20 articles published in JoVE

• Genetics

  
  High-throughput DNA Extraction and Genotyping of 3dpf Zebrafish Larvae by Fin Clipping Ceres Kosuta^1,2, Kate Daniel¹, Devon L. Johnstone^1,2, Kevin Mongeon^1,2, Kevin Ban^1,2, Sophie LeBlanc¹, Stuart MacLeod¹, Karim Et-Tahiry², Marc Ekker², Alex MacKenzie¹, Izabella Pena^{1,2 1} Zebrafish have been used as reliable genetic model organisms in biomedical research, especially with the advent of gene-editing technologies. When larval phenotypes are expected, DNA extraction and genotype identification can be challenging. Here, we describe an efficient genotyping procedure for zebrafish larvae, by tail clipping, as early as 72-h post-fertilization.

• Genetics

  
  Targeted Next-generation Sequencing and Bioinformatics Pipeline to Evaluate Genetic Determinants of Constitutional Disease Allison A. Dilliott^1,2, Sali M.K. Farhan³, Mahdi Ghani⁴, Christine Sato⁴, Eric Liang⁵, Ming Zhang⁴, Adam D. McIntyre¹, Henian Cao¹, Lemuel Racacho^6,7, John F. Robinson¹, Michael J. Strong^1,8, Mario Masellis^9,10, Dennis E. Bulman^6,7, Ekaterina Rogaeva⁴, Anthony Lang^10,11, Carmela Tartaglia^4,10, Elizabeth Finger^12,13, Lorne Zinman⁹, John Turnbull¹⁴, Morris Freedman^10,15, Rick Swartz⁹, Sandra E. Black^9,16, Robert A. Hegele^{1,2 1}Robarts 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.

• Cancer Research

  
  Genome-wide RNAi Screening to Identify Host Factors That Modulate Oncolytic Virus Therapy Kristina J. Allan^1,2, Douglas J. Mahoney^1,4, Stephen D. Baird¹, Charles A. Lefebvre¹, David F. Stojdl^{1,2,3 1} 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.

• Engineering

  
  All-electronic Nanosecond-resolved Scanning Tunneling Microscopy: Facilitating the Investigation of Single Dopant Charge Dynamics Mohammad Rashidi^1,2, Wyatt Vine¹, Jacob A.J. Burgess^3,4,5, Marco Taucer^1,2,6, Roshan Achal¹, Jason L. Pitters², Sebastian Loth^3,4, Robert A. Wolkow^{1,2 1}Department 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.

• Neuroscience

  
  Modeling Neuronal Death and Degeneration in Mouse Primary Cerebellar Granule Neurons Matthew Laaper^1,2, Takrima Haque¹, Ruth S. Slack³, Arezu Jahani-Asl^{1,2,4 1}Lady 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.

• Genetics

  
  Inducible T7 RNA Polymerase-mediated Multigene Expression System, pMGX Mohamed I. Hassan*¹, Fern R. McSorley*¹, Kinya Hotta², Christopher N. Boddy^{1 1}Department of Chemistry and Biomolecular Sciences, University of Ottawa, ²School of Biosciences, The University of Nottingham Malaysia Campus This study describes methods for the T7-mediated co-expression of multiple genes from a single plasmid in Escherichia coli using the pMGX plasmid system.

• Biology

  
  Isolation of CD146⁺ Resident Lung Mesenchymal Stromal Cells from Rat Lungs Jennifer J.P. Collins^1,2, Marius A. Möbius^1,3,4, Bernard Thébaud^{1,2,5 1}Sinclair Centre for Regenerative Medicine, Ottawa Hospital Research Institute, ²University of Ottawa, ³Department of Neonatology and Pediatric Critical Care Medicine, Medical Faculty and University Hospital Carl Gustav Carus, Technische Universität Dresden, ⁴DFG Research Center and Cluster of Excellence for Regenerative Therapies (CRTD), Technische Universität, Dresden, ⁵ This protocol describes an isolation technique for obtaining primary lung resident mesenchymal stromal cells from rats, through the use of enzymatic digestion, density gradient separation, plastic adherence and CD146⁺ magnetic bead selection.

• Behavior

  
  Substantiating Appropriate Motion Capture Techniques for the Assessment of Nordic Walking Gait and Posture in Older Adults Christopher M. Dalton¹, Julie Nantel^{1 1}School of Human Kinetics, Faculty of Health Sciences, University of Ottawa The aim was to substantiate optimal use of data collection techniques for Nordic walking gait and posture analysis. Three-dimensional motion capture should be used during short duration analysis (i.e. single gait cycle), while accelerometry should be employed for longer duration analysis (i.e. repeated cycles) like a 6 Minute Walk Test.

