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University of Virginia School of Medicine

24 ARTICLES PUBLISHED IN JoVE

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Neuroscience

RNAi-mediated Double Gene Knockdown and Gustatory Perception Measurement in Honey Bees (Apis mellifera)
Ying Wang 1, Nicholas Baker 1, Gro V. Amdam 1,2
1School of Life Sciences, Arizona State University , 2Department of Chemistry, Biotechnology, and Food Science, Norwegian University of Life Sciences

In this protocol, we describe two strategies that simultaneously suppress two genes (double gene knockdown) in honey bees. Then we present how to use the proboscis extension response (PER) assay to study the effect of double gene knockdown on honey bee gustatory perception.

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Medicine

A Thrombotic Stroke Model Based On Transient Cerebral Hypoxia-ischemia
Yu-Yo Sun 1, Chia-Yi Kuan 1
1Department of Pediatrics, Division of Neurology, The Emory University School of Medicine

Thromboembolic stroke models are vital tools for optimizing the recanalization therapy. Here we report a murine thrombotic stroke model based on transient cerebral hypoxic-ischemic (tHI) insult, which triggers thrombosis and infarction, and responds favorably to tissue plasminogen activator (tPA)-mediated fibrinolysis in a therapeutic window similar to those in stroke patients.

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Developmental Biology

Isolation of Murine Embryonic Hemogenic Endothelial Cells
Jennifer S. Fang *1, Emily C. Gritz *2, Kathrina L. Marcelo 3, Karen K. Hirschi 1
1Departments of Medicine, Genetics and Biomedical Engineering, Yale Cardiovascular Research Center, Vascular Biology and Therapeutics Program, Yale Stem Cell Center, Yale University School of Medicine, 2Department of Pediatrics, Section of Neonatal-Perinatal Medicine, Yale University School of Medicine, 3Department of Molecular and Cellular Biology, Baylor College of Medicine

Hematopoietic stem and progenitor cells (HSPC) derive from specialized (hemogenic) endothelial cells during development, yet little is known about the process by which some endothelial cells specify to become blood forming. We demonstrate a flow-cytometry based method allowing simultaneous isolation of hemogenic endothelial cells and HSPC from murine embryonic tissues.

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Immunology and Infection

In Vivo Investigation of Antimicrobial Blue Light Therapy for Multidrug-resistant Acinetobacter baumannii Burn Infections Using Bioluminescence Imaging
Yucheng Wang 1,2,3, Olivia D. Harrington 1, Ying Wang 1, Clinton K. Murray 4, Michael R. Hamblin 1, Tianhong Dai 1
1Wellman Center for Photomedicine, Massachusetts General Hospital, Harvard Medical School, 2Department of Medical Oncology, Beijing Institute of Translational Medicine, Chinese Academy of Sciences, 3Cancer Center, Aviation General Hospital, Beijing, 4Infectious Disease Service, Brooke Army Medical Center

Infections caused by multidrug-resistant (MDR) bacterial strains have emerged as a serious threat to public health, necessitating the development of alternative therapeutics. We present a protocol to evaluate the effectiveness of antimicrobial blue light (aBL) therapy for MDR Acinetobacter baumannii infections in mouse burns by using bioluminescence imaging.

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Medicine

A Cell Culture Model of Resistance Arteries
Lauren A. Biwer 1,2, Christophe Lechauve 3, Sheri Vanhoose 4, Mitchell J. Weiss 3, Brant E. Isakson 1,2
1Department of Molecular Physiology and Biophysics, University of Virginia School of Medicine, 2Robert M. Berne Cardiovascular Research Center, University of Virginia School of Medicine, 3Department of Hematology, St. Jude Children's Research Hospital, 4Research Histology Core, University of Virginia School of Medicine

A cell culture model of resistance arteries is described, allowing for the dissection of signaling pathways in endothelium, smooth muscle, or between endothelium and smooth muscle (the myoendothelial junction). The selective application of agonists or protein isolation, electron microscopy, or immunofluorescence can be utilized using this cell culture model.

