University of North Carolina

18 ARTICLES PUBLISHED IN JoVE

Biology

The Production of C. elegans Transgenes via Recombineering with the galK Selectable Marker

Yue Zhang^*1, Luv Kashyap^*2, Annabel A. Ferguson², Alfred L. Fisher²

¹Department of Radiation Oncology, Beth Israel Deaconess Medical Center, Harvard Medical School, ²Department of Medicine, Division of Geriatric Medicine and Pittsburgh Institute for Neurodegenerative Diseases, University of Pittsburgh

The ability to produce transgenes for Caenorhabditis elegans using genomic DNA carried by fosmids is particularly attractive as all of the native regulatory elements are retained. Described is a simple and robust procedure for the production of transgenes via recombineering with the galK selectable marker.

Neuroscience

Deep Brain Stimulation with Simultaneous fMRI in Rodents

John Robert Younce^1,2,5, Daniel L Albaugh^1,2,4, Yen-Yu Ian Shih^1,2,3,4

¹Department of Neurology, University of North Carolina, ²Biomedical Research Imaging Center, University of North Carolina, ³Department of Biomedical Engineering, University of North Carolina, ⁴Curriculum in Neurobiology, University of North Carolina, ⁵School of Medicine, University of North Carolina

This protocol describes a standard method for simultaneous functional magnetic resonance imaging and deep brain stimulation in the rodent. The combined use of these experimental tools allows for the exploration of global downstream activity in response to electrical stimulation at virtually any brain target.

Medicine

Rapid Fractionation and Isolation of Whole Blood Components in Samples Obtained from a Community-based Setting

Amy Weckle^1,2, Allison E. Aiello³, Monica Uddin^1,4, Sandro Galea⁵, Rebecca M. Coulborn⁶, Richelo Soliven⁷, Helen Meier⁶, Derek E. Wildman^1,2

¹Carl R. Woese Institute for Genomic Biology, University of Illinois at Urbana-Champaign, ²Department of Molecular and Integrative Physiology, University of Illinois at Urbana-Champaign, ³Department of Epidemiology, Gillings School of Global Public Health, University of North Carolina, ⁴Department of Psychology, University of Illinois at Urbana-Champaign, ⁵Department of Epidemiology, Mailman School of Public Health, Columbia University, ⁶Department of Epidemiology, University of Michigan School of Public Health, ⁷Center for Molecular Medicine and Genetics, Wayne State University School of Medicine

We outline a methodology for the processing of whole blood to obtain a variety of components for further analysis. We have optimized a streamlined protocol that enables rapid, high-throughput simultaneous processing of whole blood samples in a non-clinical setting.

Biology

Open Source High Content Analysis Utilizing Automated Fluorescence Lifetime Imaging Microscopy

Frederik Görlitz^*1, Douglas J. Kelly^*1, Sean C. Warren¹, Dominic Alibhai², Lucien West³, Sunil Kumar¹, Yuriy Alexandrov¹, Ian Munro¹, Edwin Garcia¹, James McGinty¹, Clifford Talbot¹, Remigiusz A. Serwa⁴, Emmanuelle Thinon⁴, Vincenzo da Paola³, Edward J. Murray⁵, Frank Stuhmeier⁶, Mark A. A. Neil¹, Edward W. Tate⁴, Christopher Dunsby^1,7, Paul M. W. French¹

¹Photonics Group, Department of Physics, Imperial College London, ²Institute for Chemical Biology, Department of Chemistry, Imperial College London, ³MRC Clinical Sciences Centre, Hammersmith Hospital, ⁴Chemical Biology Section, Department of Chemistry, Imperial College London, ⁵Retroscreen Virology Ltd, ⁶Pfizer Global Research and Development, Pfizer Limited, Sandwich, Kent, UK, ⁷Centre for Histopathology, Imperial College London

We present an open source high content analysis (HCA) instrument utilizing automated fluorescence lifetime imaging (FLIM) for assaying protein interactions using Förster resonance energy transfer (FRET) based readouts. Data acquisition for this openFLIM-HCA instrument is controlled by software written in µManager and data analysis is undertaken in FLIMfit.

Environment

Production and Measurement of Organic Particulate Matter in a Flow Tube Reactor

Yue Zhang^1,2, Pengfei Liu¹, Zhaoheng Gong¹, Franz M. Geiger³, Scot T. Martin^1,4

¹School of Engineering and Applied Sciences, Harvard University, ²Department of Environmental Science and Engineering, Gillings School of Global Public Health, University of North Carolina, Chapel Hill, ³Department of Chemistry, Northwestern University, ⁴Department of Earth and Planetary Sciences, Harvard University

This paper describes the operation procedure for the flow tube reactor and related data collection. It shows the protocols for setting the experiments, recording data and generating the number-diameter distribution as well as the particle mass information, which gives useful information about chemical and physical properties of the organic aerosols.

