University of Massachusetts Medical School View Institution's Website 25 articles published in JoVE Genetics Capturing Chromosome Conformation Across Length Scales Liyan Yang1, Betul Akgol Oksuz1, Job Dekker1,2, Johan Harmen Gibcus1 1Department of Systems Biology, University of Massachusetts Medical School, 2Howard Hughes Medical Institute Hi-C 3.0 is an improved Hi-C protocol that combines formaldehyde and disuccinimidyl glutarate crosslinkers with a cocktail of DpnII and DdeI restriction enzymes to increase the signal-to-noise ratio and the resolution of chromatin interaction detection. Neuroscience Single-Cell Electroporation across Different Organotypic Slice Culture of Mouse Hippocampal Excitatory and Class-Specific Inhibitory Neurons David G. Keener*1,2, Amy Cheung*1,3, Kensuke Futai1 1Brudnick Neuropsychiatric Research Institute, Department of Neurobiology, University of Massachusetts Medical School, 2Interdisciplinary Graduate Program, University of Massachusetts Medical School, 3UMMS MD/PhD Program, University of Massachusetts Medical School Presented here is a protocol for single-cell electroporation that can deliver genes in both excitatory and inhibitory neurons across a range of in vitro hippocampal slice culture ages. Our approach provides precise and efficient expression of genes in individual cells, which can be used to examine cell-autonomous and intercellular functions. Medicine Sucrose Preference and Novelty-Induced Hypophagia Tests in Rats using an Automated Food Intake Monitoring System Martha Anna Schalla*1, Stephanie Gladys Kühne*1, Tiemo Friedrich1, Vivien Hanel1, Peter Kobelt1, Miriam Goebel-Stengel1,2,3, Matthias Rose1,4, Andreas Stengel1,3 1Charité Center for Internal Medicine and Dermatology, Department for Psychosomatic Medicine, Charité-Universitätsmedizin, Corporate Member of Freie Universität Berlin, Humboldt-Universität zu Berlin and Berlin Institute of Health, 2Department of Internal Medicine, Helios Clinic, 3Department of Psychosomatic Medicine and Psychotherapy, University Hospital Tübingen, 4Department of Quantitative Health Sciences, University of Massachusetts Medical School Presented here is a protocol to study depression-like and anhedonic behavior in rats. It combines two well-established behavioral methods, the sucrose preference and novelty-induced hypophagia tests, with an automated food and liquid intake monitoring system, to indirectly investigate rodent behavior using surrogate parameters. Biochemistry Atomic Absorbance Spectroscopy to Measure Intracellular Zinc Pools in Mammalian Cells Shellaina J.V. Gordon*1, Yao Xiao*2, Amanda L. Paskavitz3, Napoleón Navarro-Tito4, Juan G. Navea2, Teresita Padilla-Benavides1 1Department of Biochemistry and Molecular Pharmacology, University of Massachusetts Medical School, 2Department of Chemistry, Skidmore College, 3Candiac MR Center, Beth Israel Deaconess Medical Center, 4Facultad de Ciencias Químico Biológicas, Universidad Autónoma de Guerrero Cultured primary or established cell lines are commonly used to address fundamental biological and mechanistic questions as an initial approach before using animal models. This protocol describes how to prepare whole cell extracts and subcellular fractions for studies of zinc (Zn) and other trace elements with atomic absorbance spectroscopy. Immunology and Infection Production of Pseudotyped Particles to Study Highly Pathogenic Coronaviruses in a Biosafety Level 2 Setting Jean K. Millet1,2, Tiffany Tang3, Lakshmi Nathan3, Javier A. Jaimes4, Hung-Lun Hsu3,5, Susan Daniel3, Gary R. Whittaker1 1Department of Microbiology and Immunology, College of Veterinary Medicine, Cornell University, 2INRA, Virologie et Immunologie Moléculaires, 3Robert Frederick Smith School of Chemical and Biomolecular Engineering, Cornell University, 4Department of Microbiology, College of Agricultural and Life Sciences, Cornell University, 5Horae Gene Therapy Center, University of Massachusetts Medical School Here, we present a protocol to generate pseudotyped particles in a BSL-2 setting incorporating the spike protein of the highly pathogenic viruses Middle East respiratory syndrome and severe acute respiratory syndrome coronaviruses. These pseudotyped particles contain a luciferase reporter gene allowing quantification of virus entry into target host cells. Neuroscience Direct Intrathecal Injection of Recombinant Adeno-associated Viruses in