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Hokkaido University

10 ARTICLES PUBLISHED IN JoVE

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Biology

Radioactive in situ Hybridization for Detecting Diverse Gene Expression Patterns in Tissue
Chun-Chun Chen 1, Kazuhiro Wada 2, Erich D. Jarvis 1
1Howard Hughes Medical Institute, Department of Neurobiology, Duke University , 2Department of Biological Sciences, Hokkaido University

This protocol is successfully used to quantitatively detect levels and spatial patterns of mRNA expression in multiple tissue types across vertebrate species. The method can detect low abundance transcripts and allows processing of hundreds of slides simultaneously. We present this protocol using expression profiling of avian embryonic brain formation as an example.

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Chemistry

Synthesis of Cyclic Polymers and Characterization of Their Diffusive Motion in the Melt State at the Single Molecule Level
Satoshi Habuchi 1, Takuya Yamamoto 2,3, Yasuyuki Tezuka 3
1Biological and Environmental Sciences and Engineering Division, King Abdullah University of Science and Technology (KAUST), 2Division of Applied Chemistry, Faculty of Engineering, Hokkaido University, 3Department of Organic and Polymeric Materials, Tokyo Institute of Technology

A protocol for the synthesis and characterization of diffusive motion of cyclic polymers at the single molecule level is presented.

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Biology

Methods for Staging Pupal Periods and Measurement of Wing Pigmentation of Drosophila guttifera
Yuichi Fukutomi 1, Keiji Matsumoto 1,2, Noriko Funayama 1, Shigeyuki Koshikawa 1,3,4
1Graduate School of Science, Kyoto University, 2Graduate School of Science, Osaka City University, 3The Hakubi Center for Advanced Research, Kyoto University, 4Graduate School of Environmental Science, Hokkaido University

Protocols for staging pupal periods and measurement of wing pigmentation of Drosophila guttifera are described. Staging and quantification of pigmentation provide a solid basis for studying developmental mechanisms of adult traits and enable interspecific comparison of trait development.

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JoVE Core

TChIP-Seq: Cell-Type-Specific Epigenome Profiling
Mari Mito 1,2, Mitsutaka Kadota 3, Shinichi Nakagawa 1,4, Shintaro Iwasaki 2,5
1RNA Biology Laboratory, RIKEN, 2RNA Systems Biochemistry Laboratory, RIKEN Cluster for Pioneering Research, 3Laboratory for Phyloinformatics, RIKEN Center for Biosystems Dynamics Research, 4RNA Biology Laboratory, Faculty of Pharmaceutical Sciences, Hokkaido University, 5Department of Computational Biology and Medical Sciences, Graduate School of Frontier Sciences, University of Tokyo

We describe a step-by-step protocol for tandem chromatin immunoprecipitation sequencing (tChIP-Seq) that enables the analysis of cell-type-specific genome-wide histone modification.

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Immunology and Infection

Identification of Mouse and Human Antibody Repertoires by Next-Generation Sequencing
Lin Sun 1, Naoko Kono 2, Hiroyuki Toh 3, Hanbing Xue 1, Kaori Sano 4,5, Tadaki Suzuki 4, Akira Ainai 4, Yasuko Orba 6, Junya Yamagishi 7,8, Hideki Hasegawa 4,5, Yoshimasa Takahashi 9, Shigeyuki Itamura 2, Kazuo Ohnishi 9,10
1Graduate School of Life and Environmental Sciences, University of Tsukuba, 2Center for Influenza Virus Research, National Institute of Infectious Diseases, 3School of Science and Technology, Kwansei Gakuin University, 4Department of Pathology, National Institute of Infectious Diseases, 5Division of Infectious Diseases Pathology, Department of Global Infectious Diseases, Tohoku University Graduate School of Medicine, 6Division of Molecular Pathobiology, Research Center for Zoonosis Control, Hokkaido University, 7Division of Collaboration and Education, Research Center for Zoonosis Control, Hokkaido University, 8Global Station for Zoonosis Control, GI-CoRE, Hokkaido University, 9Department of Immunology, National Institute of Infectious Diseases, 10Faculty of Life and Environmental Sciences, University of Tsukuba

Here, we describe protocols for the analysis and visualization of the structure and constitution of whole antibody repertoires. This involves the acquisition of vast sequences of antibody RNA using next-generation sequencing.

