Yale University School of Medicine 45 articles published in JoVE Neuroscience Intrathecal Injection of Newborn Mouse for Genome Editing and Drug Delivery Xiaona Lu1, Yong-hui Jiang1,2,3 1Department of Genetics, Yale University School of Medicine, 2Department of Neuroscience, Yale University School of Medicine, 3Department of Pediatrics, Yale University School of Medicine The present protocol outlines step-by-step instructions for performing intrathecal injections in neonatal mice for gene editing and drug delivery. Neuroscience Simultaneous Data Collection of fMRI and fNIRS Measurements Using a Whole-Head Optode Array and Short-Distance Channels Sara Sanchez-Alonso1, Rebecca R. Canale2, Isabel F. Nichoson1, Richard N. Aslin1,2,3 1Child Study Center, Yale University School of Medicine, 2Department of Psychology, University of Connecticut, 3Department of Psychology, Yale University We present a method for simultaneously collecting fMRI and fNIRS signals from the same subjects with whole-head fNIRS coverage. The protocol has been tested with three young adults and can be adapted for data collection for developmental studies and clinical populations. Medicine Multiple Intravenous Bolus Dosing and Invasive Hemodynamic Assessment in a Hypoxia-Induced Mouse Pulmonary Artery Hypertension Model Lingfeng Qin1, Bo Jiang2, Krisztina Zsebo3, Henricus J. Duckers3, Michael Simons4,5, Pei-Yu Chen4 1Department of Surgery, Yale University School of Medicine, 2Department of Vascular Surgery, The First Hospital of China Medical University, Key Laboratory of Pathogenesis, Prevention, and Therapeutics of Aortic Aneurysm, 3VasoRx Inc., 4Yale Cardiovascular Research Center, Department of Internal Medicine, Yale University School of Medicine, 5Department of Cell Biology, Yale University School of Medicine This protocol provides a step-by-step procedure for executing multiple intravenous bolus dose administration and invasive hemodynamic monitoring in mice. Investigators can use this protocol for future therapeutic compound screening for pulmonary artery hypertension. Medicine Imaging and Analysis of Oil Red O-Stained Whole Aorta Lesions in an Aneurysm Hyperlipidemia Mouse Model Pei-Yu Chen1, Lingfeng Qin2, Michael Simons1,3 1Yale Cardiovascular Research Center, Department of Internal Medicine, Yale University School of Medicine, 2Department of Surgery, Yale University School of Medicine, 3Department of Cell Biology, Yale University School of Medicine This protocol provides a step-by-step procedure to analyze atherosclerotic burden in mice. Investigators can use this protocol to compare the abundance, location, and size of atherosclerotic lesions in different animals. Developmental Biology Isolation of Murine Retinal Endothelial Cells for Next-Generation Sequencing Nicholas W. Chavkin1,2, Shelby Cain1, Kenneth Walsh2,3,4, Karen K. Hirschi1,2,4,5 1Department of Cell Biology, University of Virginia School of Medicine, 2Robert M. Berne Cardiovascular Research Center, University of Virginia School of Medicine, 3Department of Cardiology, University of Virginia School of Medicine, 4Hematovascular Biology Center, University of Virginia School of Medicine, 5Yale Cardiovascular Research Center, Yale University School of Medicine This protocol describes a method for the isolation of murine postnatal retinal endothelial cells optimized for cell yield, purity, and viability. These cells are suitable for next-generation sequencing approaches. Developmental Biology Directed Differentiation of Hemogenic Endothelial Cells from Human Pluripotent Stem Cells Elizabeth A. Nelson1,2, Jingyao Qiu3,4, Nicholas W. Chavkin1,2, Karen K. Hirschi1,2,3,4,5 1Department of Cell Biology, University of Virginia, 2Cardiovascular Research Center, University of Virginia, 3Department of Medicine, Yale University School of Medicine, 4Department of Genetics, Yale University School of Medicine, 5Yale Cardiovascular Research Center, Yale University School of Medicine Presented here is a simple protocol for the directed differentiation of hemogenic endothelial cells from human pluripotent stem