Adult Mice Dongxiao Li*1, Yundu Li*2, Yunyun Tian1, Zuoshang Xu3, Yansu Guo1,4 1Department of Neurology, Second Hospital of Hebei Medical University, 2No.2 Middle School of Shijiazhuang, 3Department of Biochemistry and Molecular Pharmacology, University of Massachusetts Medical School, 4Key Laboratory of Hebei Neurology Here we present a direct intrathecal injection technique using 1% lidocaine hydrochloride in a viral solution to ensure efficient adeno-associated virus delivery to small animals and establish a scoring system to predict transduction efficiency in the central nervous system according to the degree of transient weakness induced by lidocaine. Biology Whole Cell Electrophysiology of Primary Cultured Murine Enterochromaffin Cells Katilyn Knutson*1, Peter R. Strege*1, Joyce Li2, Andrew B. Leiter2, Gianrico Farrugia1, Arthur Beyder1 1Enteric Neuroscience Program, Division of Gastroenterology & Hepatology,Department of Physiology & Biomedical Engineering, Mayo Clinic, 2Division of Gastroenterology, Department of Medicine, University of Massachusetts Medical School Enterochromaffin (EC) cells comprise a small subset of gastrointestinal epithelial cells. EC cells are electrically excitable and release serotonin, yet difficulties in culturing and identifying EC cells have limited physiological studies. The method presented here establishes a primary culture model amenable to examination of single EC cells by electrophysiology. Biology Pancreatic Islet Embedding for Paraffin Sections Yahui Kong1, Pantea Ebrahimpour1,2, Yu Liu3, Chaoxing Yang1, Laura C. Alonso1 1Diabetes Center of Excellence, UMass Medical School, 2Department of Medicine, Saint Vincent Hospital, 3Department of Pathology, Morphology Core, UMass Medical School Ex vivo pancreatic islet studies are important for diabetes research. Existing techniques to study cultured islets in their native 3-dimensional architecture are time consuming, inefficient, and infrequently used. This work describes a new, simple, and efficient method for generating high-quality paraffin sections of whole cultured islets. Biochemistry Horizontal Gel Electrophoresis for Enhanced Detection of Protein-RNA Complexes Megan E. Dowdle1, Susanne Blaser Imboden1, Sookhee Park1, Sean P. Ryder2, Michael D. Sheets1 1Department of Biomolecular Chemistry, University of Wisconsin School of Medicine and Public Health, 2Department of Biochemistry and Molecular Pharmacology, University of Massachusetts Medical School Native polyacrylamide gel electrophoresis is a fundamental tool for analyzing RNA-protein interactions. Traditionally most experiments have used vertical gels. However, horizontal gels provide several advantages, such as the opportunity to monitor complexes during electrophoresis. We provide a detailed protocol for generating and using horizontal native gel electrophoresis. Developmental Biology Ploidy Manipulation of Zebrafish Embryos with Heat Shock 2 Treatment Destiny L. Baars1, Kendra A. Takle*1,2, Jonathon Heier*1,3, Francisco Pelegri1 1Laboratory of Genetics, University of Wisconsin, 2Department of Neurobiology, University of Massachusetts Medical School, 3Interdisciplinary Biomedical Graduate Program, University of Pittsburgh School of Medicine A modified protocol for ploidy manipulation uses a heat shock to induce a one-cycle stall in cytokinesis in the early embryo. This protocol is demonstrated in the zebrafish but may be applicable to other species. Neuroscience A Behavioral Assay for Mechanosensation of MARCM-based Clones in Drosophila melanogaster Timothy P. Murphy1,2, Dan D. Luu1, Jessica A. DeSimone1,3, Thomas C. O'Brien1,4, Christopher J. Lally1,5, Jillian J. Lindblad1,6, Sarah M. Webster1 1Department of Biology, College of the Holy Cross, 2School of Medicine, Georgetown University, 3Department of Biochemistry, Giesel School of Medicine, Dartmouth College, 4School of Medicine, Tufts University, 5Transgenomic Inc., 6Department of Molecular, Cell and Cancer Biology, UMass Medical School In order to identify novel mutations affecting mechanosensation, we designed an assay that measures the behavioral response to tactile stimulation of fly bristles in mutant clones generated by the MARCM method. The combination of techniques allows for the identification of mechanosensitive mutations that would otherwise be lethal. Biology Determination of Fatty Acid Oxidation and Lipogenesis in Mouse Primary