Japan Science and Technology Agency View Institution's Website 11 articles published in JoVE Genetics An Optogenetic Method to Control and Analyze Gene Expression Patterns in Cell-to-cell Interactions Akihiro Isomura1,2, Ryoichiro Kageyama1,3,4,5 1Institute for Frontier Life and Medical Sciences, Kyoto University, 2Japan Science and Technology Agency, PRESTO, 3Institute for Integrated Cell-Material Sciences, Kyoto University, 4Graduate School of Medicine, Kyoto University, 5Graduate School of Biostudies, Kyoto University Here, we present a protocol to analyze cell-to-cell transfer of oscillatory information by optogenetic control and live monitoring of gene expression. This approach provides a unique platform to test a functional significance of dynamic gene expression programs in multicellular systems. Biology Real-time In Vivo Recording of Arabidopsis Calcium Signals During Insect Feeding Using a Fluorescent Biosensor Thomas R. Vincent1, James Canham1, Masatsugu Toyota2,3,4, Marieta Avramova1, Sam T. Mugford5, Simon Gilroy2, Anthony J. Miller1, Saskia Hogenhout5, Dale Sanders1 1Department of Metabolic Biology, John Innes Centre, Norwich Research Park, 2Department of Botany, University of Wisconsin, Madison, 3Department of Biochemistry and Molecular Biology, Saitama University, 4Precursory Research for Embryonic Science and Technology (PRESTO), Japan Science and Technology Agency (JST), 5Department of Cell and Developmental Biology, John Innes Centre, Norwich Research Park This protocol outlines a simple method for analyzing calcium signals in plants generated by feeding hemipteran insects, such as aphids. Arabidopsis thaliana transformed with the GFP calcium biosensor GCaMP3 allow for the real-time in vivo imaging of calcium dynamics with a high temporal and spatial resolution. Neuroscience Quadruple Immunostaining of the Olfactory Bulb for Visualization of Olfactory Sensory Axon Molecular Identity Codes Naoki Ihara1, Yuji Ikegaya1,2, Haruki Takeuchi1,3 1Laboratory of Chemical Pharmacology, Graduate School of Pharmaceutical Sciences, The University of Tokyo, 2Center for Information and Neural Networks, National Institute of Information and Communications Technology, 3PRESTO, Japan Science and Technology Agency (JST) Olfactory sensory neurons express a wide variety of axon-sorting molecules to establish proper neural circuitry. This protocol describes an immunohistochemical staining method to visualize combinatorial expressions of axon-sorting molecules at the axon termini of olfactory sensory neurons. Engineering Capillary-based Centrifugal Microfluidic Device for Size-controllable Formation of Monodisperse Microdroplets Masamune Morita1, Hitoyoshi Yamashita1,2, Masayuki Hayakawa1, Hiroaki Onoe3, Masahiro Takinoue1,4 1Department of Computational Intelligence and Systems Science, Interdisciplinary Graduate School of Science and Engineering, Tokyo Institute of Technology, 2Graduate School of Bioscience and Biotechnology, Tokyo Institute of Technology, 3Department of Mechanical Engineering, Keio University, 4PRESTO, Japan Science and Technology Agency Here, we demonstrate a simple production method for size-controllable, monodisperse, water-in-oil (W/O) microdroplets using a capillary-based centrifugal microfluidic device. This method requires only a small sample volume and enables high-yield production. We expect this method will be useful for rapid biochemical and cellular analyses. Behavior Concurrent Electroencephalography Recording During Transcranial Alternating Current Stimulation (tACS) Kristoffer D. Fehér1, Yosuke Morishima1,2 1Division of Systems Neuroscience of Psychopathology, Translational Research Center, University Hospital of Psychiatry, University of Bern, 2Japan Science and Technology Agency, PRESTO In this article we explain how to set up a concurrent transcranial alternating current stimulation and EEG experiment. Bioengineering Photopatterning Proteins and Cells in Aqueous Environment Using TiO2 Photocatalysis Hideaki Yamamoto1,2, Takanori Demura3, Kohei Sekine3, Sho Kono3, Michio Niwano2,4, Ayumi Hirano-Iwata2,5, Takashi Tanii6 1Frontier Research Institute for Interdisciplinary Sciences, Tohoku University, 2CREST, Japan Science and Technology Agency, 3School of Fundamental Science and Engineering, Waseda