Osaka University 15 articles published in JoVE Bioengineering Amplification of Escherichia coli in a Continuous-Flow-PCR Microfluidic Chip and Its Detection with a Capillary Electrophoresis System Wenwen Dong1, Chunxian Tao1, Bo Yang1, Erika Miyake2, Zhenqing Li1, Dawei Zhang1, Yoshinori Yamaguchi1,2 1Shanghai Key Lab of Modern Optical System, University of Shanghai for Science and Technology, 2Graduate School of Engineering, Osaka University This protocol describes how to build a continuous-flow-polymerase chain system based on a microfluidic chip and how to build a capillary electrophoresis system in the lab. It presents a simple method for the analysis of nucleic acids in the lab. 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 Ikawa1,2 1Laboratory 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 A Tissue Clearing Method for Neuronal Imaging from Mesoscopic to Microscopic Scales Kenta Yamauchi1,2, Shinichiro Okamoto1,2,3, Megumu Takahashi1,2,4,5, Masato Koike2,3, Takahiro Furuta6, Hiroyuki Hioki1,2,7 1Department of Neuroanatomy, Juntendo University Graduate School of Medicine, 2Department of Cell Biology and Neuroscience, Juntendo University Graduate School of Medicine, 3Advanced Research Institute for Health Sciences, Juntendo University, 4Department of Neuroscience, Graduate School of Medicine, Kyoto University, 5Japan Society for the Promotion of Science, 6Department of Oral Anatomy and Neurobiology, Graduate School of Dentistry, Osaka University, 7Department of Multi-Scale Brain Structure Imaging, Juntendo University Graduate School of Medicine The protocol provides a detailed method of neuronal imaging in brain slice using a tissue clearing method, ScaleSF. The protocol includes brain tissue preparation, tissue clarification, handling of cleared slices and confocal laser scanning microscopy imaging of neuronal structures from mesoscopic to microscopic levels. Biology High-Accuracy Correction of 3D Chromatic Shifts in the Age of Super-Resolution Biological Imaging Using Chromagnon Atsushi Matsuda1,2, Takako Koujin1, Lothar Schermelleh3, Tokuko Haraguchi1,2, Yasushi Hiraoka1,2 1Advanced ICT Research Institute Kobe, National Institute of Information and Communications Technology, 2Graduate School of Frontier Biosciences, Osaka University, 3Micron Advanced Bioimaging Unit, Department of Biochemistry, University of Oxford Correction of chromatic shifts in three-dimensional (3D) multicolor fluorescence microscopy images is crucial for quantitative data analyses. This protocol is developed to measure and correct chromatic shifts in biological samples through acquisition of suitable reference images and processing with the open-source software, Chromagnon. Engineering Hydrogen Charging of Aluminum using Friction in Water Keitaro Horikawa1, Hidetoshi Kobayashi1 1Department of Mechanical Science and Bioengineering, Osaka University In order to introduce high amounts of hydrogen in aluminum and aluminum alloys, a new method of hydrogen charging was developed, called the friction in water procedure. Bioengineering An Orbital Shaking Culture of Mammalian Cells in O-shaped Vessels to Produce Uniform Aggregates Ikki Horiguchi1,2, Ikumi Suzuki3, Takashi Morimura3, Yasuyuki Sakai1,4 1Department of Chemical System Engineering, University of Tokyo, 2Department of Biotechnology, Osaka University, 3Biotech Business Unit, Fukoku Co. Ltd, 4Institute of Industrial Science, University of Tokyo Here we present a protocol for using O-shaped vessels, specialized for suspension cultures of cellular aggregates, with orbital shaking. The HEK293 cells grown in this bag form more homogeneous aggregates than those grown in conventional culture vessels. Engineering Generation and Control of Electrohydrodynamic Flows in Aqueous Electrolyte Solutions Kentaro Doi1, Fumika Nito1, Ayako Yano2, Ryo Nagura1, Satoyuki Kawano1 1Department of Mechanical Science and Bioengineering, Graduate School of Engineering Science, Osaka University, 2Division of Mechanical Science and Technology, Faculty of Science and Technology, GUNMA University The rectification of ion transport pathways is an effective method to generate one-directional ion-dragged electrohydrodynamic flows. By setting an ion-exchange membrane in a flow channel, an electrically polarized condition is generated and causes a liquid flow to be driven when an electric field is externally applied. Engineering Measuring Magnetically-Tuned Ferroelectric Polarization in Liquid Crystals Hiroki Ueda1, Takuya Akita2, Yoshiaki Uchida2, Tsuyoshi Kimura3 1Division of Materials Physics, Graduate School of Engineering Science, Osaka University, 2Division of Chemical Engineering, Graduate School of Engineering Science, Osaka University, 3Department of Advance Materials Science, University of Tokyo In this report, we present a protocol to examine direct magnetoelectric effects, i.e., induction of ferroelectric polarization by applying magnetic fields, in liquid crystals. This protocol