National Institute of Environmental Health and Sciences 12 articles published in JoVE Biochemistry Preparation of Nucleosome Core Particles Complexed with DNA Repair Factors for Cryo-Electron Microscopy Structural Determination Yesenia Rodriguez1, Kevin John Butay*1, Kedar Sharma*1, Elizabeth Viverette*1, Samuel H. Wilson1 1Genome Integrity and Structural Biology Laboratory, National Institute of Environmental Health Sciences, National Institutes of Health This protocol outlines in detail the preparation of nucleosomal complexes using two methods of sample preparation for freezing TEM grids. Biochemistry Removal and Replacement of Endogenous Ligands from Lipid-Bound Proteins and Allergens Alexander C. Y. Foo1, Peter M. Thompson1, Geoffrey A. Mueller1 1Nuclear Magnetic Resonance Group, National Institute of Environmental Health Sciences This protocol describes the removal of endogenous lipids from allergens, and their replacement with user-specified ligands through reverse-phase HPLC coupled with thermal annealing. 31P-NMR and circular dichroism allow for the rapid confirmation of ligand removal/loading, and the recovery of native allergen structure. Genetics Use of Frozen Tissue in the Comet Assay for the Evaluation of DNA Damage Cheryl A. Hobbs*1, Leslie Recio1, John Winters1, Kristine L. Witt2 1Toxicology Program, ILS, Inc., 2Division of the National Toxicology Program, National Institute of Environmental Health Sciences This protocol describes several procedures for preparing high quality frozen tissue samples at the time of necropsy for use in the comet assay to assess DNA damage: 1) minced tissue, 2) scraped epithelial cells from the gastrointestinal tract, and 3) cubed tissue samples, requiring homogenization using a tissue mincing device. Immunology and Infection In Vivo Assessment of Alveolar Macrophage Efferocytosis Following Ozone Exposure Myles X. Hodge1, Sky W. Reece1, Jennifer H. Madenspacher2, Kymberly M. Gowdy1 1Department of Pharmacology and Toxicology, East Carolina University, 2Research Triangle Park, National Institute of Environmental Health and Sciences This manuscript describes a protocol for determining whether exposure to ozone, a criteria air pollutant, impairs alveolar macrophage efferocytosis in vivo. This protocol utilizes commonly used reagents and techniques and can be adapted to multiple models of pulmonary injury to determine effects on alveolar macrophage efferocytosis. Biology Laser-assisted Lentiviral Gene Delivery to Mouse Fertilized Eggs Negin P. Martin*1, Page Myers*2, Eugenia Goulding1, Shih-Heng Chen1, Mitzie Walker1, Thomas M. Porter1, Lucas Van Gorder1, Amanda Mathew1, Artiom Gruzdev3, Erica Scappini4, Charles Romeo1 1Neurobiology Laboratory, National Institute of Environmental Health Sciences, 2Comparative Medicine Branch, National Institute of Environmental Health Sciences, 3Reproductive and Developmental Biology Laboratory, National Institute of Environmental Health Sciences, 4Signal Transduction Laboratory, National Institute of Environmental Health Sciences Mouse fertilized eggs and early stage embryos are protected by the zona pellucida, a glycoprotein matrix that forms a barrier against gene delivery. This article describes a protocol for perforating the zona with a laser to transduce embryonic cells with lentiviral vectors and to create transgenic mice. Biology Studying Ribonucleotide Incorporation: Strand-specific Detection of Ribonucleotides in the Yeast Genome and Measuring Ribonucleotide-induced Mutagenesis Zhi-Xiong Zhou*1, Jessica S. Williams*1, Thomas A. Kunkel1 1Genome Integrity and Structural Biology Laboratory, National Institute of Environmental Health Sciences, NIH, DHHS Ribonucleotides are among the most abundant non-canonical nucleotides incorporated into the genome during eukaryotic nuclear DNA replication. If not properly removed, ribonucleotides can cause DNA damage and mutagenesis. Here, we present two experimental approaches that are used to assess the abundance of ribonucleotide incorporation into DNA and its mutagenic effects. Biology Laser Capture Microdissection of Highly Pure Trabecular Meshwork from Mouse Eyes for Gene Expression Analysis Caleb Sutherland*1, Yu Wang*2, Robert V. Brown*1, Julie Foley2, Beth Mahler2, Kyathanahalli S. Janardhan2,3, Ramesh C. Kovi2,4, Anton M. Jetten1 1Immunity, Inflammation, and Disease Laboratory, Division of Intramural Research, National