Hepatocytes Thomas E. Akie1, Marcus P. Cooper1 1Division of Cardiovascular Medicine, Department of Medicine, University of Massachusetts Medical School De novo lipogenesis and β-fatty acid oxidation constitute key metabolic pathways in hepatocyte, pathways that are perturbed in several metabolic disorders, including fatty liver disease. Here we demonstrate isolation of mouse primary hepatocytes and describe quantification of β-fatty acid oxidation and lipogenesis. Medicine Utilization of the Soft Agar Colony Formation Assay to Identify Inhibitors of Tumorigenicity in Breast Cancer Cells Sachi Horibata1, Tommy V. Vo2, Venkataraman Subramanian3, Paul R. Thompson3, Scott A. Coonrod1 1Department of Baker Institute for Animal Health, Cornell University, 2Department of Molecular Biology and Genetics, Cornell University, 3Department of Biochemistry and Molecular Pharmacology, University of Massachusetts Medical School Here, we document the use of the soft agar colony formation assay to test the effects of a peptidylarginine deiminase (PADI) enzyme inhibitor, BB-Cl-amidine, on breast cancer tumorigenicity in vitro. Neuroscience Brain Slice Biotinylation: An Ex Vivo Approach to Measure Region-specific Plasma Membrane Protein Trafficking in Adult Neurons Luke R. Gabriel1, Sijia Wu1, Haley E. Melikian2 1Program in Neuroscience, Graduate School of Biomedical Sciences, University of Massachusetts Medical School, 2Brudnick Neuropsychiatric Research Institute, Department of Psychiatry, University of Massachusetts Medical School Neuronal membrane trafficking dynamically controls plasma membrane protein availability and significantly impacts neurotransmission. To date, it has been challenging to measure neuronal endocytic trafficking in adult neurons. Here, we describe a highly effective, quantitative method to measure rapid changes in surface protein expression ex vivo in acute brain slices. Medicine Screening for Melanoma Modifiers using a Zebrafish Autochthonous Tumor Model Sharanya Iyengar1, Yariv Houvras2,3, Craig J. Ceol1 1Program in Molecular Medicine and Department of Cancer Biology, University of Massachusetts Medical School, 2Departments of Surgery and Medicine, Weill Cornell Medical College, 3Departments of Surgery and Medicine, New York Presbyterian Hospital A rapid way to screen for melanoma modifiers using a zebrafish autochthonous tumor model is presented. It takes advantage of the miniCoopR vector which allows for expression of candidate melanoma genes in melanocytes. A method to obtain melanoma-free survival curves, an invasion assay, a protocol for antibody staining of scale melanocytes and a melanoma transplantation assay are described. Neuroscience Simultaneous Recording of Calcium Signals from Identified Neurons and Feeding Behavior of Drosophila melanogaster Motojiro Yoshihara1 1Department of Neurobiology, University of Massachusetts Medical School The fruit fly, Drosophila melanogaster, extends its proboscis for feeding, responding to a sugar stimulus from its proboscis or tarsus. I have combined observations of the proboscis extension response (PER) with a calcium imaging technique, allowing us to monitor the activity of neurons in the brain, simultaneously with behavioral observation. Biology Identification and Analysis of Mouse Erythroid Progenitors using the CD71/TER119 Flow-cytometric Assay Miroslav Koulnis*1, Ramona Pop*1, Ermelinda Porpiglia*1, Jeffrey R. Shearstone*1, Daniel Hidalgo1, Merav Socolovsky1 1Department of Pediatrics and Department of Cancer Biology, University of Massachusetts Medical School A flow-cytometric method for identification and molecular analysis of differentiation-stage-specific murine erythroid progenitors and precursors, directly in freshly –harvested mouse bone marrow, spleen or fetal liver. The assay relies on cell-surface markers CD71, Ter119, and cell size. Medicine Quantitative Autonomic Testing Peter Novak1 1Department of Neurology, University of Massachusetts Medical School Standardized, comprehensive and fully quantitative testing of autonomic functions is described. The autonomic tests consist of evaluation of all three major autonomic domains including cardiovagal, adrenergic and sudomotor. The severity and distribution of dysautonomia is quantitated using Composite Autonomic Severity Scores. Biology Isolation of Drosophila melanogaster Testes Phillip D. Zamore1, Shengmei Ma1 1Department