene-Manipulated Mouse Models Manabu Ozawa*¹, Chihiro Emori*², Masahito Ikawa^{1,2 1}Laboratory of Reproductive Systems Biology, Center for Experimental Medicine and Systems Biology, The Institute of Medical Science, The University of Tokyo, ²Research Institute for Microbial Diseases, Osaka University Here we present a protocol for developing genetically modified mouse models using embryonic stem cells, especially for large DNA knock-in (KI). This protocol is tuned up using CRISPR/Cas9 genome editing, resulting in significantly improved KI efficiency compared with the conventional homologous recombination-mediated linearized DNA targeting method.

• Neuroscience

  
  Simultaneous Imaging of Microglial Dynamics and Neuronal Activity in Awake Mice Hisato Maruoka¹, Ryosuke Kamei¹, Shunsuke Mizutani¹, Qingrui Liu¹, Shigeo Okabe^{1 1}Department of Cellular Neurobiology, Graduate School of Medicine and Faculty of Medicine, The University of Tokyo Here, we describe a protocol combining adeno-associated virus injection with cranial window implantation for simultaneous imaging of microglial dynamics and neuronal activity in awake mice.

• Neuroscience

  
  In Vivo Wide-Field and Two-Photon Calcium Imaging from a Mouse Using a Large Cranial Window Satoshi Manita¹, Eiji Shigetomi^2,3, Haruhiko Bito⁴, Schuichi Koizumi^2,3, Kazuo Kitamura^{1 1}Department of Neurophysiology, Faculty of Medicine, University of Yamanashi, ²Department of Neuropharmacology, Faculty of Medicine, University of Yamanashi, ³Yamanashi GLIA center, Interdisciplinary Graduate School of Medicine, University of Yamanashi, ⁴Department of Neurochemistry, Graduate School of Medicine, The University of Tokyo The present protocol describes making a large (6 x 3 mm²) cranial window using food wrap, transparent silicone, and cover glass. This cranial window allows in vivo wide-field and two-photon calcium imaging experiments in the same mouse.

• Developmental Biology

  
  Fluorescent In Situ Hybridization and 5-Ethynyl-2'-Deoxyuridine Labeling for Stem-Like Cells in the Hydrozoan Jellyfish Cladonema pacificum Sosuke Fujita¹, Erina Kuranaga¹, Masayuki Miura², Yu-ichiro Nakajima^{2 1}Graduate School of Life Sciences, Tohoku University, ²Graduate School of Pharmaceutical Sciences, The University of Tokyo Here, we describe a protocol for visualizing stem-like proliferating cells in the jellyfish Cladonema. Whole-mount fluorescent in situ hybridization with a stem cell marker allows for the detection of stem-like cells, and 5-ethynyl-2'-deoxyuridine labeling enables the identification of proliferating cells. Together, actively proliferating stem-like cells can be detected. 

• Medicine

  
  Whole-Kidney Three-Dimensional Staining with CUBIC Sho Hasegawa¹, Masaomi Nangaku^{1 1}Division of Nephrology and Endocrinology, The University of Tokyo The present protocol describes a tissue clearing method and whole-mount immunofluorescent staining for three-dimensional (3D) kidney imaging. This technique can offer macroscopic perspectives in kidney pathology, leading to new biological discoveries.

• Neuroscience

  
  In Vivo Chronic Two-Photon Imaging of Microglia in the Mouse Hippocampus Ryosuke Kamei¹, Shinji Urata¹, Hisato Maruoka¹, Shigeo Okabe^{1 1}Department of Cellular Neurobiology, Graduate School of Medicine, The University of Tokyo This paper describes a method for chronic in vivo observation of the resting microglia in the mouse hippocampal CA1 using precisely controlled surgery and two-photon microscopy.