cells in approximately 1 week. Neuroscience Measuring Post-Stroke Cerebral Edema, Infarct Zone and Blood-Brain Barrier Breakdown in a Single Set of Rodent Brain Samples Dmitry Frank*1, Benjamin F. Gruenbaum*2, Julia Grinshpun1, Israel Melamed3, Olena Severynovska4, Ruslan Kuts1, Michael Semyonov1, Evgeni Brotfain1, Alexander Zlotnik1, Matthew Boyko1 1Department of Anesthesiology and Critical Care, Soroka Medical Center, Ben-Gurion University of the Negev, 2Department of Anesthesiology, Yale University School of Medicine, 3Department of Neurosurgery, Soroka University Medical Center and the Faculty of Health Sciences, Ben-Gurion University of the Negev, 4Department of Physiology, Faculty of Biology, Ecology and Medicine, Dnepropetrovsk State University This protocol describes a novel technique of measuring the three most important parameters of ischemic brain injury on the same set of rodent brain samples. Using only one brain sample is highly advantageous in terms of ethical and economic costs. Neuroscience Laser-Induced Brain Injury in the Motor Cortex of Rats Ruslan Kuts*1, Israel Melamed*2, Honore N. Shiyntum3, Benjamin F. Gruenbaum4, Dmitry Frank1, Boris Knyazer5, Dmitry Natanel1, Olena Severynovska3, Max Vinokur1, Matthew Boyko1 1Division of Anesthesiology and Critical Care, Soroka University Medical Center and the Faculty of Health Sciences, Ben-Gurion University of the Negev, 2Department of Neurosurgery, Soroka University Medical Center and the Faculty of Health Sciences, Ben-Gurion University of the Negev, 3 The protocol presented here shows a technique to create a rodent model of brain injury. The method described here uses laser irradiation and targets motor cortex. Immunology and Infection Three-Dimensional Imaging of the Vertebral Lymphatic Vasculature and Drainage using iDISCO+ and Light Sheet Fluorescence Microscopy Laurent Jacob*1, Jose Brito*1,2, Jean-Leon Thomas1,3 1Université Pierre et Marie Curie Paris, Sorbonne Université, Institut du Cerveau et de la Moelle Epinière, 2Instituto de Ciências Biomédicas, Universidade Federal do Rio de Janeiro, 3Department of Neurology, Yale University School of Medicine A protocol is presented combining tissue clearing with light sheet fluorescence microscopy (LSFM) to obtain three-dimensional and cellular resolution images of the lymphatic vessels and lymph nodes (LNs) collecting the cerebrospinal fluid (CSF) and spinal epidural fluid. Neuroscience Induction of Diffuse Axonal Brain Injury in Rats Based on Rotational Acceleration Dmitry Frank*1, Israel Melamed*2, Benjamin F. Gruenbaum3, Julia Grinshpun1, Ruslan Kuts1, Rachel Shvartsur4, Abed N. Azab4, Mohamad H. Assadi5, Max Vinokur1, Matthew Boyko1 1Division of Anesthesia and Critical Care, Soroka University Medical Center and the Faculty of Health Sciences, Ben-Gurion University of the Negev, 2Department of Neurosurgery, Soroka University Medical Center and the Faculty of Health Sciences, Ben-Gurion University of the Negev, 3Department of Anesthesiology, Yale University School of Medicine, 4Recanati School for Community Health Professions, Faculty of Health Sciences, Ben-Gurion University of the Negev, 5Department of microbiology and immunology, Faculty of Health Sciences, Ben-Gurion University of the Negev This protocol validates a reliable, easy-to-perform and reproducible rodent model of brain diffuse axonal injury (DAI) that induces widespread white matter damage without skull fractures or contusions. Medicine Inducing Acute Liver Injury in Rats via Carbon Tetrachloride (CCl4) Exposure Through an Orogastric Tube Dmitry Frank*1, Shiri Savir*1, Benjamin F. Gruenbaum2, Israel Melamed3, Julia Grinshpun1, Ruslan Kuts1, Boris Knyazer4, Alexander Zlotnik1, Max Vinokur1, Matthew Boyko1 1Department of Anesthesiology and Critical Care, Soroka Medical Center, Ben-Gurion University of the Negev, 2Department of Anesthesiology, Yale University School of Medicine, 3Department of Neurosurgery, Soroka University Medical Center and the Faculty of Health Sciences, Ben-Gurion University of the Negev, 4Department of Ophthalmology, Soroka