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21 ARTICLES PUBLISHED IN JoVE

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Bioengineering

Spatio-Temporal Manipulation of Small GTPase Activity at Subcellular Level and on Timescale of Seconds in Living Cells
Robert DeRose 1, Christopher Pohlmeyer 1, Nobuhiro Umeda 1,2, Tasuku Ueno 1,2, Tetsuo Nagano 2, Scot Kuo 1,3, Takanari Inoue 1
1Department of Cell Biology, Center for Cell Dynamics, Johns Hopkins University, 2Graduate School of Pharmaceutical Sciences, University of Tokyo, 3Biomedical Engineering, Johns Hopkins University

A method for spatio-temporal control of small GTPase activity by light is described. This method is based on rapamycin-induced FKBP-FRB heterodimerization and photo-caging systems. Optimization of light-irradiation enables the spatio-temporally controlled activation of small GTPases at the subcellular level.

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Biology

The Green Monster Process for the Generation of Yeast Strains Carrying Multiple Gene Deletions
Yo Suzuki 1, Jason Stam 1, Mark Novotny 2, Nozomu Yachie 3, Roger S. Lasken 2, Frederick P. Roth 3,4
1Department of Synthetic Biology and Bioenergy, J. Craig Venter Institute, 2Department of Microbial and Environmental Genomics, J. Craig Venter Institute, 3Donnelly Centre & Department of Molecular Genetics, University of Toronto, 4Lunenfeld Research Institute, Mt Sinai Hospital

The Green Monster method enables the rapid assembly of multiple deletions marked with a reporter gene encoding green fluorescent protein. This method is based on driving yeast strains through repeated cycles of sexual assortment of deletions and fluorescence-based enrichment of cells carrying more deletions.

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Medicine

Technical Aspects of the Mouse Aortocaval Fistula
Kota Yamamoto 1,2, Xin Li 1,3, Chang Shu 3, Tetsuro Miyata 2, Alan Dardik 1,4
1The 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.

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Developmental Biology

Alginate Encapsulation of Pluripotent Stem Cells Using a Co-axial Nozzle
Ikki Horiguchi 1, Yasuyuki Sakai 1
1Center for International Research on Integrative Biomedical System, Institute of Industrial Science, The University of Tokyo

We established a method of encapsulating pluripotent stem cells (PS cells) into alginate hydrogel capsules using a co-axial nozzle. This prevents cells from aggregating excessively and limits the shear stress experienced by cells in suspension culture. The technique is applicable to the mass production of PS cells as well as research on stem cell niche.

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Medicine

Intraluminal Drug Delivery to the Mouse Arteriovenous Fistula Endothelium
Takuya Hashimoto 1,2,3, Kota Yamamoto 1,2,3, Trenton Foster 1, Hualong Bai 1, Kunihiro Shigematsu 4, Alan Dardik 1,3
1Department of Surgery and the Vascular Biology and Therapeutics Program, Yale University, 2Department of Vascular Surgery, University of Tokyo, 3Department of Vascular Surgery, VA Connecticut Healthcare Systems, 4Department of Vascular Surgery, International University of Health and Welfare Mita Hospital

After puncturing the aorta through the inferior vena cava (IVC) to create an aorto-caval fistula in the mouse, solution containing a drug is infused into the IVC via the same needle, followed by incubation. This method enables more robust drug delivery to the venous endothelium compared to the external route.

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JoVE Core

Measurement of Particle Size Distribution in Turbid Solutions by Dynamic Light Scattering Microscopy
Takashi Hiroi 1, Mitsuhiro Shibayama 2
1Department of Chemistry, School of Science, University of Tokyo, 2Institute for Solid State Physics, University of Tokyo

A protocol for the direct measurement of particle size distribution in concentrated solutions using dynamic light scattering microscopy is presented.

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Medicine

Patch Angioplasty in the Rat Aorta or Inferior Vena Cava
Hualong Bai 1,2,3,4,5, Xin Li 6, Takuya Hashimoto 1,2,5, Haidi Hu 1,2,5, Trenton R. Foster 1,2,5, Jesse J. Hanisch 1,2,5, Jeans M. Santana 1,2,5, Alan Dardik 1,2,5
1Department of Surgery, Yale University, 2Vascular Biology and Therapeutics Program, Yale University, 3Department of Vascular Surgery, First Affiliated Hospital of Zhengzhou University, 4Basic Medical College of Zhengzhou University, 5VA Connecticut Healthcare Systems, West Haven, CT, 6Department of Vascular Surgery, Xiangya Second Hospital of Central South University, Changsha, China

We have established a model of pericardial patch angioplasty that can be used in either small-diameter veins or arteries. This model can be used to compare venous and arterial neointimal hyperplasia formation.

