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BTX

In utero and ex vivo Electroporation for Gene Expression in Mouse Retinal Ganglion Cells

Timothy J Petros1, Alexandra Rebsam1, Carol A Mason2, 1
1Departments of Pathology and Cell Biology, and Neuroscience, Columbia University College of Physicians and Surgeons, 2Department of Ophthalmology, Columbia University College of Physicians and Surgeons


Here we present two techniques for manipulating gene expression in murine retinal ganglion cells (RGCs) by in utero and ex vivo electroporation. These techniques enable one to examine how alterations in gene expression affect RGC development, axon guidance, and functional properties.


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  • Electroporation Products


  • In Utero Intraventricular Injection and Electroporation of E16 Rat Embryos

    William Walantus, Laura Elias, Arnold Kriegstein
    Institute for Regeneration Medicine, University of California, San Francisco


    In-utero in-vivo injection and electroporation of the embryonic rat neocortex provides a powerful tool for the manipulation of individual progenitors lining the walls of the lateral ventricle. This technique is now widely used to study the processes involved in corticogenesis by over-expressing or knocking down genes and observing the effects on cellular proliferation, migration, and differentiation. In comparison to traditional knockout strategies, in-utero electroporation provides a rapid means to manipulate a population of cells during a specific temporal window. In this video protocol, we outline the experimental methodology for preparing rats for surgery, exposing the uterine horns through laporatomy, injecting DNA into the lateral ventricles of the developing embryo, electroporating DNA into the progenitors lining the lateral wall, and caring for animals post-surgery. Our laboratory uses this protocol for surgeries on E15-E21 rats, however it is most commonly performed at E16 as shown in this video.


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  • In Ovo Electroporations of HH Stage 10 Chicken Embryos

    Marissa C. Blank1, Victor Chizhikov2, Kathleen J. Millen2
    1Department of Molecular Genetics and Cell Biology, University of Chicago, 2Department of Human Genetics, University of Chicago


    Chick in ovo electroporation is a technique which allows genetic manipulation of the avian embryo. Common applications of this technique include functional analysis of genes and putative enhancer elements. This video demonstrates neural tube electroporation in HH 10 chick embryos. Injection technique and proper egg handling are discussed.


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  • Warner Instruments

    Dopamine Release at Individual Presynaptic Terminals Visualized with FFNs

    Hui Zhang1, 2, Niko G. Gubernator3, 4, Minerva Yue1, Roland G. W. Staal1, Eugene V. Mosharov1, Daniela Pereira1, Vojtech Balsanek3, Paul A. Vadola3, Bipasha Mukherjee5, Robert H. Edwards5, David Sulzer1, 2, 6, Dalibor Sames3
    1Departments of Neurology, Columbia University, 2Departments of Psychiatry and Pharmacology, Columbia University, 3Department of Chemistry, Columbia University, 4eMolecules, Inc., 5Departments of Neurology and Physiology, University of California School of Medicine, San Francisco, 6Division of Molecular Therapeutics, New York Psychiatric Institute


    A new means to measure neurotransmission optically using fluorescent dopamine analogs.


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  • Recording Chamber


  • Measuring Near Plasma Membrane and Global Intracellular Calcium Dynamics in Astrocytes

    Eiji Shigetomi, Baljit S. Khakh
    Departments of Physiology and Neurobiology, David Geffen School of Medicine, University of California, Los Angeles



    We describe how to measure near membrane and global intracellular calcium dynamics in cultured astrocytes using total internal reflection and epifluorescence microscopy.


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  • Imaging Chamber


  • Calcium Imaging of Cortical Neurons using Fura-2 AM

    Odmara L Barreto-Chang, Ricardo E Dolmetsch
    Department of Neurobiology, Stanford University


    Calcium signals play a key role in many cellular processes including gene expression, survival and differentiation. Here we demonstrate how to perform calcium imaging using Fura-2 AM. Calcium imaging is a valuable tool to study the regulation of intracellular calcium in real time and its regulation of signaling cascades.


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  • Brain Slice Stimulation Using a Microfluidic Network and Standard Perfusion Chamber

    Javeed Shaikh Mohammed1, Hugo Caicedo1, Christopher P. Fall2, David T. Eddington1
    1Dept. of Bioengineering, University of Illinois, Chicago, 2Department of Anatomy and Cell Biology, University of Illinois, Chicago


    We demonstrate fabrication of a simple microfluidic device that can be integrated with standard electrophysiology setups to expose microscale surfaces of a brain slice in a well controlled manner to different neurotransmitters.