• Bioengineering

  
  Controlled Microfluidic Environment for Dynamic Investigation of Red Blood Cell Aggregation Rym Mehri¹, Catherine Mavriplis¹, Marianne Fenech^{1 1}Department of Mechanical Engineering, University of Ottawa The protocol described details an experimental procedure to quantify Red Blood Cell (RBC) aggregates under a controlled and constant shear rate, based on image processing techniques. The goal of this protocol is to relate RBC aggregate sizes to the corresponding shear rate in a controlled microfluidic environment.

• Developmental Biology

  
  A Time Differential Staining Technique Coupled with Full Bilateral Gill Denervation to Study Ionocytes in Fish Velislava Tzaneva¹, Steve F. Perry^{1 1}Department of Biology, University of Ottawa This manuscript describes a protocol to track the re-distribution of branchial ionocytes and their innervation using a time differential staining technique coupled with full bilateral gill denervation.

• Biology

  
  Assessment of Selective mRNA Translation in Mammalian Cells by Polysome Profiling Mame Daro Faye¹, Tyson E Graber², Martin Holcik^{3 1}Apoptosis Research Centre, Children's Hospital of Eastern Ontario Research Institute and Department of Biochemistry, Microbiology and Immunology, University of Ottawa, ²Montreal Neurological Institute, ³Apoptosis Research Centre, Children's Hospital of Eastern Ontario Research Institute and Department of Pediatrics, University of Ottawa The ability of cells to adapt to stress is crucial for their survival. Regulation of mRNA translation is one such adaptation strategy, providing for rapid regulation of the proteome. Here, we provide a standardized polysome profiling protocol to identify specific mRNAs that are selectively translated under stress conditions.

• Neuroscience

  
  Long-term Behavioral Tracking of Freely Swimming Weakly Electric Fish James J. Jun^1,2,3, André Longtin^1,2,3, Leonard Maler^{2,3 1}Department 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.

• Engineering

  
  Fine-tuning the Size and Minimizing the Noise of Solid-state Nanopores Eric Beamish¹, Harold Kwok¹, Vincent Tabard-Cossa¹, Michel Godin^{1,2 1}Department of Physics, University of Ottawa, ²Ottawa-Carleton Institute of Biomedical Engineering, University of Ottawa A methodology for preparing solid-state nanopores in solution for biomolecular translocation experiments is presented. By applying short pulses of high electric fields, the nanopore diameter can be controllably enlarged with subnanometer precision and its electrical noise characteristics significantly improved. This procedure is performed in situ using standard laboratory equipment under experimental conditions.

• Bioengineering

  
  Micro-particle Image Velocimetry for Velocity Profile Measurements of Micro Blood Flows Katie L. Pitts¹, Marianne Fenech^{1,2 1}Department 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.

• Biology

  
  Isolation and Culture of Individual Myofibers and their Satellite Cells from Adult Skeletal Muscle Alessandra Pasut^1,2, Andrew E. Jones^1,2, Michael A. Rudnicki^{1,2 1}Sprott 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.

• Biology

  
  Performing Vaginal Lavage, Crystal Violet Staining, and Vaginal Cytological Evaluation for Mouse Estrous Cycle Staging Identification Ashleigh C. McLean^1,2,3, Nicolas Valenzuela^3,4, Stephen Fai^3,4, Steffany A.L. Bennett^{1,3 1}Department 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.

• Neuroscience

  
  Derivation of Enriched Oligodendrocyte Cultures and Oligodendrocyte/Neuron Myelinating Co-cultures from Post-natal Murine Tissues Ryan W. O'Meara^1,2, Scott D. Ryan¹, Holly Colognato³, Rashmi Kothary^{1,2,4 1}Regenerative 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).

• Biology

  
  Lentiviral-mediated Knockdown During Ex Vivo Erythropoiesis of Human Hematopoietic Stem Cells Carmen G. Palii¹, Roya Pasha¹, Marjorie Brand^{1,2 1}The Sprott Center for Stem Cell Research, Regenerative Medicine Program, Ottawa Hospital Research Institute, ²Department of Cellular and Molecular Medicine, University of Ottawa An ex vivo protocol to generate mature human red blood cells from hematopoietic stem/progenitors is described. Additionally we describe an efficient lentiviral-delivery method to knockdown the transcription factor TAL1 in primary erythroid cells. The efficiency of lentivirus mediated gene delivery is demonstrated using GFP expressing viruses.

• Immunology and Infection

  
  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 Hashem^1,3,4, Bozena Jaentschke¹, Gary Van Domselaar⁵, Runtao He⁵, Xuguang Li^{1,3 1}Centre 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.

• Neuroscience

  
  Localizing Protein in 3D Neural Stem Cell Culture: a Hybrid Visualization Methodology Sophie Imbeault*¹, Nicolas Valenzuela*², Stephen Fai², Steffany A.L. Bennett^{1 1}Neural 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.