University, 4Research Institute of Electrical Communication, Tohoku University, 5Graduate School of Biomedical Engineering, Tohoku University, 6Faculty of Science and Engineering, Waseda University We describe a protocol for modifying cell affinity of a scaffold surface in aqueous environment. The method takes advantage of titanium dioxide photocatalysis to decompose organic film in the photo-irradiated region. We show that it can be used to create microdomains of scaffolding proteins, both ex situ and in situ. Biology Long-term, High-resolution Confocal Time Lapse Imaging of Arabidopsis Cotyledon Epidermis during Germination Kylee M. Peterson1, Keiko U. Torii1,2,3 1Department of Biology, University of Washington, 2Howard Hughes Medical Institute, University of Washington, 3PRESTO, Japan Science and Technology Agency We describe a protocol using chamber slides and media to immobilize plant cotyledons for confocal imaging of the epidermis over several days of development, documenting stomatal differentiation. Fluorophore-tagged proteins can be tracked dynamically by expression and subcellular localization, increasing understanding of their possible roles during cell division and cell-type differentiation. Medicine Visualization and Analysis of Blood Flow and Oxygen Consumption in Hepatic Microcirculation: Application to an Acute Hepatitis Model Kosuke Tsukada1, Makoto Suematsu2,3 1Department of Applied Physics and Physico-Informatics, Keio University, 2Department of Biochemistry, School of Medicine, Keio University, 3Exploratory Research for Advanced Technology (ERATO), Suematsu Gas Biology Project, Japan Science and Technology Agency (JST) An optical system was developed to visualize hepatic microcirculation with FITC-labeled erythrocytes and to measure the partial pressure of oxygen in the microvessels with laser-assisted phosphorimetry. This method can be used to investigate physiological and pathological mechanisms by analyzing microvascular structure, diameter, blood flow velocity, and oxygen tension. Neuroscience T-maze Forced Alternation and Left-right Discrimination Tasks for Assessing Working and Reference Memory in Mice Hirotaka Shoji1,2, Hideo Hagihara1, Keizo Takao3, Satoko Hattori1,2,3, Tsuyoshi Miyakawa1,2,3 1Division of Systems Medical Science, Institute for Comprehensive Medical Science, Fujita Health University, 2Japan Science and Technology Agency, Core Research for Evolutionary Science and Technology (CREST), 3Center for Genetic Analysis of Behavior, National Institute for Physiological Sciences, National Institutes of Natural Sciences This article presents the protocol of T-maze tests using a modified automated apparatus for assessing the learning and memory functions in mice. Biology The Method of Rodent Whole Embryo Culture using the Rotator-type Bottle Culture System Masanori Takahashi1, Noriko Osumi1,2 1Division of Developmental Neuroscience, United Centers for Advanced Research and Translational Medicine (ART), Tohoku University Graduate School of Medicine, 2The Core Research for Evolutional Science and Technology (CREST), Japan Science and Technology Corporation (JST) Whole embryo culture technique allows us to culture mouse and rat embryos ex vivo condition during limited periods corresponding to midgestation stages. In this video protocol, we demonstrate our standard procedures of rat whole embryo culture after E12.5 using the rotator-type bottle culture system. Biology Dissection of Hippocampal Dentate Gyrus from Adult Mouse Hideo Hagihara1,2, Keiko Toyama1,2, Nobuyuki Yamasaki1,3, Tsuyoshi Miyakawa1,2,4,5 1Japan Science and Technology Agency, Core Research for Evolutionary Science and Technology (CREST), 2Division of Systems Medical Science, Institute for Comprehensive Medical Science, Fujita Health University, 3Department of Psychiatry, Graduate School of Medicine, Kyoto University, 4Genetic Engineering and Functional Genomics Group, Horizontal Medical Research Organization, Graduate School of Medicine, Kyoto University, 5Center for Genetic Analysis of Behavior, National Institute for Physiological Sciences, National Institutes of Natural Sciences A dissection technique for removal of the dentate gyrus from adult mouse under a stereomicroscope was demonstrated in this video-recorded protocol.