University of Waterloo View Institution's Website 16 articles published in JoVE Bioengineering Agrobacterium tumefaciens-Mediated Genetic Engineering of Green Microalgae, Chlorella vulgaris M. Reza Roushan1, Chuchi Chen1, Parisa Ahmadi1, Valerie C. A. Ward1 1Department of Chemical Engineering, University of Waterloo This protocol outlines the utilization of Agrobacterium tumefaciens-mediated transformation (AMT) for integrating gene(s) of interest into the nuclear genome of the green microalgae Chlorella vulgaris, leading to the production of stable transformants. Neuroscience Combined Peripheral Nerve Stimulation and Controllable Pulse Parameter Transcranial Magnetic Stimulation to Probe Sensorimotor Control and Learning Kylee R. Graham*1, Kara D. Hayes*1, Sean K. Meehan1 1Department of Kinesiology and Health Sciences, University of Waterloo Short-latency afferent inhibition (SAI) is a transcranial magnetic stimulation protocol to probe sensorimotor integration. This article describes how SAI can be used to study the convergent sensorimotor loops in the motor cortex during sensorimotor behavior. Biology Determining the Toxicity of UV Radiation and Chemicals on Primary and Immortalized Human Corneal Epithelial Cells Jaclyn M. L. Chang1, Junghee Seo1, Maggie Miu Yee Kwan1, Sarah Oh1, David J. McCanna1, Lakshman Subbaraman1, Lyndon Jones1,2 1Centre for Ocular Research & Education (CORE), School of Optometry & Vision Science, University of Waterloo, 2Centre for Eye and Vision Research (CEVR) This article describes the procedures used to evaluate the toxicity of UV radiation and chemical toxins on a primary and immortalized cell line. Biochemistry Optimized Incorporation of Alkynyl Fatty Acid Analogs for the Detection of Fatty Acylated Proteins using Click Chemistry Lucia M. Q. Liao1, Rachel A. V. Gray1, Dale D. O. Martin1 1Department of Biology, University of Waterloo The study of fatty acylation has important implications in cellular protein interactions and diseases. Presented here is a modified protocol to improve click chemistry detection of fatty acylated proteins, which can be applied in various cell types and combined with other assays, including pulse-chase and mass spectrometry. Behavior Tactile Vibrating Toolkit and Driving Simulation Platform for Driving-Related Research Ao Zhu1, Annebella Tsz Ho Choi1, Ko-Hsuan Ma1, Shi Cao2, Han Yao1, Jian Wu3, Jibo He1 1Psychology Department, School of Social Sciences, Tsinghua University, 2Department of Systems Design Engineering, University of Waterloo, 3Haier Innovation Design Center, Haier Company This protocol describes a driving simulation platform and a tactile vibrating toolkit for the investigation of driving-related research. An exemplar experiment exploring the effectiveness of tactile warnings is also presented. Medicine Effect of Artificial Tear Formulations on the Metabolic Activity of Human Corneal Epithelial Cells after Exposure to Desiccation Rekha Rangarajan1, Howard A. Ketelson1, Richard Do2, David J. McCanna2, Adeline Suko2, Daryl Enstone2, Lakshman N. Subbaraman1, Jaya Dantam2, Lyndon W. Jones2 1Alcon Vision, LLC, 2Centre for Ocular Research and Education (CORE), School of Optometry & Vision Science, University of Waterloo The purpose of this protocol is to evaluate if different artificial tear formulations can protect human corneal epithelial cells from desiccation using an in vitro model. After exposure to artificial tear formulations and desiccation, human corneal epithelial cells are assessed for metabolic activity. Behavior Classical Short-Delay Eyeblink Conditioning in One-Year-Old Children Lucy K. Goodman1, Nicola S. Anstice1,2, Suzanne Stevens3, Benjamin Thompson1,4, Trecia A. Wouldes3 1School of Optometry and Vision Science, The University of Auckland, 2Discipline of Optometry, University of Canberra, 3Department of Psychological Medicine, The University of Auckland, 4School of Optometry and Vision Science, University of Waterloo This protocol describes an eyeblink conditioning paradigm appropriate for experiments with one-year-old infants. Commercial or custom-made equipment can be used to deliver the stimuli, and data collection and analysis should be performed on the video recordings. Medicine Impression Cytology of the Lid Wiper Area Alex Muntz1, Kevin van Doorn1, Lakshman N. Subbaraman1, Lyndon W. Jones1 1Centre for Contact Lens Research, University of Waterloo We collected, stained and imaged cells from the conjunctiva of the inner eyelid margin of human subjects. By characterizing cell morphology and metabolic activity, this method may further our understanding of dry eye and the role that friction