University of Maryland, College Park View Institution's Website 13 articles published in JoVE Developmental Biology In Ovo Intravascular Injection in Chicken Embryos Kai Jin1,2, Jing Zhou1,2, Gaoyuan Wu1,2, Ziyi Lian1,2, Zongyi Zhao1,2, Shujian Zhou1,2, Chen Chen1,2, Hongyan Sun1,2, Yingjie Niu1,2, Qishenng Zuo1,2, Yani Zhang1,2, Jiuzhou Song3, Guohong Chen1,2, Bichun Li1,2,4 1Joint International Research Laboratory of Agriculture and Agri-Product Safety of Ministry of Education of China, Yangzhou University, 2Key Laboratory of Animal Breeding Reproduction and Molecular Design for Jiangsu Province, College of Animal Science and Technology, Yangzhou University, 3Animal & Avian Sciences, University of Maryland, College Park, 4College of Biotechnology, Jiangsu University of Science and Technology The overall goal of this paper is to describe how to perform in ovo intracellular injection of exogenous materials into chicken embryos. This approach is very useful to study the developmental biology of chicken embryos. Biology An Easy and Flexible Inoculation Method for Accurately Assessing Powdery Mildew-Infection Phenotypes of Arabidopsis and Other Plants Ying Wu*1, Darwin Diaz*1, Jian Yin1, David Bloodgood1, William Sexton1, Cheng-I Wei2, Shunyuan Xiao1,3 1Institute for Bioscience and Biotechnology Research, University of Maryland, 2Department of Nutrition and Food Science, University of Maryland College Park, 3Department of Plant Sciences and Landscape Architecture, University of Maryland College Park We present a protocol for constructing a simple spore-distribution system consisting of an inoculation box with a ~50 µm mesh and a transparent plastic chamber. This can be used to evenly inoculate plants with powdery mildew spores, thereby enabling accurate and reproducible assessment of disease phenotypes of plants under study. Developmental Biology Development of Targeting Induced Local Lesions IN Genomes (TILLING) Populations in Small Grain Crops by Ethyl Methanesulfonate Mutagenesis Lovepreet Singh1, Adam Schoen1, Alexander Mahlandt1, Bhavit Chhabra1, James Steadham1, Vijay Tiwari1, Nidhi Rawat1 1Department of Plant Science and Landscape Architecture, University of Maryland, College Park Described is a protocol for developing a Targeting Induced Local Lesions IN Genomes (TILLING) population in small grain crops with use of ethyl methanesulfonate (EMS) as a mutagen. Also provided is a protocol for mutation detection using the Cel-1 assay. Engineering Cutting Procedures, Tensile Testing, and Ageing of Flexible Unidirectional Composite Laminates Amy Engelbrecht-Wiggans1,2, Ajay Krishnamurthy1,2, Faraz Burni1,3, William Osborn1, Amanda L. Forster1 1Material Measurement Laboratory, National Institute of Standards and Technology, 2Theiss Research, 3Chemical and Biomolecular Engineering Department, University of Maryland The goal of the study was to develop protocols to prepare consistent specimens for accurate mechanical testing of high-strength aramid or ultra-high-molar-mass polyethylene-based flexible unidirectional composite laminate materials and to describe protocols for performing artificial ageing on these materials. Immunology and Infection Assessing the Cellular Immune Response of the Fruit Fly, Drosophila melanogaster, Using an In Vivo Phagocytosis Assay Ashley E. Nazario-Toole1,2, Louisa P. Wu2,3 1Department of Biology, University of Maryland, 2Department of Cell Biology and Molecular Genetics, University of Maryland, 3Institute for Bioscience and Biotechnology Research, University of Maryland This protocol describes an in vivo phagocytosis assay in adult Drosophila melanogaster to quantify phagocyte recognition and clearance of microbial infections. Developmental Biology An Efficient Strategy for Generating Tissue-specific Binary Transcription Systems in Drosophila by Genome Editing Lijuan Du1, Amy Zhou1, Alex Sohr1, Sougata Roy1 1Department of Cell Biology and Molecular Genetics, University of Maryland Here, we present a method for generating tissue-specific binary transcription systems in Drosophila by replacing the first coding exon of genes with transcription drivers. The CRISPR/Cas9-based method places a transactivator sequence under the endogenous regulation of a replaced gene, and consequently facilitates transctivator expression exclusively in gene-specific spatiotemporal patterns. Chemistry Disentangling High Strength Copolymer Aramid Fibers to Enable the Determination of Their Mechanical Properties Amanda L. Forster1, Viviana Rodriguez Cardenas1, Ajay Krishnamurthy1,2, Zois Tsinas3, Amy Engelbrecht-Wiggans1,2, Nolan Gonzalez1 1Material Measurement Laboratory, National Institute of Standards and Technology, 2Theiss Research, 3University of Maryland The primary