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Education

Gain-compensation Methodology for a Sinusoidal Scan of a Galvanometer Mirror in Proportional-Integral-Differential Control Using Pre-emphasis Techniques
Tomohiko Hayakawa 1, Takanoshin Watanabe 2, Taku Senoo 1, Masatoshi Ishikawa 1
1Department of Creative Informatics, University of Tokyo, 2Hitachi Industry & Control Solutions, Ltd.

We propose a method to extend the corresponding frequency by using a pre-emphasis technique. This method compensates for the gain reduction of a galvanometer mirror in sine-wave path tracking using proportional-integral-differential control.

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Behavior

Recording Horizontal Saccade Performances Accurately in Neurological Patients Using Electro-oculogram
Yasuo Terao 1, Hideki Fukuda 2, Yusuke Sugiyama 3, Satomi Inomata-Terada 1, Shin-ichi Tokushige 4, Masashi Hamada 3, Yoshikazu Ugawa 5
1Department of Cell Physiology, Kyorin University, 2Segawa Memorial Neurological Clinic for Children, 3Department of Neurology, University of Tokyo, 4Department of Neurology, Kyorin University, 5Department of Neurology, Fukushima Medical University

The article describes a practical method for recording horizontal eye movements with high accuracy by electro-oculogram in neurological patients, using a cup Ag-AgCl electrode with a wide plastic fringe. Stable measurement requires proper selection and fixation of electrodes, taking sufficient time for light adaptation to occur, and re-calibration as needed.

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Developmental Biology

Zygotic Fluorescence Recovery After Photo-bleaching Analysis for Chromatin Looseness That Allows Full-term Development
Masatoshi Ooga 1,3, Satoshi Funaya 2, Fugaku Aoki 2, Teruhiko Wakayama 1,3
1Faculty of Life and Environmental Sciences, Department of Biotechnology, University of Yamanashi, 2Department of Integrated Bioscience, Graduate School of Frontier Sciences, University of Tokyo, 3Advanced Biotechnology Center, University of Yamanashi

Chromatin looseness appears to be involved in the developmental potential of blastomeres. However, it is not known whether chromatin looseness can be used as a reliable index for the developmental potential for embryos. Here, an experimental system in which chromatin looseness-evaluated zygotes can develop to full term has been described.

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Engineering

Experimental Methods for Spin- and Angle-Resolved Photoemission Spectroscopy Combined with Polarization-Variable Laser
Kenta Kuroda 1, Koichiro Yaji 1, Ayumi Harasawa 1, Ryo Noguchi 1, Takeshi Kondo 1, Fumio Komori 1, Shik Shin 1
1Institute for Solid State Physics, University of Tokyo

Here, we combine polarization-variable 7-eV laser with spin- and angle-resolved photoemission technique to visualize the spin-orbital coupling effect in solid states.

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Biochemistry

Electrochemical Detection of Deuterium Kinetic Isotope Effect on Extracellular Electron Transport in Shewanella oneidensis MR-1
Yoshihide Tokunou 1, Kazuhito Hashimoto 2, Akihiro Okamoto 2
1Department of Applied Chemistry, University of Tokyo, 2Global Research Center for Environment and Energy based on Nanomaterials Science, National Institute for Materials Science

Here we present a protocol of whole-cell electrochemical experiments to study the contribution of proton transport to the rate of extracellular electron transport via the outer-membrane cytochromes complex in Shewanella oneidensis MR-1.

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Neuroscience

In Vivo Microdialysis Method to Collect Large Extracellular Proteins from Brain Interstitial Fluid with High-molecular Weight Cut-off Probes
Kaoru Yamada 1
1Department of Neuropathology, Graduate School of Medicine, University of Tokyo

In vivo microdialysis has enabled collection of molecules present in brain interstitial fluid (ISF) from awake, freely-behaving animals. In order to analyze relatively large molecules in ISF, the current article specifically focuses on the microdialysis protocol using probes with high molecular weight cut off membranes.

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Bioengineering

An Orbital Shaking Culture of Mammalian Cells in O-shaped Vessels to Produce Uniform Aggregates
Ikki Horiguchi 1,2, Ikumi Suzuki 3, Takashi Morimura 3, Yasuyuki Sakai 1,4
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.

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Engineering

Measuring Magnetically-Tuned Ferroelectric Polarization in Liquid Crystals
Hiroki Ueda 1, Takuya Akita 2, Yoshiaki Uchida 2, Tsuyoshi Kimura 3
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.