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Cancer Research

A Comprehensive Procedure to Evaluate the In Vitro Performance of the Putative Hemangioblastoma Neovascularization Using the Spheroid Sprouting Assay
Ying Wang 1, DanQi Chen 1, MingYu Chen 1, KaiYuan Ji 1, DeXuan Ma 1, LiangFu Zhou 1
1Department of Neurosurgery, Huashan Hospital, Fudan University

This paper presents a comprehensive procedure to evaluate in vitro whether classic tumor angiogenesis exists in hemangioblastomas (HBs) and its role in HBs. The results highlight the complexity of HB-neovascularization and suggest that this common form of angiogenesis is only a complementary mechanism in the HB-neovascularization.

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Engineering

Radio Frequency Magnetron Sputtering of GdBa2Cu3O7δ/ La0.67Sr0.33MnO3 Quasi-bilayer Films on SrTiO3 (STO) Single-crystal Substrates
Ying Wang 1,2, Zhen Li 1,2, YongSheng Liu 1, Yijie Li 2, Linfei Liu 2, Da Xu 2, Xiaojing Luo 1, Tian Gao 1,3, Yanyan Zhu 1,4, Luozeng Zhou 3, Jianming Xu 3
1Department of Physics, Mathematics, Shanghai Key Laboratory of Materials Protection and Advanced Materials in Electric Power, Shanghai University of Electric Power, 2Key Laboratory of Artificial Structure and Quantum Control, Ministry of Education, Department of Physics, Shanghai Jiao Tong University, 3Shanghai Institute of Space Power-sources, 4Shanghai Key Laboratory of High Temperature Superconductors, Shanghai University

Here, we present a protocol to grow LSMO nanoparticles and (Gd) BCO films on (001) SrTiO3 (STO) single-crystal substrates by radio frequency (RF)-sputtering.

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Immunology and Infection

Human Serum Anti-aquaporin-4 Immunoglobulin G Detection by Cell-based Assay
Caiyun Liu 1, Mingqin Zhu 1, Ying Wang 1
1Department of Neurology and Neuroscience Center, The First Hospital of Jilin University

Cell-based assay is a widely used method to detect serum anti-aquaporin-4 immunoglobulin G. This method could be applied to clinical diagnosis and scientific researches of neuromyelitis optical spectrum disorders.

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Biochemistry

Single-Molecule Tracking Microscopy - A Tool for Determining the Diffusive States of Cytosolic Molecules
Julian M Rocha 1, Andreas Gahlmann 1,2
1Department of Chemistry, University of Virginia, 2Department of Molecular Physiology & Biological Physics, University of Virginia School of Medicine

3D single-molecule localization microscopy is utilized to probe the spatial positions and motion trajectories of fluorescently labeled proteins in living bacterial cells. The experimental and data analysis protocol described herein determines the prevalent diffusive behaviors of cytosolic proteins based on pooled single-molecule trajectories.

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JoVE Journal

A Non-random Mouse Model for Pharmacological Reactivation of Mecp2 on the Inactive X Chromosome
Piotr Przanowski 1, Zeming Zheng 1, Urszula Wasko 1, Sanchita Bhatnagar 1,2
1Department of Biochemistry and Molecular Genetics, University of Virginia School of Medicine, 2Department of Neuroscience, University of Virginia School of Medicine

Here, we describe a protocol to generate a viable female murine model with non-random X chromosome inactivation, i.e., the maternally-inherited X chromosome is inactive in 100% of the cells. We also describe a protocol to test feasibility, tolerability, and safety of pharmacological reactivation of the inactive X chromosome in vivo.