between the ocular surfaces may play in perceiving ocular discomfort. Bioengineering Development of an In Vitro Ocular Platform to Test Contact Lenses Chau-Minh Phan1, Hendrik Walther1, Huayi Gao2, Jordan Rossy1, Lakshman N. Subbaraman1, Lyndon Jones1 1School of Optometry and Vision Science, University of Waterloo, 2Medella Health Current in vitro models for evaluating contact lenses (CLs) and other eye-related applications are severely limited. The presented ocular platform simulates physiological tear flow, tear volume, air exposure and mechanical wear. This system is highly versatile and can be applied to various in vitro analyses with CLs. Biology Production of Double-stranded DNA Ministrings Shirley Wong1, Peggy Lam1, Nafiseh Nafissi1, Steven Denniss2, Roderick Slavcev1 1School of Pharmacy, University of Waterloo, 2Sgd Health Innovation The linear covalently closed (LCC) DNA minivector (DNA ministring) is a non-viral gene delivery vector offering high transfection efficiency and is relevant to any DNA delivery application. The production system is simple, rapid, and scalable in vivo. The following protocol provides visual step-by-step instructions for the production of DNA ministrings. Biology A Method for Mouse Pancreatic Islet Isolation and Intracellular cAMP Determination Joshua C. Neuman1, Nathan A. Truchan2, Jamie W. Joseph3, Michelle E. Kimple2 1Department of Nutrional Sciences, University of Wisconsin-Madison, 2Department of Medicine, Division of Endocrinology, Diabetes, and Metabolism, University of Wisconsin-Madison, 3School of Pharmacy, University of Waterloo Assaying in vitro β-cell function using isolated mouse islets of Langerhans is an important component in the study of diabetes pathophysiology and therapeutics. While many downstream applications are available, this protocol specifically describes the measurement of intracellular cyclic adenosine monophosphate (cAMP) as an essential parameter determining β-cell function. Bioengineering Site-specific Bacterial Chromosome Engineering: ΦC31 Integrase Mediated Cassette Exchange (IMCE) John R. Heil1, Jiujun Cheng1, Trevor C. Charles1 1Biology, University of Waterloo A quick and efficient method to integrate foreign DNA of interest into pre-made acceptor strains, termed landing pad strains, is described. The method allows site-specific integration of a DNA cassette into the engineered landing pad locus of a given strain, through conjugation and expression of the ΦC31 integrase. Biology Optimized Transfection Strategy for Expression and Electrophysiological Recording of Recombinant Voltage-Gated Ion Channels in HEK-293T Cells Adriano Senatore1, Adrienne N. Boone1, J. David Spafford1 1Department of Biology, University of Waterloo Reliable method for highly efficient in vitro expression and subsequent electrophysiological recording of recombinant voltage-gated ion channels in cultured human embryonic kidney cells (HEK-293T). Biology DNA Stable-Isotope Probing (DNA-SIP) Eric A. Dunford1, Josh D. Neufeld1 1Department of Biology, University of Waterloo DNA stable-isotope probing is a cultivation-independent method to identify and characterize active communities of microorganisms that are capable of utilizing specific substrates. Assimilation of substrate enriched in heavy isotope leads to incorporation of labelled atoms into microbial biomass. Density gradient ultracentrifugation retrieves labelled DNA for downstream molecular analyses. Biology The Preparation of Drosophila Embryos for Live-Imaging Using the Hanging Drop Protocol Bruce H. Reed1, Stephanie C. McMillan1, Roopali Chaudhary1 1Department of Biology, University of Waterloo A simple, inexpensive, and effective method of preparing Drosophila embryos for live-imaging analysis is presented. Our protocol provides humidity and gas exchange and does not compress the Drosophila embryo. This method is suitable for GFP-based live imaging of Drosophila embryos using a stereomicroscope or upright compound microscope. Biology Culture of myeloid dendritic cells from bone marrow precursors Jeanette Boudreau1,2, Sandeep Koshy2,3, Derek Cummings2, Yonghong Wan2 1Medical Sciences Program, McMaster University, 2Centre for Gene Therapeutics, McMaster University, 3Department of Chemical Engineering, University of Waterloo This video demonstrates the procedure for differentiating myeloid dendritic cells from mouse bone marrow. Isolation of mouse tibia and femur, and processing of bone marrow are demonstrated. Pictures demonstrating cell morphology before and after differentiation, and figures depicting cell phenotype and IL-12 production following maturation using CpG are shown.