• Biology

  
  Optical Clearing of Plant Tissues for Fluorescence Imaging Daisuke Kurihara¹, Yoko Mizuta^1,2, Shiori Nagahara¹, Yoshikatsu Sato^1,3, Tetsuya Higashiyama^{1,3,4 1}Institute of Transformative Bio-Molecules (ITbM), Nagoya University, ²Institute for Advanced Research (IAR), Nagoya University, ³Division of Biological Science, Graduate School of Science, Nagoya University, ⁴Department of Biological Sciences, Graduate School of Science, The University of Tokyo Here, a method is described for making plant tissues transparent while maintaining the stability of fluorescent proteins. This technique facilitates deep imaging of cleared plant tissues without physical sectioning.

• Developmental Biology

  
  Electroporation-mediated RNA Interference Method in Odonata Genta Okude^1,2, Takema Fukatsu^1,2,3, Ryo Futahashi^{2 1}Department of Biological Sciences, Graduate School of Science, The University of Tokyo, ²Bioproduction Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), ³Graduate School of Life and Environmental Sciences, University of Tsukuba We provide a detailed protocol for electroporation-mediated RNA interference in insects of the order Odonata (dragonflies and damselflies) using the blue-tailed damselfly (Ischnura senegalensis: Coenagironidae: Zygoptera) and the pied skimmer dragonfly (Pseudothemis zonata: Libellulidae: Anisoptera).

• Biology

  
  Gonadectomy and Blood Sampling Procedures in the Small Size Teleost Model Japanese Medaka (Oryzias latipes) Muhammad Rahmad Royan¹, Shinji Kanda², Daichi Kayo³, Weiyi Song⁴, Wei Ge⁴, Finn-Arne Weltzien¹, Romain Fontaine^{1 1}Physiology Unit, Faculty of Veterinary Medicine, Norwegian University of Life Sciences, ²Laboratory of Physiology, Atmosphere and Ocean Research Institute, The University of Tokyo, ³Department of Biological Sciences, Graduate School of Science, The University of Tokyo, ⁴Centre of Reproduction, Development and Aging (CRDA), Faculty of Health Sciences, University of Macau The article describes a quick protocol to gonadectomize and sample blood from the small teleost fish, using Japanese medaka (Oryzias latipes) as a model, to investigate the role of sex steroids in animal physiology.

• Neuroscience

  
  Three-Dimensional Motor Nerve Organoid Generation Tatsuya Osaki^1,2, Siu Yu A. Chow^1,2, Yui Nakanishi^1,2, Joel Hernández^1,3, Jiro Kawada⁴, Teruo Fujii¹, Yoshiho Ikeuchi^{1,2 1}Institute of Industrial Science, The University of Tokyo, ²Department of Chemistry and Biotechnology, School of Engineering, The University of Tokyo, ³Faculty of Science and Engineering, Tecnologico de Monterrey, ⁴Jiksak Bioengineering, Inc. This protocol provides a comprehensive procedure to fabricate human iPS cell-derived motor nerve organoid through spontaneous assembly of a robust bundle of axons extended from a spheroid in a tissue culture chip.

• Medicine

  
  Identifying Inhibitors of the HBx-DDB1 Interaction Using a Split Luciferase Assay System Kazuma Sekiba^1,2, Motoyuki Otsuka¹, Kazuhiko Koike^{1 1}Department of Gastroenterology, Graduate School of Medicine, The University of Tokyo, ²Research Fellow of Japan Society for the Promotion of Science Here, we present a method for screening anti-hepatitis B viral agents that inhibit the HBx-DDB1 interaction using a split luciferase assay system. This system allows easy detection of protein-protein interactions and is suitable for identifying inhibitors of such interactions.

• Medicine

  
  Technical Aspects of the Mouse Aortocaval Fistula Kota Yamamoto^1,2, Xin Li^1,3, Chang Shu³, Tetsuro Miyata², Alan Dardik^{1,4 1}The Department of Surgery and the Interdepartmental Program in Vascular Biology and Therapeutics, Yale University, ²Department of Vascular Surgery, The University of Tokyo, ³Department of Vascular Surgery, Central South University, ⁴VA Connecticut Healthcare Systems The goal is to produce an arteriovenous fistula that is simple and reproducible. This method does not use sutures or glue adhesive. Therefore the samples can be used with the least amount of foreign materials for analysis.