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14 articles published in JoVE
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Targeted Next-generation Sequencing and Bioinformatics Pipeline to Evaluate Genetic Determinants of Constitutional Disease
Allison A. Dilliott1,2, Sali M.K. Farhan3, Mahdi Ghani4, Christine Sato4, Eric Liang5, Ming Zhang4, Adam D. McIntyre1, Henian Cao1, Lemuel Racacho6,7, John F. Robinson1, Michael J. Strong1,8, Mario Masellis9,10, Dennis E. Bulman6,7, Ekaterina Rogaeva4, Anthony Lang10,11, Carmela Tartaglia4,10, Elizabeth Finger12,13, Lorne Zinman9, John Turnbull14, Morris Freedman10,15, Rick Swartz9, Sandra E. Black9,16, Robert A. Hegele1,2
1Robarts Research Institute, Schulich School of Medicine and Dentistry, Western University, 2Department of Biochemistry, Schulich School of Medicine and Dentistry, Western University, 3Analytic and Translational Genetics Unit, Center for Genomic Medicine, Harvard Medical School, Massachusetts General Hospital, Stanley Centre for Psychiatric Research, Broad Institute of MIT and Harvard, 4Tanz Centre for Research in Neurodegenerative Diseases, University of Toronto, 5
Targeted next-generation sequencing is a time- and cost-efficient approach that is becoming increasingly popular in both disease research and clinical diagnostics. The protocol described here presents the complex workflow required for sequencing and the bioinformatics process used to identify genetic variants that contribute to disease.
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Genome-wide RNAi Screening to Identify Host Factors That Modulate Oncolytic Virus Therapy
Kristina J. Allan1,2, Douglas J. Mahoney1,4, Stephen D. Baird1, Charles A. Lefebvre1, David F. Stojdl1,2,3
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Here we describe a protocol for employing high-throughput RNAi screening to uncover host targets that can be manipulated to enhance oncolytic virus therapy, specifically rhabodvirus and vaccinia virus therapy, but it can be readily adapted to other oncolytic virus platforms or for discovering host genes that modulate virus replication generally.
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All-electronic Nanosecond-resolved Scanning Tunneling Microscopy: Facilitating the Investigation of Single Dopant Charge Dynamics
Mohammad Rashidi1,2, Wyatt Vine1, Jacob A.J. Burgess3,4,5, Marco Taucer1,2,6, Roshan Achal1, Jason L. Pitters2, Sebastian Loth3,4, Robert A. Wolkow1,2
1Department of Physics, University of Alberta, 2National Institute for Nanotechnology, National Research Council of Canada, Edmonton, 3Max Planck Institute for the Structure and Dynamics of Matter, 4Max Planck Institute for Solid State Research, 5Department of Physics and Astronomy, University of Manitoba, 6Joint Attosecond Science Laboratory, University of Ottawa
We demonstrate an all-electronic method to observe nanosecond-resolved charge dynamics of dopant atoms in silicon with a scanning tunneling microscope.
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Modeling Neuronal Death and Degeneration in Mouse Primary Cerebellar Granule Neurons
Matthew Laaper1,2, Takrima Haque1, Ruth S. Slack3, Arezu Jahani-Asl1,2,4
1Lady Davis Institute for Medical Research, Jewish General Hospital, 2Integrated Program in Neuroscience, McGill University, 3Department of Cellular and Molecular Medicine, University of Ottawa, 4Department of Oncology, Faculty of Medicine, McGill University
This protocol describes a simple method for isolating and culturing primary mouse cerebral granule neurons (CGNs) from 6-7 day old pups, efficient transduction of CGNs for loss and gain of function studies, and modelling NMDA-induced neuronal excitotoxicity, low-potassium-induced cell death, DNA-damage, and oxidative stress using the same culture model.
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Long-term Behavioral Tracking of Freely Swimming Weakly Electric Fish
James J. Jun1,2,3, André Longtin1,2,3, Leonard Maler2,3
1Department of Physics, University of Ottawa, 2Department of Cellular and Molecular Medicine, University of Ottawa, 3Centre for Neural Dynamics, University of Ottawa
We describe a set of techniques for studying spontaneous behavior of freely swimming weakly electric fish over an extended period of time, by synchronously measuring the animal's electric organ discharge timing, body position and posture both accurately and reliably in a specially designed aquarium tank inside a sensory isolation chamber.
