Keio University View Institution's Website 13 articles published in JoVE Genetics Use of Freeze-thawed Embryos for High-efficiency Production of Genetically Modified Mice Hirofumi Nishizono*1,2,3, Mohamed Darwish*4,5, Hideki Uosaki6,7, Nanami Masuyama8,9,10, Motoaki Seki8,11, Hiroyuki Abe3, Nozomu Yachie8,9,10,12,13, Ryohei Yasuda1 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. Neuroscience Chronic Implantation of Whole-cortical Electrocorticographic Array in the Common Marmoset Misako Komatsu1, Takaaki Kaneko2,3, Hideyuki Okano2,3, Noritaka Ichinohe1,4 1Laboratory for Molecular Analysis of Higher Brain Function, RIKEN Center for Brain Science, 2Laboratory for Marmoset Neural Architecture, RIKEN Center for Brain Science, 3Department of Physiology, Keio University School of Medicine, 4Department of Ultrastructural Research, National Center of Neurology and Psychiatry We have developed a whole-cortical electrocorticographic array for the common marmoset that continuously covers almost the entire lateral surface of cortex, from the occipital pole to the temporal and frontal poles. This protocol describes a chronic implantation procedure of the array in the epidural space of the marmoset brain. Neuroscience Inducement and Evaluation of a Murine Model of Experimental Myopia Xiaoyan Jiang1,2, Toshihide Kurihara1,2, Shin-ichi Ikeda1,2, Hiromitsu Kunimi1,2, Kiwako Mori1,2, Hidemasa Torii1,2, Kazuo Tsubota2 1Laboratory of Photobiology, Keio University School of Medicine, 2Department of Ophthalmology, Keio University School of Medicine In this protocol, we describe the full process of experimental myopia inducement in mice using newly designed eyeglasses and the technic needed for achieving stable and reproducible results in ocular parameter measurements. Behavior A Method to Study Adaptation to Left-Right Reversed Audition Atsushi Aoyama1,2 1Faculty of Environment and Information Studies, Shonan Fujisawa Campus (SFC), Keio University, 2School of Information Environment, Tokyo Denki University The present study proposes a protocol to investigate the adaptation to left-right reversed audition achieved only by wearable devices, using neuroimaging, which can be an effective tool for uncovering the adaptability of humans to a novel environment in the auditory domain. Genetics Ultralow Input Genome Sequencing Library Preparation from a Single Tardigrade Specimen Yuki Yoshida1,2, Sayuri Konno1,3, Ryousuke Nishino1,3, Yumi Murai1,3, Masaru Tomita1,2,3, Kazuharu Arakawa1,2,3 1Institute for Advanced Biosciences, Keio University, 2Systems Biology Program, Graduate School of Media and Governance, Keio University, 3Faculty of Environment and Information Studies, Keio University Contamination during the genomic sequencing of microscopic organisms remains a large problem. Here, we show a method to sequence the genome of a tardigrade from a single specimen with as little as 50 pg of genomic DNA without whole genome amplification to minimize the risk of contamination. Medicine A Surgical Procedure for the Administration of Drugs to the Inner Ear in a Non-Human Primate Common Marmoset (Callithrix jacchus) Sho Kurihara*1,2, Masato Fujioka*3, Tomohiko Yoshida*1,2, Makoto Koizumi4, Kaoru Ogawa3, Hiromi Kojima2, Hirotaka James Okano1 1Division of Regenerative Medicine, Jikei University School of Medicine, 2Department of Otorhinolaryngology, Jikei University School of Medicine, 3Department of Otorhinolaryngology, Head and Neck Surgery, Keio University School of Medicine, 4Laboratory Animal Facilities, Jikei University School of Medicine We report a surgical method to administrate drugs to the inner ear of a non-human primate, the common marmoset (Callithrix jacchus), via the round window membrane. Cancer Research Induction and Diagnosis of Tumors in Drosophila Imaginal Disc Epithelia Kenta Morimoto1,2, Yoichiro Tamori1 1Structural Biology Center, National Institute of Genetics and Department of Genetics, School of Life Science, SOKENDAI, 2Graduate School of Media and Governance, Keio University Mosaic clone analysis in Drosophila imaginal disc epithelia is a powerful model system to study the genetic and cellular mechanisms of tumorigenesis. Here we describe a protocol to induce tumors in Drosophila wing imaginal discs using the GAL4-UAS system, and introduce a diagnosis method to classify the tumor phenotypes. Biology Dissection of the Auditory Bulla in Postnatal Mice: Isolation of the Middle Ear Bones and Histological Analysis Ayako Sakamoto1, Yukiko Kuroda1, Sho Kanzaki2, Koichi Matsuo1 1Laboratory of Cell and Tissue Biology, Keio University School of Medicine, 2Department of Otolaryngology Head and Neck Surgery, Keio University School of Medicine We present a protocol to isolate the auditory bulla, capsule, and ossicles from postnatal mice for whole mount and histological analysis. Developmental Biology Generation of Induced Pluripotent Stem Cells from Human Melanoma Tumor-infiltrating Lymphocytes Hidehito Saito1,2, Kumiko Iwabuchi3, Noemi Fusaki4,5, Fumito Ito3,6 1Department of Surgery, University of Michigan, 2Department of Biochemistry II, Kanazawa Medical University, 3Center for Immunotherapy, Roswell Park Cancer Institute, 4DNAVEC Corporation, 5Department of Ophthalmology, Keio University School of Medicine, 6Department of Surgical Oncology, Roswell Park Cancer Institute The goal of this protocol is to show the protocol for reprogramming melanoma tumor-infiltrating lymphocytes into induced pluripotent stem cells. Medicine Using Micro-computed Tomography for the Assessment of Tumor Development and Follow-up of Response to Treatment in a Mouse Model of Lung Cancer Ahmed E. Hegab1, Naofumi Kameyama1, Aoi Kuroda1, Shizuko Kagawa1, Yongjun Yin2, David Ornitz2, Tomoko Betsuyaku1 1Division of Pulmonary Medicine, Keio University School of Medicine, 2Department of Developmental Biology, Washington University School of Medicine We describe a method for the detection of tumor nodule development in the lungs of an adenocarcinoma mouse model using micro-computed tomography and its use for monitoring changes in nodule size over time and in response to treatment. The accuracy of the assessment was confirmed with end-point histological quantification. 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. Developmental Biology Generation of Induced Pluripotent Stem Cells from Human Peripheral T Cells Using Sendai Virus in Feeder-free Conditions Yoshikazu Kishino1, Tomohisa Seki2, Shinsuke Yuasa1, Jun Fujita1, Keiichi Fukuda1 1Department of Cardiology, Keio University School of Medicine, 2Department of Emergency and Critical Care Medicine, Keio University School of Medicine This protocol describes how to generate induced pluripotent stem cells (iPSCs) from human peripheral T cells in feeder-free conditions using a combination of matrigel and Sendai virus vectors containing reprogramming factors. 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.