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  • Medical Systems Research

    Zebrafish Brain Ventricle Injection

    Jennifer H. Gutzman1, Hazel Sive1, 2
    1Whitehead Institute for Biochemical Research, 2Massachusetts Institute of Technology


    After neural tube formation, the neuroepithelium constricts and folds while the tube fills with embryonic cerebrospinal fluid (eCSF) to form the embryonic brain ventricles. We developed this ventricle injection technique to better visualize the fluid filled space in contrast to the neuroepithelial shape in a live embryo.


    View the full movie here


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  • PLI-100


  • Live Imaging of the Zebrafish Embryonic Brain by Confocal Microscopy

    Ellie Graeden1, 2, Hazel Sive1, 2
    1Department of Biology, Massachusetts Institute of Technology, 2Whitehead Institute for Biomedical Research, Massachusetts Institute of Technology


    In this video, we demonstrate a method by which to analyze the developing vertebrate brain in live zebrafish embryos at single cell resolution by confocal microscopy. This includes the method by which we inject the single-cell zebrafish embryo and subsequently mount and image the developing brain.


    View the full movie here


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  • PLI-100


  • Microinjection of Zebrafish Embryos to Analyze Gene Function

    Jonathan N. Rosen1, 2, Michael F. Sweeney1, John D. Mably1, 2
    1Department of Genetics, Harvard Medical School, 2Department of Cardiology, Children's Hospital Boston


    This video shows how morpholino or mRNA can be injected into zebrafish embryos at the one-cell stage to decrease or increase the level of specific gene products during subsequent development.


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  • PLI-100


  • Harvard Apparatus

    Analysis of Physiologic E-Selectin-Mediated Leukocyte Rolling on Microvascular Endothelium

    Georg Wiese1, Steven R. Barthel2, Charles J. Dimitroff2
    1Department of Dermatology, Brigham and Women's Hospital, 2Department of Dermatology, Brigham and Women's Hospital and Harvard Medical School


    This report provides a visual depiction of parallel-plate flow chamber analysis for studying leukocyte endothelial interactions under physiologic shear stress. This method is particularly useful for investigating the role of endothelial (E)-selectin and leukocyte E-selectin ligands that trigger leukocyte rolling on endothelial cell surfaces.


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  • PHD PUMP


  • Implantation of Engineered Tissue in the Rat Heart

    Bjoern Sill, Ivan V. Alpatov, Christina A. Pacak, Douglas B. Cowan
    Department of Anesthesiology, Perioperative and Pain Medicine, Children?s Hospital Boston and Harvard Medical School


    Here, we describe a cardiac surgical procedure to implant engineered tissue in the atrioventricular (AV)-groove of an adult Lewis rat.


    View the full movie here


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  • Inspira


  • Visualizing Single-molecule DNA Replication with Fluorescence Microscopy

    Nathan A. Tanner, Joseph J. Loparo, Antoine M. van Oijen
    Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School


    This protocol demonstrates a simple single-molecule fluorescence microscopy technique for visualizing DNA replication by individual replisomes in real time.


    View the full movie here


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  • Model 11 Syringe Pump


  • Chemotactic Response of Marine Micro-Organisms to Micro-Scale Nutrient Layers

    Justin R. Seymour, Marcos, Roman Stocker
    Environmental Microfluidics Group, Massachusetts Institute of Technology


    The fabrication of microfluidic channels and their implementation in experiments for studying the chemotactic foraging behaviour of marine microbes within a patchy nutrient seascape and the swimming behaviour of bacteria within shear flow are described.


    View the full movie here


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  • PHD PUMP


  • Method for Measurement of Viral Fusion Kinetics at the Single Particle Level

    Daniel L. Floyd1, Stephen C. Harrison1, 2, Antoine M. van Oijen1
    1Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, 2Howard Hughes Medical Institute, Harvard Medical School


    We present an in vitro, two-color fluorescence assay to visualize the fusion of single virus particles with a fluid target bilayer. By labeling viral particles with fluorophores that differentially stain the viral membrane and its interior, we are able to monitor the kinetics of hemifusion and pore formation.


    View the full movie here


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  • PHD PUMP


  • Automated Microfluidic Blood Lysis Protocol for Enrichment of Circulating Nucleated Cells

    William N. White1, Palaniappan Sethu2
    1Department of Mechanical Engineering, University of Louisville, 2Department of Bioengineering, University of Louisville


    An automated microfluidic device was developed for circulating nucleated cell enrichment from peripheral blood via erythrocytelysis that ensures isolation of high quality sample without cell loss.


    View the full movie here


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  • PHD and Pico Plus Pump


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