goal of the study is to develop a protocol to prepare consistent specimens for accurate mechanical testing of high strength copolymer aramid fibers, by removing a coating and disentangling the individual fiber strands without introducing significant chemical or physical degradation. Environment A Video Surveillance System to Monitor Breeding Colonies of Common Terns (Sterna Hirundo) Jennifer Lynn Wall*1, Paul R. Marbán*2, David F. Brinker3, Jeffery D. Sullivan4, Mia Zimnik5, Jennifer L. Murrow6, Peter C. McGowan7, Carl R. Callahan7, Diann J. Prosser8 1Chesapeake Conservation Corps, Chesapeake Bay Trust, 2Department of Marine, Estuarine, and Environmental Science, University of Maryland, 3Natural Heritage Program, Maryland Department of Natural Resources, 4Natural Systems Analyst, 5Department of Biology, Hood College, 6Department of Environmental Science and Technology, University of Maryland, 7U.S. Fish and Wildlife Service Chesapeake Bay Field Office, 8U.S. Geological Survey Patuxent Wildlife Research Center This paper describes a protocol that uses a remote video monitoring surveillance system to continuously monitor breeding colonies of ground-nesting waterbirds. The system includes five cameras monitoring individual nests and one camera monitoring the colony as a whole, and is powered by car batteries that are recharged via solar panels. Behavior The "Motor" in Implicit Motor Sequence Learning: A Foot-stepping Serial Reaction Time Task Yue Du1, Jane E. Clark1,2 1Department of Kinesiology, University of Maryland, College Park, 2The Neuroscience and Cognitive Science Program, University of Maryland, College Park We introduce the foot-stepping serial reaction time (SRT) task. This modified SRT task, complementing the classic SRT task that involves only finger-pressing movement, better approximates daily sequenced activities and allows researchers to study the dynamic processes underlying discrete response measures and disentangle the explicit process operating in implicit sequence learning. Immunology and Infection Quantification of Intracellular Growth Inside Macrophages is a Fast and Reliable Method for Assessing the Virulence of Leishmania Parasites Amrita Sarkar1, Yousuf A. Khan1, Maria Fernanda Laranjeira-Silva1, Norma W. Andrews1, Bidyottam Mittra1 1Department of Cell Biology and Molecular Genetics, University of Maryland All pathogenic Leishmania species reside and replicate inside macrophages of their vertebrate hosts. Here, we present a protocol to infect murine bone marrow-derived macrophages in culture with Leishmania, followed by precise quantification of intracellular growth kinetics. This method is useful for studying individual factors influencing host-pathogen interaction and Leishmania virulence. Chemistry Microprobe Capillary Electrophoresis Mass Spectrometry for Single-cell Metabolomics in Live Frog (Xenopus laevis) Embryos Rosemary M. Onjiko1, Erika P. Portero1, Sally A. Moody2, Peter Nemes1,3 1Department of Chemistry, George Washington University, 2Department of Anatomy & Regenerative Biology, George Washington University, 3Department of Chemistry & Biochemistry, University of Maryland, College Park We describe steps that enable fast in situ sampling of a small portion of an individual cell with high precision and minimal invasion using capillary-based micro-sampling, to facilitate chemical characterization of a snapshot of metabolic activity in live embryos using a custom-built single cell capillary electrophoresis and mass spectrometry platform. Bioengineering Efficient Generation and Editing of Feeder-free IPSCs from Human Pancreatic Cells Using the CRISPR-Cas9 System Anjali Nandal1,2, Barbara Mallon3, Bhanu P. Telugu1,2,4 1Department of Animal and Avian Sciences, University of Maryland, 2Animal Bioscience and Biotechnology Laboratory, ARS, USDA, 3NIH Stem Cell Unit, Bethesda, National Institutes of Health, 4RenOVAte Biosciences Inc This protocol describes in detail the generation of footprint-free induced pluripotent stem cells (iPSCs) from human pancreatic cells in feeder-free conditions, followed by editing using CRISPR/Cas9 ribonucleoproteins and characterization of the modified single-cell clones. Biology Floral-dip Transformation of Arabidopsis thaliana to Examine pTSO2::β-glucuronidase Reporter Gene Expression Chloe Mara1, Boyana Grigorova1, Zhongchi Liu1 1Department of Cell Biology and Molecular Genetics, University of Maryland College Park This article illustrates the floral-dip method of Agrobacterium tumefaciens -mediated transformation of Arabidopsis thaliana. By introducing a cell-cycle regulated promoter-reporter, pTSO2::β-glucuronidase (GUS), into Arabidopsis, we illustrates how one detects GUS reporter expression in transgenic seedlings.