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Genetics

A Bioinformatics Pipeline to Accurately and Efficiently Analyze the MicroRNA Transcriptomes in Plants
Ying Wang *1,2, Zheng Kuang *1,2, Lei Li 2, Xiaozeng Yang 1
1Beijing Key Laboratory of Agricultural Genetic Resources and Biotechnology, Beijing Agro-Biotechnology Research Center, Beijing Academy of Agriculture and Forestry Sciences, 2State Key Laboratory of Protein and Plant Gene Research, Peking-Tsinghua Center for Life Sciences, School of Advanced Agricultural Sciences and School of Life Sciences, Peking University

A bioinformatics pipeline, namely miRDeep-P2 (miRDP2 for short), with updated plant miRNA criteria and an overhauled algorithm, could accurately and efficiently analyze microRNA transcriptomes in plants, especially for species with complex and large genomes.

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Immunology and Infection

Lentiviral CRISPR/Cas9-Mediated Genome Editing for the Study of Hematopoietic Cells in Disease Models
Soichi Sano 1, Ying Wang 1, Megan A. Evans 1, Yoshimitsu Yura 1, Miho Sano 1, Hayato Ogawa 1, Keita Horitani 1, Heather Doviak 1, Kenneth Walsh 1
1Hematovascular Biology Center, Robert M. Berne Cardiovascular Research Center, University of Virginia School of Medicine

Described are protocols for the highly efficient genome editing of murine hematopoietic stem and progenitor cells (HSPC) by the CRISPR/Cas9 system to rapidly develop mouse model systems with hematopoietic system-specific gene modifications.

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Medicine

Murine Surgical Model of Topical Elastase Induced Descending Thoracic Aortic Aneurysm
Zachary Tyerman 1, Jolian Dahl 1, Alexander Shannon 1, W. Forrest Johnston 2, Nicholas H. Pope 1, Guanyi Lu 3, Gilbert R. Upchurch Jr. 3, Gorav Ailawadi 1,4, Morgan Salmon 1
1Department of Surgery, University of Virginia School of Medicine, 2Department of Surgery, Ochsner Medical Center, 3Department of Surgery, University of Florida, 4The Robert M. Berne Cardiovascular Research Center, University of Virginia School of Medicine

We describe a surgical protocol to consistently induce robust descending thoracic aortic aneurysms in mice. The procedure involves left thoracotomy, thoracic aorta exposure, and placement of a sponge soaked in porcine pancreatic elastase on the aortic wall.

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Medicine

Porcine Model of Infrarenal Abdominal Aortic Aneurysm
Alexander H. Shannon 1, J. Michael Cullen 1, Jolian J. Dahl 1, Erik J. Scott 1, Zachary Tyerman 1, Michael D. Spinosa 1, William G. Montgomery 1, W. Forrest Johnston 2, Guanyi Lu 3, Morgan Salmon *1, Gorav Ailawadi *1,4, Gilbert R. Upchurch Jr. *3
1Department of Surgery, University of Virginia School of Medicine, 2Department of Surgery, Ochsner Medical Center, 3Department of Surgery, University of Florida, 4The Robert M. Berne Cardiovascular Research Center, University of Virginia School of Medicine

This novel model creates robust infrarenal abdominal aortic aneurysms in swine using a combination of balloon angioplasty, elastase/collagenase perfusion, topical elastase application, and oral compound β-aminopropionitrile administration, which interferes with collagen cross-linking.

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Cancer Research

Analyzing Tumor and Tissue Distribution of Target Antigen Specific Therapeutic Antibody
Gururaj Shivange *1,2, Tanmoy Mondal *1,2, Evan Lyerly 1,2, Jeremy Gatesman 3, Jogender Tushir-Singh 1,2
1Laboratory of Novel Biologics, University of Virginia School of Medicine, 2Department of Biochemistry and Molecular Genetics, UVA Cancer Center, University of Virginia School of Medicine, 3Center for Comparative Medicine, University of Virginia

Here we present a protocol to study the in vivo localization of antibodies in mice tumor xenograft models.