Environment

Production and Measurement of Organic Particulate Matter in the Harvard Environmental Chamber

Yue Zhang^1,2, Zhaoheng Gong¹, Suzane de Sa¹, Adam P. Bateman¹, Yingjun Liu¹, Yongjie Li¹, Franz M. Geiger³, Scot T. Martin^1,4

¹School of Engineering and Applied Sciences, Harvard University, ²Department of Environmental Science and Engineering, Gillings School of Global Public Health, University of North Carolina, ³Department of Chemistry, Northwestern University, ⁴Department of Earth and Planetary Sciences, Harvard University

This paper describes operation procedures for the Harvard Environmental Chamber (HEC) and related instrumentation for measuring gaseous and particle species. The environmental chamber is used to produce and study secondary organic species produced from the organic precursors, especially related to atmospheric organic particulate matter.

Developmental Biology

Establishment of Larval Zebrafish as an Animal Model to Investigate Trypanosoma cruzi Motility In Vivo

Veronica Akle¹, Nathalie Agudelo-Dueñas^*1,2, Maria A. Molina-Rodriguez^*1, Laurel Brianne Kartchner^1,3,4,6, Annette Marie Ruth^1,3,5,6, John M. González³, Manu Forero-Shelton²

¹Laboratory of Neurosciences and Circadian Rhythms, School of Medicine, Universidad de los Andes, ²Biophysics Group, Department of Physics, Universidad de los Andes, ³Laboratory of Basic Medical Sciences, School of Medicine, Universidad de los Andes, ⁴Department of Microbiology and Immunology, University of North Carolina, ⁵Notre Dame Initiative for Global Development, University of Notre Dame, ⁶USAID Research and Innovation Fellowship program

In this protocol, fluorescently labeled T. cruzi were injected into transparent zebrafish larvae, and parasite motility was observed in vivo using light sheet fluorescence microscopy.

Immunology and Infection

Generation of Monoclonal Antibodies Against Natural Products

Yue Zhang¹, Peng Cao², Fang Lu³, Xin Yan⁴, Bingqian Jiang³, Jinjun Cheng³, Huihua Qu⁵

¹School of Life Science, Beijing University of Chinese Medicine, ²Third Affiliated Hospital, Beijing University of Chinese Medicine, ³School of Basic Medical Sciences, Beijing University of Chinese Medicine, ⁴School of Chinese Materia Medica, Beijing University of Chinese Medicine, ⁵Center of Scientific Experiment, Beijing University of Chinese Medicine

This article provides a detailed protocol for the preparation and evaluation of monoclonal antibodies against natural products for use in various immunoassays. This procedure includes immunization, cell fusion, indirect competitive ELISA for positive clone screening, and monoclonal hybridoma preparation. The specifications for antibody characterization using MALDI-TOF-MS and ELISA analyses are also provided.

Chemistry

Luminophore Formation in Various Conformations of Bovine Serum Albumin by Binding of Gold(III)

Jacob M. Dixon¹, Shunji Egusa¹

¹Department of Physics and Optical Science, Center for Biomedical Engineering & Science, University of North Carolina

The protocols for studying the binding of gold cations (Au(III)) to various conformations of bovine serum albumin (BSA) as well as for characterizing the conformational dependent unique BSA-Au fluorescence are presented.

JoVE Journal

Repressing Gene Transcription by Redirecting Cellular Machinery with Chemical Epigenetic Modifiers

Anna M. Chiarella¹, Tiffany A. Wang², Kyle V. Butler³, Jian Jin³, Nathaniel A. Hathaway⁴

¹Chemical Biology and Medicinal Chemistry, Center for Integrative Chemical Biology and Drug Discovery, Curriculum in Genetics and Molecular Biology, University of North Carolina, ²College of Arts and Sciences, Chemical Biology and Medicinal Chemistry, Center for Integrative Chemical Biology and Drug Discovery, University of North Carolina, ³Chemical Biology and Drug Discovery, Pharmacological Sciences and Oncological Sciences, Icahn School of Medicine at Mount Sinai, ⁴Chemical Biology and Medicinal Chemistry, Center for Integrative Chemical Biology and Drug Discovery, Curriculum in Genetics and Molecular Biology, Lineberger Comprehensive Cancer Center, University of North Carolina

Regulation of the chromatin environment is an essential process required for proper gene expression. Here, we describe a method for controlling gene expression through the recruitment of chromatin-modifying machinery in a gene-specific and reversible manner.

Immunology and Infection

Antigenic Liposomes for Generation of Disease-specific Antibodies

Kyle J. Bednar^*1, Lakeya Hardy^*2,3, Johanna Smeekens^*3,4, Dharmendra Raghuwanshi⁵, Shiteng Duan⁶, Mike D. Kulis^3,4, Matthew S. Macauley^5,7

¹Janssen R&D, ²Department of Microbiology and Immunology, University of North Carolina, ³UNC Food Allergy Initiative, University of North Carolina, ⁴Department of Pediatrics, University of North Carolina, ⁵Department of Chemistry, University of Alberta, ⁶Department of Molecular Medicine, Scripps Research Institute, ⁷Department of Medical Microbiology and Immunology, University of Alberta

Described is the preparation of antigenic liposomal nanoparticles and their use in stimulating B-cell activation in vitro and in vivo. Consistent and robust antibody responses led to the development of a new peanut allergy model. The protocol for generating antigenic liposomes can be extended to different antigens and immunization models.