University Medical Center and the Faculty of Health Sciences, Ben-Gurion University of the Negev This protocol describes a common and feasible method of inducing acute liver injury (ALI) via CCl4 exposure through an orogastric tube. CCl4 exposure induces ALI through the formation of reactive oxygen species during its biotransformation in the liver. This method is used to analyze the pathophysiology of ALI and examine different hepatoprotective strategies. Immunology and Infection Applying Live Cell Imaging and Cryo-Electron Tomography to Resolve Spatiotemporal Features of the Legionella pneumophila Dot/Icm Secretion System David Chetrit1, Donghyun Park1,2, Bo Hu3, Jun Liu1,2, Craig R. Roy1 1Department of Microbial Pathogenesis, Boyer Center for Molecular Medicine, Yale University School of Medicine, 2Microbial Sciences Institute, Yale University, 3Department of Microbiology and Molecular Genetics, McGovern Medical School, The University of Texas Health Science Center at Houston Imaging of bacterial cells is an emerging systems biology approach focused on defining static and dynamic processes that dictate the function of large macromolecular machines. Here, integration of quantitative live cell imaging and cryo-electron tomography is used to study Legionella pneumophila type IV secretion system architecture and functions. Developmental Biology A Static Self-Directed Method for Generating Brain Organoids from Human Embryonic Stem Cells Erin M. Boisvert1, Robert E. Means1, Michael Michaud1, Jason J. Thomson2, Joseph A. Madri1, Samuel G. Katz1 1Department of Pathology, Yale University School of Medicine, 2Stem Cell Center, Yale University School of Medicine This protocol was generated as a means to produce brain organoids in a simplified, low cost manner without exogenous growth factors or basement membrane matrix while still maintaining the diversity of brain cell types and many features of cellular organization. Genetics Expression, Purification, and Liposome Binding of Budding Yeast SNX-BAR Heterodimers Mengxiao Ma1, Shreya Goyal2, Christopher G. Burd1, Richard J. Chi2 1Department of Cell Biology, Yale School of Medicine, 2Department of Biological Sciences, University of North Carolina at Charlotte Here, we present a workflow for the expression, purification and liposome binding of SNX-BAR heterodimers in yeast. Immunology and Infection Quantitative Fluorescence In Situ Hybridization (FISH) and Immunofluorescence (IF) of Specific Gene Products in KSHV-Infected Cells Tenaya K. Vallery1, Joan A. Steitz2 1Norfolk Academy, 2Department of Molecular Biophysics and Biochemistry, Howard Hughes Medical Institute, Yale School of Medicine We describe a protocol utilizing fluorescence in situ hybridization (FISH) to visualize multiple herpesviral RNAs within lytically infected human cells, either in suspension or adherent. This protocol includes quantification of fluorescence producing a nucleocytoplasmic ratio and can be extended for simultaneous visualization of host and viral proteins with immunofluorescence (IF). Immunology and Infection Measurement of Chitinase Activity in Biological Samples Alyssa K. Amick1, Qing Liu1, Samir Gautam1, Geoffrey Chupp1, Charles S. Dela Cruz*1, Lokesh Sharma*1 1Pulmonary, Critical Care and Sleep Medicine, Yale University School of Medicine Presented here is a simple method to measure chitinase activity in biological fluids such as bronchoalveolar lavage or serum. Immunology and Infection A Positioning Device for the Placement of Mice During Intranasal siRNA Delivery to the Central Nervous System Irfan Ullah1,2, Kunho Chung1, Jagadish Beloor2, Sang-Kyung Lee1, Priti Kumar2 1Department of Bioengineering and Institute of Nanoscience and Technology, Hanyang University, 2Department of Internal Medicine, Section of Infectious Diseases, Yale University School of Medicine Here, we present a protocol using a mouse positioning device that enables the appropriate placement of mice for the intranasal administration of a brain-targeting peptide-siRNA formulation enabling effective gene silencing in the central nervous