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A Xenograft Mouse Model to Assess Efficacy of Therapeutic Agents for Human Acute Leukemia
Chandrika Gowda *1, Charyguly Annageldiyev *2,3, Pavan Kumar Dhanyamraju 1, Morgann Klink 1, Sinisa Dovat 1, Mark Kester 4,5, Thomas P. Loughran, Jr 4,6, David Claxton 2,3, Arati Sharma 1,2,7
1Department of Pediatrics, Pennsylvania State University College of Medicine, 2Penn State Hershey Cancer Institute, Pennsylvania State University College of Medicine, 3Departments of Medicine, Division of Hematology and Oncology, Pennsylvania State University College of Medicine, 4University of Virginia Cancer Center, 5nanoSTAR Institute, University of Virginia, 6Division of Hematology and Oncology, Department of Medicine, University of Virginia School of Medicine, 7Department of Pharmacology, Pennsylvania State University College of Medicine

Mouse (Mus Musculus) models are being widely used to develop xenografts using human leukemia cells. These models provide a comparable biological system to study drug efficacy, pharmacodynamics, and pharmacokinetics. Modeling acute myeloid leukemia in immunocompromised mice is described in detail using the U937 cell line xenograft as an example.

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Neuroscience

A Fibrin-Enriched and tPA-Sensitive Photothrombotic Stroke Model
Yi-Min Kuo 1, Yu-Yo Sun 2, Chia-Yi Kuan 2
1Department of Anesthesiology, Taipei Veterans General Hospital, National Yang-Ming University School of Medicine, 2Department of Neuroscience, Center for Brain Immunology and Glia (BIG), University of Virginia School of Medicine

Traditional photothrombotic stroke (PTS) models mainly induce dense platelet aggregates of a high resistance to tissue plasminogen activator (tPA)-lytic treatment. Here a modified murine PTS model is introduced by co-injecting thrombin and photosensitive dye for photoactivation. The thrombin-enhanced PTS model produces mixed platelet:fibrin clots and is highly sensitive to tPA-thrombolysis.

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JoVE Core

Bone Marrow Transplantation Procedures in Mice to Study Clonal Hematopoiesis
Eunbee Park 1, Megan A. Evans 4, Heather Doviak 4, Keita Horitani 4, Hayato Ogawa 4, Yoshimitsu Yura 4, Ying Wang 2, Soichi Sano 3,4, Kenneth Walsh 1,4
1Department of Biochemistry and Molecular Genetics, University of Virginia School of Medicine, 2Department of Cardiology, Xinqiao Hospital, Army Medical University, 3Department of Cardiology, Osaka City University Graduate School of Medicine, 4Hematovascular Biology Center, Robert M. Berne Cardiovascular Research Center, University of Virginia School of Medicine

We describe three methods of bone marrow transplantation (BMT): BMT with total-body irradiation, BMT with shielded irradiation, and BMT method with no pre-conditioning (adoptive BMT) for the study of clonal hematopoiesis in mouse models.

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Bioengineering

Preparation and Characterization of Targeted Microbubbles
Galina B. Diakova 1, Matthew Wang 1, Sunil Unnikrishnan 1, Alexander L. Klibanov 1
1Division of Cardiovascular Medicine, Department of Medicine and Robert M Berne Cardiovascular Research Center, Department of Biomedical Engineering, Department of Radiology, University of Virginia School of Medicine

The goal of this protocol is to prepare, purify and characterize gas-filled microbubbles (targeted contrast agents for ultrasound molecular imaging). Two targeting systems are described: biotinylated bubbles adherent to streptavidin, and cyclic RGD peptide microbubbles that bind to αvβ3, a known tumor neovasculature biomarker.