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Environment

Xylem Water Distribution in Woody Plants Visualized with a Cryo-scanning Electron Microscope
Kenichi Yazaki 1, Mayumi Y. Ogasa 2, Katsushi Kuroda 3, Yasuhiro Utsumi 4, Peter Kitin 5, Yuzou Sano 6
1Department of Plant Ecology, Forestry and Forest Products Research Institute (FFPRI), 2Kansai Research Center, Forestry and Forest Products Research Institute (FFPRI), 3Department of Wood Properties and Processing, Forestry and Forest Products Research Institute (FFPRI), 4Faculty of Agriculture, Kyushu University, 5Department of Bacteriology, University of Wisconsin, 6Research Faculty of Agriculture, Hokkaido University

Observing the water distribution within the xylem provides significant information regarding water flow dynamics in woody plants. In this study, we demonstrate the practical approach to observe xylem water distribution in situ by using a cryostat and cryo-SEM, which eliminates artifactual changes in the water status during sample preparation.

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JoVE Journal

Detection of Protein Aggregation using Fluorescence Correlation Spectroscopy
Akira Kitamura 1, Ai Fujimoto 1, Masataka Kinjo 1
1Laboratory for Molecular Cell Dynamics, Faculty of Advanced Life Science, Hokkaido University

We here introduce a procedure to measure protein oligomers and aggregation in cell lysate and live cells using fluorescence correlation spectroscopy.

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Biochemistry

Production of siRNA-Loaded Lipid Nanoparticles using a Microfluidic Device
Masatoshi Maeki 1,2, Yuto Okada 3, Shuya Uno 3, Ayuka Niwa 3, Akihiko Ishida 1, Hirofumi Tani 1, Manabu Tokeshi 1
1Division of Applied Chemistry, Faculty of Engineering, Hokkaido University, 2JST PRESTO, 3Graduate School of Chemical Sciences and Engineering, Hokkaido University

Microfluidic-based lipid nanoparticle (LNP) production methods have attracted attention in drug delivery systems (DDSs), including RNA delivery. This protocol describes the fabrication, LNP (siRNA-loaded LNP) production, and LNP evaluation processes using our original microfluidic device named iLiNP.

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Neuroscience

Measuring Axonal Cargo Transport in Mouse Primary Cortical Cultured Neurons
Yuriko Sobu 1,2, Toshiharu Suzuki 1,2
1Laboratory of Neuroscience, Graduate School of Pharmaceutical Sciences, Hokkaido University, 2Advanced Prevention and Research Laboratory for Dementia, Graduate School of Pharmaceutical Sciences, Hokkaido University

The present protocol describes the whole procedure of analysis for axonal transport. In particular, the calculation of transport velocity, not including the pausing, and the visualization method using open-access software "KYMOMAKER" are shown here.

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Neuroscience

Low-Cost Electroencephalographic Recording System Combined with a Millimeter-Sized Coil to Transcranially Stimulate the Mouse Brain In Vivo
Takahiro Yoshikawa *1, Hiromu Sato *1, Koki Kawakatsu 1, Takashi Tateno 2
1Bioengineering and Bioinformatics, Graduate School of Information Science and Technology, Hokkaido University, 2Bioengineering and Bioinformatics, Division of Information Science and Technology, Hokkaido University

A low-cost electroencephalographic recording system combined with a millimeter-sized coil is proposed to drive transcranial magnetic stimulation of the mouse brain in vivo. Using conventional screw electrodes with a custom-made, flexible, multielectrode array substrate, multi-site recording can be carried out from the mouse brain in response to transcranial magnetic stimulation.

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