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36 ARTICLES PUBLISHED IN JoVE

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Biology

Whole Mount in Situ Hybridization of E8.5 to E11.5 Mouse Embryos
Qiaozhi Wei 1, Nancy R. Manley 1, Brian G. Condie 1
1Department of Genetics, University of Georgia

This whole mount in situ hybridization protocol discusses critical steps that ensure reproducible high quality results for gene expression studies in E8.5-E11.5 day old mouse embryos.

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Immunology and Infection

An Allelotyping PCR for Identifying Salmonella enterica serovars Enteritidis, Hadar, Heidelberg, and Typhimurium
John J. Maurer 1, Margie D. Lee 1, Ying Cheng 1, Adriana Pedroso 1
1Department of Population Health, University of Georgia

We describe a multiplex PCR for the rapid detection of Salmonella enterica serovars Enteritidis, Hadar, Heidelberg, and Typhimurium. Specific Salmonella serovars can be identified by targeting a multiplex PCR to genes and sequences unique to the O-antigen biosynthesis cluster and flagellin of a given serovar. Serovar is assigned then to a Salmonella isolate based on the appearance of specific, size amplicons (PCR product) corresponding to the target allele.

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Immunology and Infection

A Method for Labeling Vasculature in Embryonic Mice
Jerrod L. Bryson 1, Mark C. Coles 2, Nancy R. Manley 3
1Department of Cellular Biology, University of Georgia, 2Centre for Immunology and Infection, Department of Biology and HYMS, University of York, 3Department of Genetics, University of Georgia

This article describes a method for labeling embryonic skin and thymus blood vessels.

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Environment

A Rapid and Efficient Method for Assessing Pathogenicity of Ustilago maydis on Maize and Teosinte Lines
Suchitra Chavan 1, Shavannor M. Smith 1
1Department of Plant Pathology, University of Georgia

The use of a needle injection method to inoculate maize and teosinte plants with the biotrophic pathogen Ustilago maydis is described. The needle injection inoculation method facilitates the controlled delivery of the fungal pathogen in between the plant leaves where the pathogen enters the plant through the formation of appresoria. This method is highly efficient, enabling reproducible inoculations with U. maydis.

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Biology

Mouse Embryonic Development in a Serum-free Whole Embryo Culture System
Vijay K. Kalaskar 1, James D. Lauderdale 1,2
1Neuroscience Division of the Biomedical & Health Sciences Institute, University of Georgia, 2Department of Cellular Biology, University of Georgia

Serum utilized in embryo cultures contains unknown components that can affect the outcome of experiments especially in studies involving signaling interactions. Here we utilized a serum-free oxygenated culture system and show that mid-gestation mouse embryos cultured for 16-40 hr exhibit morphological development comparable to embryos developing in utero.

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Neuroscience

Electrophysiological Recording in the Brain of Intact Adult Zebrafish
Lindsey Johnston 1, Rebecca E. Ball 2, Seth Acuff 3, John Gaudet 4, Andrew Sornborger 5, James D. Lauderdale 2
1Department of Genetics, University of Georgia, 2Department of Cellular Biology, University of Georgia, 3College of Engineering, Architecture and Technology, Oklahoma State University, 4University of Georgia, 5Department of Mathematics, University of California, Davis

This paper describes how an adult zebrafish can be immobilized, intubated, and used for in vivo electrophysiological experiments to allow recordings and manipulation of neural activity in an intact animal.

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Neuroscience

Feeder-free Derivation of Neural Crest Progenitor Cells from Human Pluripotent Stem Cells
Nadja Zeltner 1, Fabien G. Lafaille 2, Faranak Fattahi 1, Lorenz Studer 1
1Developmental Biology, Center for Stem Cell Biology, Sloan-Kettering Institute for Cancer Research, 2St. Giles Laboratory of Human Genetics of Infectious Diseases, The Rockefeller University

Neural crest (NC) cells derived from human pluripotent stem cells (hPSC) have great potential for modeling human development and disease and for cell replacement therapies. Here, a feeder-free adaptation of the currently widely used in vitro differentiation protocol for the derivation of NC cells from hPSCs is presented.

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Medicine

Developing Neuroimaging Phenotypes of the Default Mode Network in PTSD: Integrating the Resting State, Working Memory, and Structural Connectivity
Noah S. Philip 1, S. Louisa Carpenter 1, Lawrence H. Sweet 2
1Providence VA Medical Center, Department of Psychiatry & Human Behavior, Alpert Medical School, Brown University, 2Department of Psychology, University of Georgia

This protocol describes the complementary neuroimaging techniques of resting state structural connectivity, task-induced deactivation, and structural connectivity analyses to examine the default network in post-traumatic stress disorder. The use of synergistic methods could potentially lead to improved diagnostics and assessments of severity, outcome, and other relevant clinical factors.