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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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Chemistry

Spatiotemporally Controlled Nuclear Translocation of Guests in Living Cells Using Caged Molecular Glues as Photoactivatable Tags
Rina Mogaki 1, Kou Okuro 1, Akio Arisaka 1, Takuzo Aida 1,2
1Department of Chemistry and Biotechnology, School of Engineering, University of Tokyo, 2Riken Center for Emergent Matter Science

This protocol describes light-triggered nuclear translocation of guests in living cells using caged molecular glue tags. This method is promising for site-selective nuclear-targeting drug delivery.

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Chemistry

An Electrochemical Cholesteric Liquid Crystalline Device for Quick and Low-Voltage Color Modulation
Shoichi Tokunaga *1, Mengyan Zeng *1,2, Yoshimitsu Itoh 1, Fumito Araoka 3, Takuzo Aida 3
1Department of Chemistry and Biotechnology, The University of Tokyo, 2Department of Chemistry, Tsinghua University, 3RIKEN Center for Emergent Matter Science

A protocol for the fabrication of a reflective cholesteric liquid crystalline display device containing a redox-responsive chiral dopant allowing quick and low-voltage operation is presented.

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Medicine

Murine Model of Central Venous Stenosis using Aortocaval Fistula with an Outflow Stenosis
Toshihiko Isaji 1,2,3, Shun Ono 1,2,4,5, Takuya Hashimoto 1,2,3, Kota Yamamoto 1,2,3, Ryosuke Taniguchi 1,2,3, Haidi Hu 1,2, Tun Wang 1,2, Jun Koizumi 4, Toshiya Nishibe 5, Katsuyuki Hoshina 3, Alan Dardik 1,2,6
1Department of Surgery, Yale University, 2Vascular Biology and Therapeutics Program, Yale University, 3Department of Vascular Surgery, University of Tokyo, 4Department of Diagnostic Radiology, Tokai University School of Medicine, 5Department of Cardiovascular Surgery, Tokyo Medical University, 6Department of Vascular Surgery, VA Connecticut Healthcare Systems

An aortocaval fistula was created by puncturing the murine infra-renal aorta through both walls into the inferior vena cava and was followed by creation of a stenosis in its outflow via partial ligation of the inferior vena cava. This reproducible model can be used to study central venous stenosis.

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Environment

Visualization of Low-Level Gamma Radiation Sources Using a Low-Cost, High-Sensitivity, Omnidirectional Compton Camera
Hiroshi Muraishi 1, Ryoji Enomoto 2, Hideaki Katagiri 3, Mika Kagaya 4, Takara Watanabe 5, Naofumi Narita 3, Daisuke Kano 5
1School of Allied Health Sciences, Kitasato University, 2Institute for Cosmic Ray Research, University of Tokyo, 3College of Science, Ibaraki University, 4National Institute of Technology, Sendai College, 5National Cancer Center Hospital East

We present experimental protocols for visualizing various low-level gamma radiation sources in the ambient environment using a low-cost, high-sensitivity, omnidirectional, gamma-ray imaging Compton camera.

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Genetics

Use of Freeze-thawed Embryos for High-efficiency Production of Genetically Modified Mice
Hirofumi Nishizono *1,2,3, Mohamed Darwish *4,5, Hideki Uosaki 6,7, Nanami Masuyama 8,9,10, Motoaki Seki 8,11, Hiroyuki Abe 3, Nozomu Yachie 8,9,10,12,13, Ryohei Yasuda 1
1Max Planck Florida Institute for Neuroscience, 2Life Science Research Center, University of Toyama, 3Department of Biochemical Engineering, Graduate School of Science and Engineering, Yamagata University, 4Graduate School of Innovative Life Science, University of Toyama, 5Department of Biochemistry, Faculty of Pharmacy, Cairo University, 6Division of Regenerative Medicine, Center for Molecular Medicine, Jichi Medical University, 7Division of Stem Cell Research and Drug Development, Center for Development of Advanced Medical Technology, Jichi Medical University, 8Synthetic Biology Division, Research Center for Advanced Science and Technology, University of Tokyo, 9Institute for Advanced Biosciences, Keio University, 10Graduate School of Media and Governance, Keio University, 11Department of Molecular Oncology, Graduate School of Medicine, Chiba University, 12Department of Biological Sciences, School of Science, University of Tokyo, 13College of Arts and Sciences, University of Tokyo

Here, we present a modified method for cryopreservation of one-cell embryos as well as a protocol that couples the use of freeze-thawed embryos and electroporation for the efficient generation of genetically modified mice.

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