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Hospital for Sick Children

10 ARTICLES PUBLISHED IN JoVE

Neuroscience

Multiple-mouse Neuroanatomical Magnetic Resonance Imaging

Jun Dazai^*1, Shoshana Spring^*1, Lindsay S. Cahill¹, R. Mark Henkelman^1,2

¹Mouse Imaging Centre, Hospital for Sick Children, ²Department of Medical Biophysics and Medical Imaging, University of Toronto

Magnetic resonance imaging (MRI) has become an increasingly popular tool for examining the phenotype of genetically altered mice. This article illustrates the methods necessary to achieve high-throughput phenotyping of genetically altered mice using multiple-mouse MRI.

Developmental Biology

Understanding Early Organogenesis Using a Simplified In Situ Hybridization Protocol in Xenopus

Steven J. Deimling¹, Rami R. Halabi^2,3, Stephanie A. Grover⁴, Jean H. Wang^2,5, Thomas A. Drysdale^2,4,5

¹Developmental and Stem Cell Biology, Hospital for Sick Children, ²Children's Health Research Institute, University of Western Ontario, ³Department of Physiology and Pharmacology, University of Western Ontario, ⁴Neurosciences and Mental Health, Hospital for Sick Children, ⁵Department of Paediatrics, University of Western Ontario

The Xenopus laevis embryo continues to be exceptionally useful in the study of early development due to its large size and ease of manipulation. A simplified protocol for whole mount in situ hybridization protocol is provided that can be used in the identification of specific organs in this model system.

Biology

Functional Reconstitution and Channel Activity Measurements of Purified Wildtype and Mutant CFTR Protein

Paul D. W. Eckford¹, Canhui Li¹, Christine E. Bear^1,2,3

¹Programme in Molecular Structure and Function, Hospital for Sick Children, ²Department of Biochemistry, University of Toronto, ³Department of Physiology, University of Toronto

Described here is a rapid and effective procedure for functional reconstitution of purified wild-type and mutant CFTR protein that preserves activity for this chloride channel, which is defective in Cystic Fibrosis. Iodide efflux from reconstituted proteoliposomes mediated by CFTR allows studies of channel activity and the effects of small molecules.

Bioengineering

Generation of ESC-derived Mouse Airway Epithelial Cells Using Decellularized Lung Scaffolds

Sharareh Shojaie¹, Joyce Lee¹, Jinxia Wang¹, Cameron Ackerley¹, Martin Post¹

¹Department of Physiology and Experimental Medicine, Peter Gilgan Centre for Research and Learning, Hospital for Sick Children

This protocol efficiently directs mouse embryonic stem cell-derived definitive endoderm to mature airway epithelial cells. This differentiation technique uses 3-dimensional decellularized lung scaffolds to direct lung lineage specification, in a defined, serum-free culture setting.

Immunology and Infection

Visualizing the Effects of Sputum on Biofilm Development Using a Chambered Coverglass Model

Trevor Beaudoin¹, Sarah Kennedy², Yvonne Yau³, Valerie Waters⁴

¹Physiology and Experimental Medicine, Hospital for Sick Children, ²Department of Clinical Microbiology, Royal College of Surgeons in Ireland, ³Department of Laboratory Medicine and Pathobiology, University of Toronto, ⁴Department of Pediatrics, University of Toronto

This protocol describes the visualization of biofilm development following exposure to host-factors using a slide chamber model. This model allows for direct visualization of biofilm development as well as analysis of biofilm parameters using computer software programs.

Immunology and Infection

Visualization of Pseudomonas aeruginosa within the Sputum of Cystic Fibrosis Patients

Lindsay Jackson¹, William DePas², Amanda J. Morris¹, Kevin Guttman¹, Yvonne C W Yau^1,3, Valerie Waters^1,4

¹Translational Medicine, Hospital for Sick Children, ²Center for Microbial Pathogenesis, Department of Pediatrics, University of Pittsburgh, ³Microbiology, Department of Pediatric Laboratory Medicine, Hospital for Sick Children, ⁴Infectious Diseases, Department of Pediatrics, Hospital for Sick Children

This protocol provides methods for visualization of bacterial cells and polysaccharide synthesis locus (Psl) polysaccharide within the sputum of cystic fibrosis patients.

Immunology and Infection

Quantifying the Effects of Antimicrobials on In vitro Biofilm Architecture using COMSTAT Software

Amanda J. Morris¹, Alvin Li¹, Lindsay Jackson¹, Yvonne C. W. Yau^1,2, Valerie Waters^1,3

¹Translational Medicine, Hospital for Sick Children, ²Microbiology, Department of Pediatric Laboratory Medicine, Hospital for Sick Children, ³Infectious Diseases, Department of Pediatrics, Hospital for Sick Children

Antimicrobial-induced changes to Pseudomonas aeruginosa biofilm architecture differ among clinical isolates cultured from patients with cystic fibrosis and chronic pulmonary infection. Following confocal microscopy, COMSTAT software can be utilized to quantify variations in biofilm architecture (e.g., surface area, thickness, biomass) for individual isolates to assess the efficacy of anti-infective agents.

Neuroscience

Time-Lapse Imaging of Neuronal Arborization using Sparse Adeno-Associated Virus Labeling of Genetically Targeted Retinal Cell Populations

Samantha Ing-Esteves^1,2, Julie L. Lefebvre^1,2

¹Program for Neuroscience and Mental Health, Hospital for Sick Children, ²Department of Molecular Genetics, University of Toronto

Here, we present a method for investigating neurite morphogenesis in postnatal mouse retinal explants by time-lapse confocal microscopy. We describe an approach for sparse labeling and acquisition of retinal cell types and their fine processes using recombinant adeno-associated virus vectors that express membrane-targeted fluorescent proteins in a Cre-dependent manner.

Immunology and Infection

Isolation of Total RNA from Pseudomonas aeruginosa within Biofilms for Measuring Gene Expression

Kevin Guttman¹, Pauline Wang², Lindsay Jackson¹, Amanda Morris¹, Yvonne Yau³, Valerie Waters^1,4

¹Translational Medicine, Hospital for Sick Children, ²Department of Cell and Systems Biology, University of Toronto, ³Microbiology, Department of Pediatric Laboratory Medicine, Hospital for Sick Children, ⁴Infectious Diseases, Department of Pediatrics, Hospital for Sick Children

This protocol presents a method to isolate RNA from Pseudomonas aeruginosa biofilms grown in chamber slides for high throughput sequencing.

Biology

A Fluorescence-Based Assay of Membrane Potential for High-Throughput Functional Study of Two Endogenous Ion Channels in Two Epithelial Cell Lines

Sunny Xia^1,3, Michelle Di Paola³, Nicola L. Jones^2,4,5, Christine E. Bear^1,3,5

¹Molecular Medicine, Hospital for Sick Children, ²Cell Biology, Hospital for Sick Children, ³Department of Physiology, University of Toronto, ⁴Department of Paediatrics, University of Toronto, ⁵Department of Biochemistry, University of Toronto

This protocol describes a method for the study of electrogenic membrane proteins by measuring changes in membrane potential. This assay provides a platform for the functional readout of multiple ion channels endogenously expressed in epithelial cell lines.