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Hokkaido University

10 ARTICLES PUBLISHED IN JoVE

Biology

Radioactive in situ Hybridization for Detecting Diverse Gene Expression Patterns in Tissue

Chun-Chun Chen¹, Kazuhiro Wada², Erich D. Jarvis¹

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.

Chemistry

Synthesis of Cyclic Polymers and Characterization of Their Diffusive Motion in the Melt State at the Single Molecule Level

Satoshi Habuchi¹, Takuya Yamamoto^2,3, Yasuyuki Tezuka³

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.

Biology

Methods for Staging Pupal Periods and Measurement of Wing Pigmentation of Drosophila guttifera

Yuichi Fukutomi¹, Keiji Matsumoto^1,2, Noriko Funayama¹, 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.

JoVE Core

TChIP-Seq: Cell-Type-Specific Epigenome Profiling

Mari Mito^1,2, Mitsutaka Kadota³, 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.

Immunology and Infection

Identification of Mouse and Human Antibody Repertoires by Next-Generation Sequencing

Lin Sun¹, Naoko Kono², Hiroyuki Toh³, Hanbing Xue¹, Kaori Sano^4,5, Tadaki Suzuki⁴, Akira Ainai⁴, Yasuko Orba⁶, Junya Yamagishi^7,8, Hideki Hasegawa^4,5, Yoshimasa Takahashi⁹, Shigeyuki Itamura², 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.

Environment

Xylem Water Distribution in Woody Plants Visualized with a Cryo-scanning Electron Microscope

Kenichi Yazaki¹, Mayumi Y. Ogasa², Katsushi Kuroda³, Yasuhiro Utsumi⁴, Peter Kitin⁵, Yuzou Sano⁶

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.

JoVE Journal

Detection of Protein Aggregation using Fluorescence Correlation Spectroscopy

Akira Kitamura¹, Ai Fujimoto¹, Masataka Kinjo¹

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.

Biochemistry

Production of siRNA-Loaded Lipid Nanoparticles using a Microfluidic Device

Masatoshi Maeki^1,2, Yuto Okada³, Shuya Uno³, Ayuka Niwa³, Akihiko Ishida¹, Hirofumi Tani¹, Manabu Tokeshi¹

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.

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.

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 Kawakatsu¹, Takashi Tateno²

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.