Institute of Environmental Health Sciences, NIH, 2Cellular and Molecular Pathology Branch, Division of the National Toxicology Program, National Institute of Environmental Health Sciences, NIH, 3Integrated Laboratory Systems Inc., 4Experimental Pathology Laboratories Inc. Here, we describe a protocol for a reproducible laser capture microdissection (LCM) for isolating trabecular meshwork (TM) for downstream RNA analysis. The ability to analyze changes in gene expression in the TM will help in understanding the underlying molecular mechanisms of TM-related ocular diseases. Biology Trans-inner Cell Mass Injection of Embryonic Stem Cells Leads to Higher Chimerism Rates Gregory J. Scott1, Artiom Gruzdev1, Thomas B. Hagler1, Manas K. Ray1 1Knock Out Mouse Core, NIEHS/NIH Here we present a protocol to increase chimera production without the use of new equipment. A simple orientation change of the embryo for injection can increase the number of embryos produced, and potentially reduce the timeline to germline transmission. Developmental Biology A Novel Use of Three-dimensional High-frequency Ultrasonography for Early Pregnancy Characterization in the Mouse Mary C. Peavey1,4, Corey L. Reynolds2, Maria M. Szwarc2, William E. Gibbons1, Cecilia T. Valdes1, Francesco J. DeMayo*3, John P. Lydon*4 1Devision of Repreoductive Endocrinology and Infertility, Department of Obstetrics and Gynecology, Baylor College of Medicine, 2Mouse Phenotyping Core, Baylor College of Medicine, 3Reproductive and Developmental Biology Laboratory, National Institute of Environmental Health Sciences, 4Department of Molecular and Cellular Biology, Baylor College of Medicine Mice are widely used to study gestational biology. However, pregnancy termination is required for such studies which precludes longitudinal investigations and necessitates the use of large numbers of animals. Therefore, we describe a non-invasive technique of high-frequency ultrasonography for early detection and monitoring of post-implantation events in the pregnant mouse. Cancer Research Sectioning Mammary Gland Whole Mounts for Lesion Identification Deirdre K Tucker1,2, Julie F Foley3, Schantel A Bouknight4, Suzanne E Fenton2 1Curriculum in Toxicology, University of North Carolina at Chapel Hill, 2National Toxicology Program Laboratory (NTPL), DNTP, National Institute of Environmental Health Sciences, 3Cellular and Molecular Pathology Branch, DNTP, National Institute of Environmental Health Sciences, 4Charles River Laboratories Inc. We developed a method to successfully remove, process, section, and stain, for histopathological evaluation, mammary tissue that had originally been fixed on slides as whole mounts. This method may promote the collection and evaluation of mammary gland whole mounts in reproductive and developmental test guideline studies. Immunology and Infection Precision-cut Mouse Lung Slices to Visualize Live Pulmonary Dendritic Cells Miranda R. Lyons-Cohen1, Seddon Y. Thomas1, Donald N. Cook1, Hideki Nakano1 1Immunity, Inflammation, and Disease Laboratory, Division of Intramural Research, National Institute of Environmental Health Sciences, NIH We describe a method for generating Precision-cut Lung Slices (PCLS) and immunostaining them to visualize the localization of various immune cell types in the lung. Our protocol can be extended to visualize the location and function of many different cell types under a variety of conditions. Immunology and Infection Using Zebrafish Models of Human Influenza A Virus Infections to Screen Antiviral Drugs and Characterize Host Immune Cell Responses Con Sullivan1,2, Denise Jurcyzszak1, Michelle F. Goody3, Kristin A. Gabor4, Jacob R. Longfellow1, Paul J. Millard2,5, Carol H. Kim1,2 1Department of Molecular and Biomedical Sciences, University of Maine, 2Graduate School of Biomedical Sciences and Engineering, University of Maine, 3School of Biology and Ecology, University of Maine, 4Division of Intramural Research, Immunity, Inflammation and Disease Laboratory, National Institute of Environmental Health Sciences, NIH, 5Department of Chemical and Biological Engineering, University of Maine Systemic and localized zebrafish infection models for human influenza A virus are demonstrated. Using a systemic infection model, zebrafish can be used to screen antiviral drugs. Using a localized infection model, zebrafish can be used to characterize host immune cell responses.