system. Developmental Biology Isotropic Light-Sheet Microscopy and Automated Cell Lineage Analyses to Catalogue Caenorhabditis elegans Embryogenesis with Subcellular Resolution Leighton H. Duncan1,5, Mark W. Moyle1,5, Lin Shao1,5, Titas Sengupta1,5, Richard Ikegami1,5, Abhishek Kumar4,5, Min Guo4,5, Ryan Christensen4,5, Anthony Santella2,5, Zhirong Bao2,5, Hari Shroff4,5, William Mohler3,5, Daniel A. Colón-Ramos1,5,6 1Department of Neuroscience and Department of Cell Biology, Yale University School of Medicine, 2Developmental Biology Program, Sloan Kettering Institute, 3Department of Genetics and Genome Sciences and Center for Cell Analysis and Modeling, University of Connecticut Health Center, 4Section on High Resolution Optical Imaging, National Institute of Biomedical Imaging and Bioengineering, National Institutes of Health, 5WormGUIDES.org, 6Instituto de Neurobiología, Recinto de Ciencias Médicas, Universidad de Puerto Rico Here, we present a combinatorial approach using high-resolution microscopy, computational tools, and single-cell labeling in living C. elegans embryos to understand single cell dynamics during neurodevelopment. Immunology and Infection Novel Protocol for Generating Physiologic Immunogenic Dendritic Cells Alessandra Ventura*1,2, Aaron Vassall*1, Alp Yurter1, Eve Robinson1, Renata Filler1, Douglas Hanlon1, Katrina Meeth1, Harib Ezaldein1, Michael Girardi1, Olga Sobolev1, Marcus W. Bosenberg1,3, Richard L. Edelson1 1Department of Dermatology, Yale University School of Medicine, 2Dermatology Department, University of Rome Tor Vergata, 3Department of Pathology, Yale University School of Medicine The transimmunization (TI) device or plate and related protocols have been developed to replicate the key features of extracorporeal photochemotherapy (ECP), in an experimental setting, allowing for production of physiologically activated, tunable dendritic cells (DCs) for cancer immunotherapy. Bioengineering Mouse Model of Pressure Ulcers After Spinal Cord Injury Suneel Kumar1, Yuying Tan1, Martin L. Yarmush1,2, Biraja C. Dash3, Henry C. Hsia3, Francois Berthiaume1 1Department of Biomedical Engineering, Rutgers University, 2Center for Engineering in Medicine, Massachusetts General Hospital, Harvard Medical School, Shriners Hospitals for Children, 3Department of Surgery, Yale School of Medicine, Yale University Here, we describe a simple method to induce clinically relevant skin pressure ulcers (PUs) in a mouse model of spinal cord injury (SCI). This model can be used in pre-clinical studies to screen for different therapeutics for healing PUs in SCI patients. Developmental Biology In Vitro Assays to Evaluate the Migration, Invasion, and Proliferation of Immortalized Human First-trimester Trophoblast Cell Lines Edwina P. Kisanga1, Zhonghua Tang1, Seth Guller1, Shannon Whirledge1 1Department of Obstetrics, Gynecology and Reproductive Sciences, Yale School of Medicine Here, we present a highly accessible protocol for evaluating the cell movement in human trophoblast cells using three in vitro assays: the scratch assay, the transwell invasion assay, and the cell proliferation assay. Medicine Predicting Treatment Response to Image-Guided Therapies Using Machine Learning: An Example for Trans-Arterial Treatment of Hepatocellular Carcinoma Aaron Abajian1, Nikitha Murali1, Lynn Jeanette Savic1,2, Fabian Max Laage-Gaupp1, Nariman Nezami1, James S. Duncan3, Todd Schlachter1, MingDe Lin4, Jean-François Geschwind5, Julius Chapiro1 1Department of Radiology and Biomedical Imaging, Yale School of Medicine, 2Department of Diagnostic and Interventional Radiology, Universitätsmedizin Charité Berlin, 3Department of Biomedical Engineering, Yale School of Engineering and Applied Science, 4Philips Research North America, 5Prescience Labs Intra-arterial therapies are the standard of care for patients with hepatocellular carcinoma who cannot undergo surgical resection. A method for predicting response to these therapies is proposed. The technique uses pre-procedural clinical, demographic, and imaging information to train machine learning models