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JoVE Journal

Improved In-gel Reductive β-Elimination for Comprehensive O-linked and Sulfo-glycomics by Mass Spectrometry
David B. Nix 1,2, Tadahiro Kumagai 1, Toshihiko Katoh 1,3, Michael Tiemeyer 1,2, Kazuhiro Aoki 1
1Complex Carbohydrate Research Center, University of Georgia, 2Department of Biochemistry and Molecular Biology, University of Georgia, 3Research Institute for Bioresources and Biotechnology, Ishikawa Prefectural University

In order to comprehensively explore the diversity of O-linked glycans, a new procedure for in-gel reductive β-elimination, combined with permethylation and a rapid phase-partition method, is applied to the analysis of O-linked glycans directly released from glycoproteins resolved by SDS-PAGE and amenable to subsequent glycomic analysis by mass spectrometry.

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Biology

A Hybrid DNA Extraction Method for the Qualitative and Quantitative Assessment of Bacterial Communities from Poultry Production Samples
Michael J. Rothrock Jr. 1, Kelli L. Hiett 2, John Gamble 3, Andrew C. Caudill 4, Kellie M. Cicconi-Hogan 1, J. Gregory Caporaso 5
1Egg Safety and Quality Research Unit, USDA-Agricultural Research Service, 2Poultry Microbiological Safety and Processing Research Unit, USDA-Agricultural Research Service, 3Department of Biochemistry and Biophysics, Oregon State University, 4College of Public Health, University of Georgia, 5Department of Biological Sciences, Center for Microbial Genetics and Genomics, Northern Arizona University

A novel semi-automated hybrid DNA extraction method for use with environmental poultry production samples was developed and demonstrated improvements over a common mechanical and enzymatic extraction method in terms of the quantitative and qualitative estimates of the total bacterial communities.

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Immunology and Infection

High Throughput Measurement of Extracellular DNA Release and Quantitative NET Formation in Human Neutrophils In Vitro
Payel Sil *1, Dae-goon Yoo *1, Madison Floyd *1, Aaron Gingerich 1, Balazs Rada 1
1Department of Infectious Diseases, University of Georgia

High throughput assays are presented that in combination provide excellent tools to quantitate NET release from human neutrophils.

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Biology

Multifunctional, Micropipette-based Method for Incorporation And Stimulation of Bacterial Mechanosensitive Ion Channels in Droplet Interface Bilayers
Joseph S. Najem 1, Myles D. Dunlap 2, Anthony Yasmann 3, Eric C. Freeman 4, John W. Grant 5, Sergei Sukharev 3, Donald J. Leo 4
1Department of Mechanical Engineering, Virginia Polytechnic Institute and State University, 2School of Biomedical Engineering and Sciences, Virginia Polytechnic Institute and State University, 3Department of Biology, University of Maryland, 4College of Engineering, University of Georgia, 5Department of Engineering Sciences and Mechanics, Virginia Polytechnic Institute and State University

Bacterial mechanosensitive channels can be used as mechanoelectrical transducers in biomolecular devices. Droplet interface bilayers (DIBs), cell-inspired building blocks to such devices, represent new platforms to incorporate and stimulate mechanosensitive channels. Here, we demonstrate a new micropipette-based method of forming DIBs, allowing the study of mechanosensitive channels under mechanical stimulation.

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Neuroscience

A Simple Flight Mill for the Study of Tethered Flight in Insects
Alfredo Attisano 1, James T. Murphy 2, Andrew Vickers 3, Patricia J. Moore 2
1Museum and Institute of Zoology, Polish Academy of Sciences, 2Department of Entomology, University of Georgia, 3Riventa Pool Innovation Centre

Flight in insects is influenced by a number of factors and the propensity to disperse is an important variable in understanding insect ecology and biological control strategies. We describe the construction and use of a simple, relatively inexpensive, and flexible flight mill for measuring parameters of tethered flight in insects.

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Biology

High Yield Expression of Recombinant Human Proteins with the Transient Transfection of HEK293 Cells in Suspension
Ganesh P. Subedi 1, Roy W. Johnson 2, Heather A. Moniz 2, Kelley W. Moremen 2, Adam W. Barb 1
1The Roy J. Carver Department of Biochemistry, Biophysics and Molecular Biology, Iowa State University, 2Complex Carbohydrate Research Center, University of Georgia

Laboratory-scale production of eukaryotic proteins with appropriate post-translational modification represents a significant barrier. Here is a robust protocol with rapid establishment and turnaround for protein expression using a mammalian expression system. This system supports selective amino acid, selective labeling of proteins and small molecule modulators of glycan composition.

