University of Virginia School of Medicine 11 articles published in JoVE Developmental Biology Isolation of Murine Retinal Endothelial Cells for Next-Generation Sequencing Nicholas W. Chavkin1,2, Shelby Cain1, Kenneth Walsh2,3,4, Karen K. Hirschi1,2,4,5 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. Neuroscience A Fibrin-Enriched and tPA-Sensitive Photothrombotic Stroke Model Yi-Min Kuo1, Yu-Yo Sun2, Chia-Yi Kuan2 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. Immunology and Infection Bone Marrow Transplantation Procedures in Mice to Study Clonal Hematopoiesis Eunbee Park1, Megan A. Evans4, Heather Doviak4, Keita Horitani4, Hayato Ogawa4, Yoshimitsu Yura4, Ying Wang2, Soichi Sano3,4, Kenneth Walsh1,4 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. Cancer Research Analyzing Tumor and Tissue Distribution of Target Antigen Specific Therapeutic Antibody Gururaj Shivange*1,2, Tanmoy Mondal*1,2, Evan Lyerly1,2, Jeremy Gatesman3, Jogender Tushir-Singh1,2 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. Medicine Porcine Model of Infrarenal Abdominal Aortic Aneurysm Alexander H. Shannon1, J. Michael Cullen1, Jolian J. Dahl1, Erik J. Scott1, Zachary Tyerman1, Michael D. Spinosa1, William G. Montgomery1, W. Forrest Johnston2, Guanyi Lu3, 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. Immunology and Infection Lentiviral CRISPR/Cas9-Mediated Genome Editing for the Study of Hematopoietic Cells in Disease Models Soichi Sano1, Ying Wang1, Megan A. Evans1, Yoshimitsu Yura1, Miho Sano1, Hayato Ogawa1, Keita Horitani1, Heather Doviak1, Kenneth Walsh1 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. Biochemistry Single-Molecule Tracking Microscopy - A Tool for Determining the Diffusive States of Cytosolic Molecules Julian M Rocha1, Andreas Gahlmann1,2 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. Medicine Murine Surgical Model of Topical Elastase Induced Descending Thoracic Aortic Aneurysm Zachary Tyerman1, Jolian Dahl1, Alexander Shannon1, W. Forrest Johnston2, Nicholas H. Pope1, Guanyi Lu3, Gilbert R. Upchurch Jr.3, Gorav Ailawadi1,4, Morgan Salmon1 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. Genetics A Non-random Mouse Model for Pharmacological Reactivation of Mecp2 on the Inactive X Chromosome Piotr Przanowski1, Zeming Zheng1, Urszula Wasko1, Sanchita Bhatnagar1,2 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. Medicine A Cell Culture Model of Resistance Arteries Lauren A. Biwer1,2, Christophe Lechauve3, Sheri Vanhoose4, Mitchell J. Weiss3, Brant E. Isakson1,2 1Department of Molecular Physiology and Biophysics, University of Virginia School of Medicine, 2Robert M. Berne Cardiovascular Research Center, University of Virginia School of Medicine, 3 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. Biology Measurement of Carbon Dioxide Production from Radiolabeled Substrates in Drosophila melanogaster Michelle L. Bland1 1Department of Pharmacology, University of Virginia This paper describes a method for the measurement of fuel oxidation in Drosophila melanogaster in which trace amounts of specific radiolabeled metabolic substrates are fed to flies. The exhaled radiolabeled CO2 that is a produced from fuel oxidation is collected and measured.