capable of predicting response prior to treatment. Genetics Tissue-specific miRNA Expression Profiling in Mouse Heart Sections Using In Situ Hybridization Fani Memi1, Daniela Tirziu2, Irinna Papangeli3 1Department of Cell and Developmental Biology, University College London, 2Yale Cardiovascular Research Group, Section of Cardiovascular Medicine, Yale School of Medicine, 3Yale Cardiovascular Research Center, Section of Cardiovascular Medicine, Yale School of Medicine micro-RNAs (miRNAs) are short and highly homologous RNA sequences, serving as post-transcriptional regulators of messenger RNAs (mRNAs). Current miRNA detection methods vary in sensitivity and specificity. We describe a protocol that combines in situ hybridization and immunostaining for concurrent detection of miRNA and protein molecules on mouse heart tissue sections. Medicine Pre-Conditioning the Airways of Mice with Bleomycin Increases the Efficiency of Orthotopic Lung Cancer Cell Engraftment Laura E. Stevens*1, Anna Arnal-Estapé*1, Don X. Nguyen1,2 1Department of Pathology, Yale University School of Medicine, 2Department of Medical Oncology, Yale University School of Medicine We describe a method to significantly enhance orthotopic engraftment of lung cancer cells into the murine lungs by pre-conditioning the airways with injury. This approach may also be applied to study stromal interactions within the lung microenvironment, metastatic dissemination, lung cancer co-morbidities, and to more efficiently generate patient derived xenografts. Genetics Visualization of Microbiota in Tick Guts by Whole-mount In Situ Hybridization Caitlin E. Moss1, Andrew Robson2, Erol Fikrig3, Sukanya Narasimhan3 1Department of Microbial Pathogenesis, Yale University School of Medicine, 2Program in Vertebrate Developmental Biology, Departments of Pediatrics and Genetics, Yale University School of Medicine, 3Department of Internal Medicine, Yale University School of Medicine Here, we present a protocol to spatially and temporally assess the presence of viable microbiota in tick guts using a modified whole-mount in situ hybridization approach. Neuroscience Cannula Implantation into the Cisterna Magna of Rodents Anna L.R. Xavier1, Natalie Linea Hauglund1, Stephanie von Holstein-Rathlou1, Qianliang Li1, Simon Sanggaard1,2, Nanhong Lou3, Iben Lundgaard3,4, Maiken Nedergaard1,3 1Center for Translational Neuromedicine, Division of Glial Therapeutics, University of Copenhagen, 2Department of Anesthesiology, Yale School of Medicine, 3Center for Translational Neuromedicine, Division of Glial Therapeutics, University of Rochester Medical Center, 4Department of Experimental Medical Science, Wallenberg Center for Molecular Medicine, Lund University Here we describe a protocol to perform cisterna magna cannulation (CMc), a minimally invasive way to deliver tracers, substrates and signaling molecules into the cerebrospinal fluid (CSF). Combined with different imaging modalities, CMc enables glymphatic system and CSF dynamics assessment, as well as brain-wide delivery of various compounds. Developmental Biology Culturing of Retinal Pigment Epithelial Cells on an Ex Vivo Model of Aged Human Bruch's Membrane Hui Cai1, Jie Gong1, Lucian V. Del Priore1, Tongalp H. Tezel2, Mark A. Fields1 1Department of Ophthalmology and Visual Science, Yale School of Medicine, 2Edward S. Harkness Eye Institute, Columbia University School of Medicine The goal of this protocol is to demonstrate the culturing of retinal pigment epithelial (RPE) cells on aged and/or diseased human Bruch's membrane. This method is suitable to study RPE cell behavior on a compromised extracellular matrix. Behavior A Burrowing/Tunneling Assay for Detection of Hypoxia in Drosophila melanogaster Larvae Karen M. Qiang1,2, Fanli Zhou1, Kathleen M. Beckingham1 1Department of Biosciences, Rice University, 2Yale Medical School The protocol describes a simple assay to identify Drosophila melanogaster larvae that are experiencing hypoxia under normal atmospheric oxygen levels. This protocol allows hypoxic larvae to be distinguished from other mutants