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Medicine

An Orthotopic Mouse Model of Spontaneous Breast Cancer Metastasis
Amy V. Paschall 1,2,3, Kebin Liu 1,2,3
1Department of Biochemistry and Molecular Biology, Medical College of Georgia, Augusta University, 2Georgia Cancer Center, Augusta University, 3Charlie Norwood VA Medical Center

An orthotopic breast cancer primary tumor model and surgical removal of primary tumor to extend mouse life to generate spontaneous metastasis are described. The tumor growth and progression are monitored and quantified by luciferase fluorescence imaging.

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Biology

Expression and Purification of Virus-like Particles for Vaccination
Maria T. Arevalo 1, Terianne M. Wong 1, Ted M. Ross 1
1Center for Vaccines and Immunology, Department of Infectious Diseases, College of Veterinary Medicine, University of Georgia

Here, we present a protocol for synthesizing virus-like particles using either baculovirus or mammalian expression systems, and ultracentrifugation purification. This highly customizable approach is used to identify viral antigens as vaccine targets in a safe and flexible manner.

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Biology

Cryosectioning of Contiguous Regions of a Single Mouse Skeletal Muscle for Gene Expression and Histological Analyses
Aaron M. Beedle 1
1Department of Pharmaceutical and Biomedical Sciences, University of Georgia

Consecutive cryo-sections are collected to enable histological applications and enrichment of RNA for gene expression measurements using adjacent regions from a single mouse skeletal muscle. High-quality RNA is obtained from 20 - 30 mg of pooled cryosections and measurements are directly compared across applications.

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Immunology and Infection

The Ex Vivo Colon Organ Culture and Its Use in Antimicrobial Host Defense Studies
S. M. Nashir Udden 1, Sumyya Waliullah 1, Melanie Harris 1, Hasan Zaki 1
1Department of Pathology, UT Southwestern Medical Center

The ex vivo organ culture allows investigation of biological processes in the context of the intact tissue architecture. Here, we introduce a method of ex vivo culture of the mouse colon, which can be used to study innate immunity and antimicrobial host defense in the intestine.

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Neuroscience

Establishing Mouse Models for Zika Virus-induced Neurological Disorders Using Intracerebral Injection Strategies: Embryonic, Neonatal, and Adult
Stephanie A. Herrlinger 1, Qiang Shao 2, Li Ma 2, Melinda Brindley 3, Jian-Fu Chen 2
1Biomedical and Health Sciences Institute, University of Georgia, 2Center for Craniofacial Molecular Biology, University of Southern California, 3Department of Infectious Diseases, College of Veterinary Medicine, University of Georgia

Here we describe a method for establishing a model of Zika virus-induced microcephaly in mouse. This protocol includes methods for embryonic, neonatal, and adult-stage intracerebral inoculation of the Zika virus.

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Genetics

CRISPR/Cas9 Gene Editing to Make Conditional Mutants of Human Malaria Parasite P. falciparum
Heather M. Kudyba *1,2, David W. Cobb *1, Anat Florentin 1,2, Michelle Krakowiak 1, Vasant Muralidharan 1,2
1Department of Cellular Biology, University of Georgia, 2Center for Tropical and Emerging Global Diseases, University of Georgia

We describe a method for generating glmS-based conditional knockdown mutants in Plasmodium falciparum using CRISPR/Cas9 genome editing.

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JoVE Core

Sperm Collection of Differential Quality Using Density Gradient Centrifugation
Muslah Uddin Ahammad *1, Zachery Ryan Jarrell *1, Andrew Parks Benson 1
1Department of Poultry Science, University of Georgia

In this paper, we aim to describe the performance of the density gradient centrifugation technique and its application in sperm physiology research.

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Immunology and Infection

Opsonophagocytic Killing Assay to Assess Immunological Responses Against Bacterial Pathogens
Amy V. Paschall 1, Dustin R. Middleton 1, Fikri Y. Avci 1
1Department of Biochemistry and Molecular Biology, Center for Molecular Medicine and Complex Carbohydrate Research Center, University of Georgia

This opsonophagocytic killing assay is used to compare the ability of phagocytic immune cells to respond to and kill bacteria based on different treatments and/or conditions. Classically, this assay serves as the gold standard for assessing effector functions of antibodies raised against a bacterium as opsonin.