of Biochemistry & Molecular Pharmacology and Howard Hughes Medical Institute, University of Massachusetts Medical School Drosophila melanogaster testes can be rapidly and efficiently isolated from adult males using dissecting needles. With practice, one can readily isolate in one or two days an amount of testes sufficient for the analysis of DNA or RNA by high throughput sequencing or more traditional molecular biology methods or of protein for antibody- or enzyme-based assays. Biology Chromatin Immunoprecipitation Assay for Tissue-specific Genes using Early-stage Mouse Embryos Ok Hyun Cho1, Jaime A. Rivera-Pérez1, Anthony N. Imbalzano1 1Department of Cell Biology, University of Massachusetts Medical School We demonstrate a chromatin immunoprecipitation (ChIP) method to identify factor interactions at tissue-specific genes during or after the onset of tissue-specific gene expression in mouse embryonic tissue. This protocol should be widely applicable for the study of tissue-specific gene activation as it occurs during normal embryonic development. Biology Isolation and Culture of Adult Epithelial Stem Cells from Human Skin Zhiru Guo1, Kyle Draheim1, Stephen Lyle1 1Department of Cancer Biology, University of Massachusetts Medical School A rapid, robust way of isolating viable adult epithelial stem cells from human skin is described. The method utilizes enzymatic digestion of skin collagen matrix , followed by plucking of hair follicles and isolation of single cell suspensions or tissue fragments for cell culture. Biology Hi-C: A Method to Study the Three-dimensional Architecture of Genomes. Nynke L. van Berkum*1, Erez Lieberman-Aiden*2,3,4,5, Louise Williams*2, Maxim Imakaev6, Andreas Gnirke2, Leonid A. Mirny3,6, Job Dekker1, Eric S. Lander2,7,8 1Program in Gene Function and Expression, Department of Biochemistry and Molecular Pharmacology, University of Massachusetts Medical School, 2Broad Institute of Harvard and Massachusetts Institute of Technology, 3Division of Health Sciences and Technology, Massachusetts Institute of Technology, 4Program for Evolutionary Dynamics, Department of Organismic and Evolutionary Biology, Department of Mathematics, Harvard University, 5Department of Applied Mathematics, Harvard University, 6Department of Physics, Massachusetts Institute of Technology, 7Department of Systems Biology, Harvard Medical School, 8Department of Biology, Massachusetts Institute of Technology The Hi-C method allows unbiased, genome-wide identification of chromatin interactions (1). Hi-C couples proximity ligation and massively parallel sequencing. The resulting data can be used to study genomic architecture at multiple scales: initial results identified features such as chromosome territories, segregation of open and closed chromatin, and chromatin structure at the megabase scale. Biology Measuring Plasma Membrane Protein Endocytic Rates by Reversible Biotinylation Luke Gabriel1, Zachary Stevens1, Haley Melikian1 1University of Massachusetts Medical School Regulated endocytosis governs the cell surface expression levels of the majority of membrane proteins. Here we utilize reducible, membrane impermeant biotinylation reagents to measure the endocytic rate of the dopamine transporter (DAT), a polytopic membrane protein. The method facilitates a straightforward approach to measuring the endocytic rate of most plasma membrane proteins. Biology Neuronal Nuclei Isolation from Human Postmortem Brain Tissue Anouch Matevossian1, Schahram Akbarian1 1Psychiatry, Brudnick Neuropsychiatric Research Institute, University of Massachusetts Medical School The cellular heterogeneity of brain tissue poses a significant limitation for the study of epigenetic markings in chromatin because most assays lack single cell resolution. Neurons typically are intermingled with glia and other non-neuronal cells. We provide a protocol to extract and collect neuronal nuclei from human brain. Biology A Chromatin Assay for Human Brain Tissue Anouch Matevossian1, Schahram Akbarian1 1Psychiatry, Brudnick Neuropsychiatric Research Institute, University of Massachusetts Medical School Until recently, expression studies on human brain were limited to quantification of RNA or protein. With the chromatin immunoprecipitation techniques described in this paper, it will be possible to map histone methylation and other epigenetic regulators of gene expression in postmortem brain.