that show overlapping phenotypes such as sluggishness or slow growth. Cancer Research Characterization of Cell Membrane Extensions and Studying Their Roles in Cancer Cell Adhesion Dynamics Taylor C. Brown1, Norman G. Nicolson2, Joyce Cheng1, Reju Korah1, Tobias Carling1 1Department of Surgery & Yale Endocrine Neoplasia Laboratory, Yale University School of Medicine, 2Yale New Haven Hospital This study demonstrates the utility and ease of quantitative cell membrane extension measurement and its correlation to adhesive capacity of cells. As a representative example, we show here that Dickkopf-related protein 3 (DKK3) promotes increased lobopodia formation and cell adhesiveness in adrenocortical carcinoma cells in vitro. Developmental Biology Using In Vivo and Tissue and Cell Explant Approaches to Study the Morphogenesis and Pathogenesis of the Embryonic and Perinatal Aorta Ashish Misra1, Zhonghui Feng1, Jiasheng Zhang1, Zhi-Yin Lou2,3, Daniel M. Greif1 1Yale Cardiovascular Research Center, Section of Cardiovascular Medicine, Department of Internal Medicine, Yale University School of Medicine, 2Department of Neurology, Yale University School of Medicine, 3Department of Neurology, Xinhua Hospital, Shanghai Jiaotong University School of Medicine Protocols for studying the embryonic and perinatal murine aorta using in vivo clonal analysis and fate mapping, aortic explants, and isolated smooth muscle cells are detailed here. These diverse approaches facilitate the investigation of the morphogenesis of the embryonic and perinatal aorta in normal development and the pathogenesis in disease. Developmental Biology Biosensing Motor Neuron Membrane Potential in Live Zebrafish Embryos Lorena Benedetti1,2,3, Anna Ghilardi4, Laura Prosperi4, Maura Francolini*1, Luca Del Giacco*4 1Department of Medical Biotechnology and Translational Medicine, Università degli Studi di Milano, 2Department of Neuroscience; Department of Cell Biology, Howard Hughes Medical Institute, Yale University School of Medicine, 3Program in Cellular Neuroscience, Neurodegeneration and Repair, Yale University School of Medicine, 4Department of BioSciences, Università degli Studi di Milano Protocols described here allow for the study of the electrical properties of excitable cells in the most non-invasive physiological conditions by employing zebrafish embryos in an in vivo system together with a fluorescence resonance energy transfer (FRET)-based genetically encoded voltage indicator (GEVI) selectively expressed in the cell type of interest. Neuroscience SNARE-mediated Fusion of Single Proteoliposomes with Tethered Supported Bilayers in a Microfluidic Flow Cell Monitored by Polarized TIRF Microscopy Joerg Nikolaus1,2, Erdem Karatekin1,2,3,4 1Department of Cellular and Molecular Physiology, Yale University School of Medicine, 2Nanobiology Institute, Yale University, 3Department of Molecular Biophysics and Biochemistry, Yale University, 4Laboratoire de Neurophotonique, Université Paris Descartes, Faculté des Sciences Fondamentales et Biomédicales, Centre National de la Recherche Scientifique (CNRS) Here, we present a protocol to detect single, SNARE-mediated fusion events between liposomes and supported bilayers in microfluidic channels using polarized TIRFM, with single molecule sensitivity and ~15 msec time resolution. Lipid and soluble cargo release can be detected simultaneously. Liposome size, lipid diffusivity, and fusion pore properties are measured. Medicine A Phenotyping Regimen for Genetically Modified Mice Used to Study Genes Implicated in Human Diseases of Aging Victoria L. Patterson*1, Brian S. Thompson*1, Catherine Cherry*1, Shao-bin Wang2, Bo Chen2, Josephine Hoh1,2 1Department of Environmental Health Sciences, Yale University School of Medicine, 2Department of Ophthalmology, Yale University School of Medicine A reverse-genetics approach to understanding gene families associated with human disease is presented, using mouse as a model system, and the subsequent mouse phenotyping schedule is described. Because mice defective in a gene of interest, HtrA2, manifested