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Environment

Cultivation of the Marine Pelagic Tunicate Dolioletta gegenbauri (Uljanin 1884) for Experimental Studies
Tina L. Walters 1, Deidre M. Gibson 2, Marc E. Frischer 1
1Skidaway Institute of Oceanography, University of Georgia, 2Hampton University

Doliolids, including the species Dolioletta gegenbauri, are small gelatinous marine zooplankton of ecological significance found on productive subcontinental shelf systems worldwide. The difficulty of culturing these delicate organisms limits their investigation. In this study, we describe cultivation approaches for collecting, rearing, and maintaining the doliolid Dolioletta gegenbauri.

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Developmental Biology

Retrograde Tracing of Drosophila Embryonic Motor Neurons Using Lipophilic Fluorescent Dyes
Melissa Ana Inal 1, Kota Banzai 1, Daichi Kamiyama 1
1Department of Cellular Biology, University of Georgia

We describe a method for retrograde tracing of the Drosophila embryonic motor neurons using lipophilic fluorescent dyes.

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Developmental Biology

Efficient Differentiation of Postganglionic Sympathetic Neurons using Human Pluripotent Stem Cells under Feeder-free and Chemically Defined Culture Conditions
Hsueh Fu Wu 1,2, Nadja Zeltner 1,2,3
1Center for Molecular Medicine, University of Georgia, 2Department of Biochemistry and Molecular Biology, Franklin College of Arts and Sciences, University of Georgia, 3Department of Cellular Biology, Franklin College of Arts and Sciences, University of Georgia

In this protocol, we describe a stable, highly efficient differentiation strategy for the generation of postganglionic sympathetic neurons from human pluripotent stem cells. This model will make neurons available for the use of studies of multiple autonomic disorders.

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Biology

Establishment and Maintenance of Gnotobiotic American Cockroaches (Periplaneta americana)
Helen E. Dukes 1, Josey E. Dyer 1, Elizabeth A. Ottesen 1
1Department of Microbiology, University of Georgia

This protocol is used in establishing and maintaining gnotobiotic American cockroaches (Periplaneta americana) by surface sterilizing the egg cases (oothecae) prior to hatching. These gnotobiotic insects contain their vertically transmitted Blattabacterium endosymbionts but have axenic guts.

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Genetics

Culture and Assay of Large-Scale Mixed-Stage Caenorhabditis elegans Populations
Amanda O. Shaver 1, Goncalo J. Gouveia 2, Pamela S. Kirby 3, Erik C. Andersen 4, Arthur S. Edison 1,2,3
1Department of Genetics, University of Georgia, 2Department of Biochemistry and Molecular Biology, University of Georgia, 3Complex Carbohydrate Research Center, University of Georgia, 4Department of Molecular Biosciences, Northwestern University

To use Caenorhabditis elegans (C. elegans) in omics research, a method is needed to generate large populations of worms where a single sample can be measured across platforms for comparative analyses. Here, a method to culture C. elegans populations on large-scale culture plates (LSCPs) and to document population growth is presented.

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Environment

Detection of Phytophthora capsici in Irrigation Water using Loop-Mediated Isothermal Amplification
Owen Hudson 1, Sumyya Waliullah 1, Justin Hand 2, Romina Gazis-Seregina 3, Fulya Baysal-Gurel 4, Md Emran Ali 1
1Department of Plant Pathology, Coastal Plain Experiment Station, University of Georgia, 2University of Georgia Cooperative Extension, 3Department of Plant Pathology, Tropical Research & Education Center, University of Florida, 4Department of Agricultural and Environmental Sciences, Otis L. Floyd Nursery Research Center, Tennessee State University

We developed a method to detect Phytophthora capsici zoospores in water sources using a filter paper DNA extraction method coupled with a loop-mediated isothermal amplification (LAMP) assay that can be analyzed in the field or in the lab.

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Developmental Biology

Cell Dissociation from the Tongue Epithelium and Mesenchyme/Connective Tissue of Embryonic-Day 12.5 and 8-Week-Old Mice
Wenxin Yu 1, Mohamed Ishan 1, Zhonghou Wang 1, Hong-Xiang Liu 1
1Regenerative Bioscience Center; Department of Animal and Dairy Science, College of Agricultural and Environmental Sciences, University of Georgia

We have developed a generalized protocol to dissociate a large quantity of high-quality single cells from the epithelium and mesenchyme/connective tissue of embryonic and adult mouse tongues.