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Micro-particle Image Velocimetry for Velocity Profile Measurements of Micro Blood Flows
Katie L. Pitts1, Marianne Fenech1,2
1Department of Chemical and Biological Engineering, University of Ottawa, 2Department of Mechanical Engineering, University of Ottawa
Micro-particle image velocimetry (μPIV) is used to visualize paired images of micro particles seeded in blood flows which are cross-correlated to give an accurate velocity profile. Shear rate, maximum velocity, velocity profile shape, and flow rate, each of which has clinical applications, can be derived from these measurements.
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Isolation and Culture of Individual Myofibers and their Satellite Cells from Adult Skeletal Muscle
Alessandra Pasut1,2, Andrew E. Jones1,2, Michael A. Rudnicki1,2
1Sprott Center for Stem Cell Research, Ottawa Hospital Research Institute, 2Department of Cellular and Molecular Medicine, University of Ottawa
Isolation and culture of myofibers is the gold standard in vitro system to study the transition of satellite cells through quiescence, activation and differentiation. Importantly, the single myofiber culture system preserves the myofiber/stem cell association, which is an essential component of the muscle stem cell niche.
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Performing Vaginal Lavage, Crystal Violet Staining, and Vaginal Cytological Evaluation for Mouse Estrous Cycle Staging Identification
Ashleigh C. McLean1,2,3, Nicolas Valenzuela3,4, Stephen Fai3,4, Steffany A.L. Bennett1,3
1Department of Biochemistry, Microbiology and Immunology, Neural Regeneration Laboratory and Ottawa Institute of Systems Biology, 2Department of Cellular and Molecular Medicine, University of Ottawa, 3CIHR Program in Neurodegenerative Lipidomics, University of Ottawa, 4Carleton Immersive Media Studio, Azrieli School of Architecture and Urbanism
Here, we describe how to identify the stage of the murine reproductive (proestrus, estrus, metestrus, or diestrus) by simple, non-invasive collection and cytological assessment of vaginal smear samples. We further describe how vaginal cytology reflects circulating hormonal levels underlying transition through the murine reproductive cycle.
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Derivation of Enriched Oligodendrocyte Cultures and Oligodendrocyte/Neuron Myelinating Co-cultures from Post-natal Murine Tissues
Ryan W. O'Meara1,2, Scott D. Ryan1, Holly Colognato3, Rashmi Kothary1,2,4
1Regenerative Medicine Program, Ottawa Hospital Research Institute, 2Department of Cellular and Molecular Medicine, University of Ottawa, 3Department of Pharmacological Sciences, Stony Brook University, 4Department of Medicine, University of Ottawa
This article describes methods to derive enriched populations of murine oligodendrocyte precursor cells (OPCs) in primary culture, which differentiate to produce mature oligodendrocytes (OLs). In addition, this report describes techniques to produce murine myelinating co-cultures by seeding mouse OPCs onto a neurite bed of mouse dorsal root ganglion neurons (DRGNs).
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Quantitative Analyses of all Influenza Type A Viral Hemagglutinins and Neuraminidases using Universal Antibodies in Simple Slot Blot Assays
Caroline Gravel*1, Changgui Li*2, Junzhi Wang2, Anwar M Hashem1,3,4, Bozena Jaentschke1, Gary Van Domselaar5, Runtao He5, Xuguang Li1,3
1Centre for Vaccine Evaluation, Biologics and Genetic Therapies Directorate, HPFB, Health canada, 2National Institute for the Control of Pharmaceutical and Biological Products, The State Food and Drug Administration, Beijing, 3Department of Biochemistry, Microbiology and Immunology, University of Ottawa, 4Microbiology Department, Faculty of Medicine, King Abdulaziz University, 5National Microbiology Laboratory, Public Health Agency of Canada
A simple slot blot method was developed for the quantification of influenza viral hemagglutinin and neuraminidase using universal antibodies targeting their most conserved sequences identified through bioinformatics analyses. This innovative approach may provide a useful alternative to quantitative determination of all viral hemagglutinin and neuraminidase.
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Localizing Protein in 3D Neural Stem Cell Culture: a Hybrid Visualization Methodology
Sophie Imbeault*1, Nicolas Valenzuela*2, Stephen Fai2, Steffany A.L. Bennett1
1Neural Regeneration Laboratory and Ottawa Institute of Systems Biology, Department of Biochemistry, Microbiology and Immunology, University of Ottawa, 2Carleton Immersive Media Studio, Azrieli School of Architecture and Urbanism, Carleton University
Here, we describe how to produce, expand, and immunolabel postnatal hippocampal neural progenitor cells (NPCs) in three-dimensional (3D) culture. Next, using hybrid visualization technologies, we demonstrate how digital images of immunolabelled cryosections can be used to reconstruct and map the spatial position of immunopositive cells throughout the entire 3D neurosphere.
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