Simon Fraser University View Institution's Website 12 articles published in JoVE Biology CRISPR-Cas9-Mediated Precise Knock-In Edits in Zebrafish Hearts Kyle E. Simpson1, Shoaib Faizi1, Ravichandra Venkateshappa1, Mandy Yip1, Raj Johal1, Damon Poburko1, Yen May Cheng1, Diana Hunter1, Eric Lin1, Glen F. Tibbits1, Thomas W. Claydon1 1Department of Biomedical Physiology and Kinesiology, Simon Fraser University This protocol describes an approach to facilitate precise knock-in edits in zebrafish embryos using CRISPR-Cas9 technology. A phenotyping pipeline is presented to demonstrate the applicability of these techniques to model a Long QT Syndrome-associated gene variant. Biochemistry Fluorescent Visualization of Mango-tagged RNA in Polyacrylamide Gels via a Poststaining Method Iqra M. Yaseen*1, Quiana R. Ang*1, Peter J. Unrau1 1Department of Molecular Biology and Biochemistry, Simon Fraser University Here we present a sensitive, rapid, and discriminating post-gel staining method to image RNAs tagged with RNA Mango aptamers I, II, III, or IV, using either native or denaturing polyacrylamide gel electrophoresis (PAGE) gels. After running standard PAGE gels, Mango-tagged RNA can be easily stained with TO1-Biotin and then analyzed using commonly available fluorescence readers. Environment Determination of the Settling Rate of Clay/Cyanobacterial Floccules Tiffany Playter1, Kurt Konhauser1, George W. Owttrim2, Denise S. Whitford2, Tyler Warchola1, Cheryl Hodgson1,3, Aleksandra M. Mloszewska4, Bruce Sutherland1, J.-P. Zonneveld1, S. George Pemberton1, Murray K. Gingras1 1Department of Earth and Atmospheric Sciences, University of Alberta, 2Department of Biological Sciences, University of Alberta, 3Department of Earth Sciences, Simon Fraser University, 4Earth Sciences Department, University of Toronto The interaction and sedimentation of the clay and bacterial cells within the marine realm, observed in natural environments, can be best investigated in a controlled lab environment. Here, we describe a detailed protocol, which outlines a novel method for measuring the sedimentation rate of clay and cyanobacterial floccules. Environment Enhancement of the Initial Growth Rate of Agricultural Plants by Using Static Magnetic Fields Seung C. Kim1, Alex Mason2, Wooseok Im3 1College of Medicine, Yonsei University, 2Simon Fraser University, 3Biomedical Research Institute, Seoul National University Hospital The goal of this protocol is to demonstrate the acceleration of the initial growth rate of plants by applying static magnetic fields with no external energy. Chemistry A 'Plug and Play' Method to Create Water-dispersible Nanoassemblies Containing an Amphiphilic Polymer, Organic Dyes and Upconverting Nanoparticles Khaled M. Arafeh*1, Amir M. Asadirad*1, Jason Woodson Li1, Danielle Wilson*1, Tuoqi Wu*1, Neil R. Branda1 14D LABS and Department of Chemistry, Simon Fraser University Organic dye molecules and oleic acid coated upconverting nanoparticles are not water-soluble. This protocol describes a ‘plug and play’ method that enables the transfer of organic dye molecules and upconverting particles from their initial hydrophobic solvent to water. Medicine Mouse Model of Alloimmune-induced Vascular Rejection and Transplant Arteriosclerosis Winnie Enns1, Anna von Rossum1, Jonathan Choy1 1Department of Molecular Biology and Biochemistry, Simon Fraser University We describe a protocol for aortic interposition grafting in mice. The goal of the protocol is to provide a model with which to study pathological processes and therapeutic strategies relevant to alloimmune reactions in arteries and the resultant arterial changes that contribute to organ transplant failure. Medicine Busulfan as a Myelosuppressive Agent for Generating Stable High-level Bone Marrow Chimerism in Mice Kyle Peake1, John Manning1, Coral-Ann Lewis1,2, Christine Barr1, Fabio Rossi2, Charles Krieger1,3 1Department of Biomedical Physiology and Kinesiology, Simon Fraser University, 2The Biomedical Research Centre, University of British Columbia, 3Division of Neurology, Department of Medicine, Neuromuscular Disease Unit, VHHSC We describe a protocol whereby busulfan conditioning permits the bone marrow of a recipient mouse to be replaced with bone marrow cells from donor mice ubiquitously expressing green fluorescent protein, in the absence of irradiation. This technique is useful to study bone marrow cell accumulation in the central nervous system. Neuroscience Detection of In Situ Protein-protein Complexes at the Drosophila Larval Neuromuscular Junction Using Proximity Ligation Assay Simon Wang1, SooHyun Yoo1, Hae-yoon Kim1, Mannan Wang2, Clare Zheng2, Wade Parkhouse2, Charles Krieger2, Nicholas Harden1 1Department of Molecular Biology and Biochemistry, Simon Fraser University, 2Department of Biomedical Physiology and Kinesiology, Simon Fraser University This protocol demonstrates how Proximity Ligation Assay can be used to detect in situ protein-protein interactions at the Drosophila larval neuromuscular junction. With this technique, Discs large and Hu-li tai shao are shown to form a complex at the postsynaptic region, an association previously identified through co-immunoprecipitation. Biology Glycopeptide Capture for Cell Surface Proteomics M. C. Gilbert Lee1, Bingyun Sun1 1Department of Chemistry, Simon Fraser University Cell surface proteins are biologically important and widely glycosylated. We introduce here a glycopeptide-capture approach to solubilize, enrich, and deglycosylate these proteins for facile LC-MS based proteomic analyses. Medicine Tilt Testing with Combined Lower Body Negative Pressure: a "Gold Standard" for Measuring Orthostatic Tolerance Clare L. Protheroe1, Henrike (Rianne) J.C. Ravensbergen1, Jessica A. Inskip1, Victoria E. Claydon1 1Department of Biomedical Physiology and Kinesiology, Simon Fraser University We describe a "gold standard" for evaluating orthostatic tolerance (OT) using tilt testing with combined lower body negative pressure (LBNP). This can be combined with non-invasive evaluations of cardiovascular reflex control. Normal and abnormal responses are defined. Immunology and Infection Avian Influenza Surveillance with FTA Cards: Field Methods, Biosafety, and Transportation Issues Solved Robert H.S. Kraus1, Pim van Hooft1, Jonas Waldenström2, Neus Latorre-Margalef2, Ronald C. Ydenberg1,3, Herbert H.T. Prins1 1Resource Ecology Group, Wageningen University, 2Section for Zoonotic Ecology and Epidemiology, School of Natural Sciences, Linnaeus University, 3Centre for Wildlife Ecology, Simon Fraser University A method to preserve, detect and sequence RNA from Avian Influenza Viruses was validated and extended using natural faecal samples from birds. This technique removes the necessity of maintaining a cool chain and handling of infectious viruses and can be applied in a 96-well high-throughput setup. Biology Live Imaging of Dense-core Vesicles in Primary Cultured Hippocampal Neurons David M. Kwinter1, Michael A. Silverman1 1Department of Biological Sciences, Simon Fraser University Live cell imaging is of particular utility when studying the dynamics of organelle trafficking. Here we describe a protocol for live imaging of dense-core vesicles in cultured neurons using wide-field fluorescence microscopy. This protocol is flexible and can be adapted to image other organelles such as mitochondria, endosomes, and peroxisomes.