Parkinsonian symptoms, the phenotyping regimen is focused on identifying neurological defects. Immunology and Infection Intravital Imaging of Neutrophil Priming Using IL-1β Promoter-driven DsRed Reporter Mice Yi Yao1,2, Yun Liu1,3, Akira Takashima1 1Department of Medical Microbiology and Immunology, University of Toledo College of Medicine and Life Sciences, 2Department of Internal Medicine, Yale University School of Medicine, 3Department of Pathophysiology, Southern Medical University (China) This current protocol employs fluorescent reporters, in vivo labeling, and intravital imaging techniques to enable monitoring of the dynamic process of neutrophil priming in living animals. Developmental Biology Isolation of Murine Embryonic Hemogenic Endothelial Cells Jennifer S. Fang*1, Emily C. Gritz*2, Kathrina L. Marcelo3, Karen K. Hirschi1 1Departments of Medicine, Genetics and Biomedical Engineering, Yale Cardiovascular Research Center, Vascular Biology and Therapeutics Program, Yale Stem Cell Center, Yale University School of Medicine, 2Department of Pediatrics, Section of Neonatal-Perinatal Medicine, Yale University School of Medicine, 3Department of Molecular and Cellular Biology, Baylor College of Medicine Hematopoietic stem and progenitor cells (HSPC) derive from specialized (hemogenic) endothelial cells during development, yet little is known about the process by which some endothelial cells specify to become blood forming. We demonstrate a flow-cytometry based method allowing simultaneous isolation of hemogenic endothelial cells and HSPC from murine embryonic tissues. Biology DamID-seq: Genome-wide Mapping of Protein-DNA Interactions by High Throughput Sequencing of Adenine-methylated DNA Fragments Feinan Wu1, Brennan G. Olson1, Jie Yao1 1Department of Cell Biology, Yale University School of Medicine We describe herein an assay by coupling DNA adenine methyltransferase identification (DamID) to high throughput sequencing (DamID-seq). This improved method provides a higher resolution and a wider dynamic range, and allows analyzing DamID-seq data in conjunction with other high throughput sequencing data such as ChIP-seq, RNA-seq, etc. Behavior Examination of Rapid Dopamine Dynamics with Fast Scan Cyclic Voltammetry During Intra-oral Tastant Administration in Awake Rats Robert J. Wickham1, Jinwoo Park2, Eric J. Nunes3, Nii A. Addy1,3,4 1Interdepartmental Neuroscience Program, Yale University, 2Department of Biotechnical and Clinical Laboratory Sciences, School of Medicine and Biomedical Sciences, University at Buffalo, 3Department of Psychiatry, Yale School of Medicine, 4Department of Cellular and Molecular Physiology, Yale School of Medicine Rapid fluctuations in extracellular dopamine (DA) mediate both reward processing and motivated behavior in mammals. This manuscript describes the combined use of fast scan cyclic voltammetry (FSCV) and intra-oral tastant administration to determine how tastants alter rapid dopamine release in awake, freely moving rats. Neuroscience Long-term Continuous EEG Monitoring in Small Rodent Models of Human Disease Using the Epoch Wireless Transmitter System Andrew Zayachkivsky1, Mark J. Lehmkuhle2, F. Edward Dudek2 1Department of Neurosurgery, Yale University School of Medicine, 2Department of Neurosurgery, University of Utah Here we demonstrate the use of a wireless enabling technology for electroencephalogram (EEG) in neonatal rodent models of human disease. With telemetry, there are no encumbering connections, thus allowing natural behaviors. Behavior fMRI Validation of fNIRS Measurements During a Naturalistic Task J. Adam Noah1, Yumie Ono2, Yasunori Nomoto2, Sotaro Shimada2, Atsumichi Tachibana3, Xian Zhang1, Shaw Bronner1, Joy Hirsch1,5 1Department of Psychiatry, Yale School of Medicine, 2Department of Electronics and Bioinformatics, Meiji University, 3Department of Histology and Neurobiology, Dokkyo Medical University School of Medicine, 4ADAM Center, Department of Physical Therapy, Movement and Rehabilitation Sciences, Northeastern University, 5Department of Neurobiology, Yale School of