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Developmental Biology

Directed Differentiation of Hemogenic Endothelial Cells from Human Pluripotent Stem Cells
Elizabeth A. Nelson 1,2, Jingyao Qiu 3,4, Nicholas W. Chavkin 1,2, Karen K. Hirschi 1,2,3,4,5
1Department of Cell Biology, University of Virginia, 2Cardiovascular Research Center, University of Virginia, 3Department of Medicine, Yale University School of Medicine, 4Department of Genetics, Yale University School of Medicine, 5Yale Cardiovascular Research Center, Yale University School of Medicine

Presented here is a simple protocol for the directed differentiation of hemogenic endothelial cells from human pluripotent stem cells in approximately 1 week.

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Developmental Biology

Isolation of Murine Retinal Endothelial Cells for Next-Generation Sequencing
Nicholas W. Chavkin 1,2, Shelby Cain 1, Kenneth Walsh 2,3,4, Karen K. Hirschi 1,2,4,5
1Department of Cell Biology, University of Virginia School of Medicine, 2Robert M. Berne Cardiovascular Research Center, University of Virginia School of Medicine, 3Department of Cardiology, University of Virginia School of Medicine, 4Hematovascular Biology Center, University of Virginia School of Medicine, 5Yale Cardiovascular Research Center, Yale University School of Medicine

This protocol describes a method for the isolation of murine postnatal retinal endothelial cells optimized for cell yield, purity, and viability. These cells are suitable for next-generation sequencing approaches.

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Bioengineering

Fabricating Highly Open Porous Microspheres (HOPMs) via Microfluidic Technology
Sheng-Chang Luo 1,2, Ying Wang 3, Ranjith Kumar Kankala 1,2, Yu Shrike Zhang 4, Ai-Zheng Chen 1,2
1Institute of Biomaterials and Tissue Engineering, Huaqiao University, 2Fujian Provincial Key Laboratory of Biochemical Technology, Huaqiao University, 3Affiliated Dongguan Hospital, Southern Medical University, 4Division of Engineering in Medicine, Department of Medicine, Brigham and Women's Hospital, Harvard Medical School

The present protocol describes the fabrication of poly(lactic-co-glycolic acid)-based highly open porous microspheres (HOPMs) via the single-emulsion formulation based facile microfluidic technology. These microspheres have potential applications in tissue engineering and drug screening.

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Neuroscience

Using a Bipolar Electrode to Create a Temporal Lobe Epilepsy Mouse Model by Electrical Kindling of the Amygdala
Yongchang Lu *1,2, Yang Dai *1,2, Siqi Ou 1,2, Yujing Miao 1, Ying Wang 1,2, Quanlei Liu 1,2, Yihe Wang 1,2, Penghu Wei 1,2, Yongzhi Shan 1,2, Guoguang Zhao 1,2,3
1Department of Neurosurgery, Xuanwu Hospital Capital Medical University, 2China Clinical Research Center for Epilepsy Capital Medical University, 3China Beijing Municipal Geriatric Medical Research Center

The amygdala plays a key role in temporal lobe epilepsy, which originates in and propagates from this structure. This article provides a detailed description of the fabrication of deep brain electrodes with both recording and stimulating functions. It introduces a model of medial temporal lobe epilepsy originating from the amygdala.

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A Photopolymerizable Hyaluronic Acid-Collagen Model of the Invasive Glioma Microenvironment with Interstitial Flow

A Photopolymerizable Hyaluronic Acid-Collagen Model of the Invasive Glioma Microenvironment with Interstitial Flow
Samantha Howerton 1,2, Yanping Liang 1, Jennifer Hammel 1,3, Benjamin Purow 4, Jennifer Munson 1,3
1Fralin Biomedical Research Institute at Virginia Tech Carilion, 2Translational Biology, Medicine, and Health Graduate Program, Virginia Tech, 3Department of Biomedical Engineering & Mechanics, Virginia Tech, 4Department of Neurology, University of Virginia School of Medicine

We present a method for replicating the glioma tumor microenvironment at the invasive front that incorporates interstitial fluid flow. This model is a hyaluronan-collagen I hydrogel in a tissue culture insert where a fluid pressure head can be applied. Invasion can be quantified, and cells can be isolated or lysed.

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