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Behavior

Measuring the Behavioral Effects of Intraocular Scatter
Jeffrey Nightingale 1, Billy R. Hammond 1
1Vision Sciences Laboratory, UGA Psychology Department, University of Georgia

In this protocol, we outline the conceptual design elements and structural development of a glare acuity apparatus. Additionally, the design of a device for measuring positive dysphotopsia (halos, spokes) and two-point light thresholds is described.

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Biology

In Vivo Measurement of Hindlimb Dorsiflexor Isometric Torque from Pig
Benjamin T. Corona 1, Jarrod A. Call 2,3, Matthew Borkowski 4, Sarah M. Greising 5
1School of Medicine, Wake Forest University, 2Department of Kinesiology, University of Georgia, 3Regenerative Bioscience Center, University of Georgia, 4Aurora Scientific Inc., 5School of Kinesiology, University of Minnesota

The present protocol describes concise experimental details on the evaluation and interpretation of in vivo torque data obtained via electrical stimulation of the common peroneal nerve in anesthetized pigs.

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Developmental Biology

In Ovo Feeding of Commercial Broiler Eggs: An Accurate and Reproducible Method to Affect Muscle Development and Growth
Hanna M. Alcocer 1, Xiaoxing Xu 1, Morgan E. Gravely 1, John M. Gonzalez 1
1Department of Animal and Dairy Sciences, University of Georgia

A robust methodology has been developed for conducting in ovo feeding research trials utilizing unincubated commercial broiler eggs to test the ability of natural and synthetic compounds, in this case, nicotinamide riboside, to influence muscle development and growth.

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Biology

A Contrast of Three Inoculation Techniques used to Determine the Race of Unknown Fusarium oxysporum f.sp. niveum Isolates
James C. Fulton *1, Matthew A. Cullen *1, Kristin Beckham 1, Tatiana Sanchez 2, Zhuxuan Xu 1, Preston Stern 1, Gary Vallad 3, Geoffrey Meru 4,5, Cecilia McGregor 6, Nicholas S. Dufault 1
1Department of Plant Pathology, University of Florida, 2University of Florida Institute of Food and Agricultural Sciences, 3Gulf Coast Research and Education Center, University of Florida, 4Horticultural Sciences Department, University of Florida, 5Tropical Research and Education Center, University of Florida, 6Department of Horticulture, University of Georgia

Managing Fusarium wilt of watermelon requires knowledge of the pathogen races present. Here, we describe the root-dip, infested kernel seeding, and modified tray-dip inoculation methods to demonstrate their efficacy in race-typing of the pathogenic fungus Fusarium oxysporum f. sp niveum (Fon).

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Immunology and Infection

Quantitative 3D Imaging of Trypanosoma cruzi-Infected Cells, Dormant Amastigotes, and T Cells in Intact Clarified Organs
Fernando Sanchez-Valdez 1, Ángel M. Padilla 1, Juan M. Bustamante 1, Caleb W. D. Hawkins 1, Rick L. Tarleton 1,2
1Center for Tropical and Emerging Global Diseases, University of Georgia, 2Department of Cellular Biology, University of Georgia

The present protocol describes light-sheet fluorescent microscopy and automated software-assisted methods to visualize and precisely quantify proliferating and dormant Trypanosoma cruzi parasites and T cells in intact, cleared organs and tissues. These techniques provide a reliable way to evaluate treatment outcomes and offer new insights into parasite-host interactions.

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Immunology and Infection

Generating Genetically Modified Plasmodium berghei Sporozoites
Carson Bowers 1, Samarchith P. Kurup 1,2
1Center for Tropical and Emerging Global Diseases, University of Georgia, 2Department of Cellular Biology, University of Georgia

Malaria is transmitted through inoculation of the sporozoite stage of Plasmodium by infected mosquitoes. Transgenic Plasmodium has allowed us to understand the biology of malaria better and has contributed directly to malaria vaccine development efforts. Here, we describe a streamlined methodology to generate transgenic Plasmodium berghei sporozoites.

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Bioengineering

Continuous Liquid-Liquid Extraction of Medium-Chain Fatty Acids from Fermentation Broth Using Hollow-Fiber Membranes
Kayode J. Taiwo 1, Hubert Nguyen 1, Joseph G. Usack 1,2,3
1Department of Food Science and Technology, University of Georgia, 2New Materials Institute, University of Georgia, 3Institute for Integrative Agriculture, Office of Research, University of Georgia

A liquid-liquid extraction (LLE) system involving hollow-fiber membranes was developed to continuously and selectively extract medium-chain fatty acids (MCFAs) from the fermentation broth. The LLE system achieves high MCFA specificities from broths containing short-chain fatty acids and alcohols. Also, MCFAs are concentrated in a stripping solution to facilitate product recovery.

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