Behavior

A Conflict Model of Reward-seeking Behavior in Male Rats

Shaofei Jiang^1,2, Yue Zhang^1,2, Xigeng Zheng^1,2, Haoshuang Luo^1,2, Zhengkui Liu^1,2, Yunjing Bai^1,2

¹CAS Key Laboratory of Mental Health, Institute of Psychology, Chinese Academy of Sciences, ²Department of Psychology, University of Chinese Academy of Sciences

This conflict model is used to measure the impairment of inhibitory control after exposure to addictive drugs, or other factors that may influence inhibitory control. A sexual stimulus and an aversive obstacle are concurrently presented, thus male rats have to conquer the obstacle to approach the sexual reward.

Immunology and Infection

Coincubation Assay for Quantifying Competitive Interactions between Vibrio fischeri Isolates

Lauren Speare¹, Alecia N. Septer¹

¹Department of Marine Sciences, University of North Carolina, Chapel Hill

Bacteria encode diverse mechanisms for engaging in interbacterial competition. Here, we present a culture-based protocol for characterizing competitive interactions between bacterial isolates and how they impact the spatial structure of a mixed population.

A Droplet PCR-Based Next Generation Sequencing Assay to Track Plasma DNA Mutation Dynamics in Estrogen Receptor Positive Metastatic Breast Cancer

Sunil Kumar^1,2, Dennis A. Simpson¹, Gaorav P. Gupta^1,2

¹Lineberger Comprehensive Cancer Center, University of North Carolina, ²Department of Radiation Oncology, University of North Carolina

Here we describe a protocol that uses droplet polymerase chain reaction for target enrichment followed by next generation sequencing of blood plasma circulating tumor DNA. This technique was used to characterize mutations in the genes ESR1 (all coding regions), TP53 (all coding regions), PIK3CA (hotspots), PIK3R1 (hotspots) and POLE (exonuclease domain).

Genetics

Mapping Alzheimer's Disease Variants to Their Target Genes Using Computational Analysis of Chromatin Configuration

Nana Matoba^1,2, Ivana Y. Quiroga³, Douglas H. Phanstiel^*3,4, Hyejung Won^*1,2

¹Department of Genetics, University of North Carolina, ²Neuroscience Center, University of North Carolina, ³Thurston Arthritis Research Center, University of North Carolina, ⁴Department of Cell Biology and Physiology, University of North Carolina

We present a protocol to identify functional implications of non-coding variants identified by genome-wide association studies (GWAS) using three-dimensional chromatin interactions.

Biochemistry

Development of a Lateral Flow Immunochromatographic Strip for Rapid and Quantitative Detection of Small Molecule Compounds

Yue Zhang^*1, Peng Cao^*2, Fang Lu¹, Jinjun Cheng³, Huihua Qu⁴

¹School of Life Science, Beijing University of Chinese Medicine, ²Third Affiliated Hospital, Beijing University of Chinese Medicine, ³National Institute of TCM Constitution and Preventive Medicine, Beijing University of Chinese Medicine, ⁴Center of Scientific Experiment, Beijing University of Chinese Medicine

Development of a Lateral Flow Immunochromatographic Strip for Rapid and Quantitative Detection of Small Molecule Compounds

Biology

Quantification of Interbacterial Competition using Single-Cell Fluorescence Imaging

Stephanie Smith¹, Alecia N. Septer¹

¹Department of Earth, Marine, and Environmental Sciences, University of North Carolina

This manuscript describes a method for using single-cell fluorescence microscopy to visualize and quantify bacterial competition in coculture.

Neuroscience

Whole-Brain Single-Cell Imaging and Analysis of Intact Neonatal Mouse Brains Using MRI, Tissue Clearing, and Light-Sheet Microscopy

Felix A. Kyere^*1,2, Ian Curtin^*1,2, Oleh Krupa^1,2, Carolyn M. McCormick^1,2, Mustafa Dere³, Sarah Khan^3,7, Minjeong Kim⁷, Tzu-Wen Winnie Wang^4,5,6, Qiuhong He^4,5,6, Guorong Wu³, Yen-Yu Ian Shih^4,5,6, Jason L. Stein^1,2

¹UNC Neuroscience Center, University of North Carolina, Chapel Hill, ²Department of Genetics, University of North Carolina, Chapel Hill, ³Department of Psychiatry, University of North Carolina, Chapel Hill, ⁴Center for Animal Magnetic Resonance Imaging, The University of North Carolina at Chapel Hill, ⁵Biomedical Research Imaging Center, The University of North Carolina at Chapel Hill, ⁶Department of Neurology, The University of North Carolina at Chapel Hill, ⁷Department of Computer Science, The University of North Carolina at Greensboro

This protocol describes methods for conducting magnetic resonance imaging, clearing, and immunolabeling of intact mouse brains using iDISCO+, followed by a detailed description of imaging using light-sheet microscopy, and downstream analyses using NuMorph.