provides a unique approach, supported by the softness of liquid crystals, to achieve room-temperature magnetoelectrics. Engineering Electric-field Control of Electronic States in WS2 Nanodevices by Electrolyte Gating Feng Qin1, Toshiya Ideue1, Wu Shi2, Yijin Zhang3,4, Ryuji Suzuki1, Masaro Yoshida5, Yu Saito1, Yoshihiro Iwasa1,5 1Quantum-Phase Electronics Center (QPEC) and Department of Applied Physics, The University of Tokyo, 2Materials Sciences Division, Lawrence Berkeley National Laboratory, 3Institute of Scientific and Industrial Research, Osaka University, 4Max Planck Institute for Solid State Research, 5RIKEN Center for Emergent Matter Science (CEMS) Here, we present a protocol to control the carrier number in solids by using the electrolyte. Neuroscience Visualization of Thalamocortical Axon Branching and Synapse Formation in Organotypic Cocultures Naoyuki Matsumoto1,2, Nobuhiko Yamamoto2 1Department of Medical Neuroscience, Graduate School of Medical Sciences, Kanazawa University, 2Neuroscience Laboratories, Graduate School of Frontier Biosciences, Osaka University This protocol describes a method for simultaneous imaging of thalamocortical axon branching and synapse formation in organotypic cocultures of the thalamus and cerebral cortex. Individual thalamocortical axons and their presynaptic terminals are visualized by a single cell electroporation technique with DsRed and GFP-tagged synaptophysin. Biochemistry Real-time Tracking of DNA Fragment Separation by Smartphone Chunxian Tao1, Bo Yang1, Zhenqing Li1, Dawei Zhang1, Yoshinori Yamaguchi1,2,3 1Shanghai Key Lab of Modern Optical System, University of Shanghai for Science and Technology, 2Department of Applied Physics, Graduate School of Engineering, Osaka University, 3East China University of Science and Technology, Department of Physics Faculty of Science Traditional slab gel electrophoresis (SGE) experiments require a complicated apparatus and high chemical consumption. This work presents a protocol that describes a low-cost method to separate DNA fragments within a short timeframe. Biochemistry A Protein Preparation Method for the High-throughput Identification of Proteins Interacting with a Nuclear Cofactor Using LC-MS/MS Analysis Megumi Tsuchiya*1, M. Rezaul Karim*2, Taro Matsumoto3, Hidesato Ogawa1, Hiroaki Taniguchi3,4,5 1Graduate School of Frontier Biosciences, Osaka University, 2Department of Biotechnology and Genetic Engineering, Jahangirnagar University, 3Division of Cell Regeneration and Transplantation, School of Medicine, Nihon University, 4Institute of Genetics and Animal Breeding of the Polish Academy of Sciences, 5Graduate School of Life and Medical Sciences, Doshisha University We have established a method for the purification of coregulatory interaction proteins using the LC-MS/MS system. Chemistry Construction and Systematical Symmetric Studies of a Series of Supramolecular Clusters with Binary or Ternary Ammonium Triphenylacetates Toshiyuki Sasaki1, Yoko Ida1, Tetsuharu Yuge1, Atsushi Yamamoto1, Ichiro Hisaki1, Norimitsu Tohnai1, Mikiji Miyata1 1Department of Material and Life Science, Graduate School of Engineering, Osaka University This article describes construction of a series of hydrogen-bonding supramolecular clusters in crystals using primary ammonium triphenylacetates, which are recrystallized from non-polar solvents. This selective construction of the supramolecular clusters leads to effective systematical symmetric studies about a correlation between the supramolecular clusters and their components. Biology Isolation of Specific Genomic Regions and Identification of Associated Molecules by enChIP Toshitsugu Fujita1, Hodaka Fujii1 1Chromatin Biochemistry Research Group, Combined Program on Microbiology and Immunology, Research Institute for Microbial Diseases, Osaka University The identification of molecules associated with specific genomic regions of interest is required to understand the mechanisms of regulation of the functions of these regions. This protocol describes procedures to perform engineered DNA-binding molecule-mediated chromatin imunoprecipitation (enChIP) for identification of proteins and RNAs associated with a specific genomic region. Engineering Measurement of Scattering Nonlinearities from a Single Plasmonic Nanoparticle Hsuan Lee1, Kuan-Yu Li1, Yen-Ta Huang1, Po-Ting Shen1, Gitanjal Deka1, Ryosuke Oketani2, Yasuo Yonemaru2, Masahito Yamanaka2, Katsumasa Fujita2, Shi-Wei Chu1,3 1Department of Physics, National Taiwan University, 2Department of Applied Physics, Osaka University, 3Molecular Imaging Center, National Taiwan University Saturable and reverse saturable scattering were discovered in isolated plasmonic particles and adopted as a novel non-bleaching contrast method in super-resolution microscopy. Here the experimental procedures of detecting and extracting nonlinear scattering are explained in detail, as well as how to enhance resolution with the aid of saturated excitation microscopy.