10 articles published in JoVE

• 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.

• 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.