Medicine We present a method to compare functional brain activity recorded during a naturalistic task using fNIRS with activity recorded during fMRI. Neuroscience Isolation, Culture and Long-Term Maintenance of Primary Mesencephalic Dopaminergic Neurons From Embryonic Rodent Brains Maria Weinert1, Tharakeswari Selvakumar2, Travis S. Tierney2, Kambiz N. Alavian1,3 1Division of Brain Sciences, Department of Medicine, Imperial College London, 2 The causes of degeneration of midbrain dopaminergic neurons during Parkinson’s disease are not fully understood. Cellular culture systems provide an essential tool for study of the neurophysiological properties of these neurons. Here we present an optimized protocol, which can be utilized for in vitro modeling of neurodegeneration. Medicine Murine Model for Non-invasive Imaging to Detect and Monitor Ovarian Cancer Recurrence Natalia J. Sumi1, Eydis Lima1, John Pizzonia2, Sean P. Orton3, Vinicius Craveiro1, Wonduk Joo1, Jennie C. Holmberg1, Marta Gurrea1, Yang Yang-Hartwich1, Ayesha Alvero1, Gil Mor1 1Department of Obstetrics, Gynecology and Reproductive Sciences, Reproductive Immunology Unit, Yale University School of Medicine, 2NatureMost Laboratories, 3Bruker Preclinical Imaging We describe a non-invasive animal imaging platform that allows the detection, quantification, and monitoring of ovarian cancer growth and recurrence. This intra-peritoneal xenograft model mimics the clinical profile of patients with ovarian cancer. Biology Generation of Transgenic Hydra by Embryo Microinjection Celina E. Juliano1, Haifan Lin1, Robert E. Steele2 1Yale Stem Cell Center and Department of Cell Biology, Yale University School of Medicine, 2Department of Biological Chemistry and the Developmental Biology Center, University of California, Irvine Stably transgenic Hydra are made by microinjection of plasmid DNA into embryos followed by random genomic integration and asexual propagation to establish a uniform line. Transgenic Hydra are used to track cell movements, overexpress genes, study promoter function, or knock down gene expression using RNAi. Neuroscience Organotypic Slice Cultures to Study Oligodendrocyte Dynamics and Myelination Robert A. Hill1,3, Jelena Medved1, Kiran D. Patel1, Akiko Nishiyama1,2 1Department of Physiology and Neurobiology, University of Connecticut, 2Stem Cell Institute, University of Connecticut, 3Department of Neurology, Yale University School of Medicine A technique to study NG2 cells and oligodendrocytes using a slice culture system of the forebrain and cerebellum is described. This method allows examination of the dynamics of proliferation and differentiation of cells within the oligodendrocyte lineage where the extracellular environment can be easily manipulated while maintaining tissue cytoarchitecture. Bioengineering Use of Label-free Optical Biosensors to Detect Modulation of Potassium Channels by G-protein Coupled Receptors Matthew R. Fleming1, Steven M. Shamah3, Leonard K. Kaczmarek1,2 1Department of Pharmacology, Yale School of Medicine, 2Department of Cellular and Molecular Physiology, Yale School of Medicine, 3X-BODY Biosciences Optical biosensor techniques can detect changes in mass near the plasma membrane in living cells and allow one to follow cellular responses in both individual cells and populations of cells. This protocol will describe detection of the modulation of potassium channels by G-protein coupled receptors in intact cells using this approach. Neuroscience Production of RNA for Transcriptomic Analysis from Mouse Spinal Cord Motor Neuron Cell Bodies by Laser Capture Microdissection Urmi Bandyopadhyay1,2, Wayne A. Fenton1, Arthur L. Horwich1,2, Maria Nagy1,2 1Department of Genetics, Yale School of Medicine, 2Howard Hughes Medical Institute High-quality total RNA has been prepared from cell bodies of mouse spinal cord motor neurons by laser capture microdissection after staining spinal cord sections with Azure B in 70% ethanol. Sufficient RNA (~40-60 ng) is recovered from 3,000-4,000 motor neurons to allow downstream RNA analysis by RNA-seq and qRT-PCR.
