Adenoviridae:
A family of non-enveloped viruses infecting mammals (Mastadenovirus) and birds (Aviadenovirus) or both (Atadenovirus). Infections may be asymptomatic or result in a variety of diseases.
JoVE Clinical and Translational Medicine
Assessing Replication and Beta Cell Function in Adenovirally-transduced Isolated Rodent Islets
1Department of Pediatrics, Indiana University School of Medicine, 2Department of Cellular & Integrative Physiology, Indiana University School of Medicine
This protocol allows one to identify factors that modulate functional beta cell mass to find potential therapeutic targets for the treatment of diabetes. The protocol consists of a streamlined method to assess islet replication and beta cell function in isolated rat islets following manipulation of gene expression with adenoviruses.
JoVE General
Preparation of Cell-lines for Conditional Knockdown of Gene Expression and Measurement of the Knockdown Effects on E4orf4-Induced Cell Death
Department of Molecular Microbiology, Faculty of Medicine, Technion - Israel Institute of Technology
Contribution of the ACF chromatin remodeling factor to E4orf4-induced cell death was measured. The protocol includes selection of cell clones in which doxycycline treatment induces conditional knockdown of the ACF subunits Acf1 and SNF2h, and use of the DAPI assay to measure E4orf4-induced cell death in the inducible cell lines.
JoVE Neuroscience
Dissection of Adult Mouse Utricle and Adenovirus-mediated Supporting-cell Infection
1Department of Pathology and Laboratory Medicine, Medical University of South Carolina, 2Department of Microbiology & Immunology, Medical University of South Carolina, 3National Institute on Deafness and Other Communication Disorders, National Institutes of Health
Mechanosensory hair cells are the receptor cells of the inner ear. The best-characterized in vitro model system of mature mammalian hair cells utilizes organ cultures of utricles from adult mice. We present the dissection of the adult mouse utricle, and we demonstrate adenovirus-mediated infection of supporting cells in cultured utricles.
JoVE Immunology and Infection
Chemoselective Modification of Viral Surfaces via Bioorthogonal Click Chemistry
Department of Chemistry, Stony Brook University
Adenovirus particles are engineered to contain either the unnatural amino acid analogue azidohomoalanine or the azido sugar O-GlcNAz. The azide group of each is chemoselectively ligated via "click" chemistry reactions as a means of viral surface modification.
JoVE Immunology and Infection
Engineering and Evolution of Synthetic Adeno-Associated Virus (AAV) Gene Therapy Vectors via DNA Family Shuffling
1Cluster of Excellence CellNetworks, Department of Infectious Diseases, Virology, Heidelberg University, 2Department of Infectious Diseases, Virology, Heidelberg University
We demonstrate the basic technique to molecularly engineer and evolve synthetic Adeno-associated viral (AAV) gene therapy vectors via DNA family shuffling. Moreover, we provide general guidelines and representative examples for selection and analysis of individual chimeric capsids with enhanced properties on target cells in culture or in mice.
JoVE Immunology and Infection
Efficient Recombinant Parvovirus Production with the Help of Adenovirus-derived Systems
1Tumour Virology Division F010, German Cancer Research Center (DKFZ), 2Inserm Unit 701, German Cancer Research Center (DKFZ)
Here we describe a protocol based only on cell infection, which improves the efficiency of recombinant parvovirus production by more than 100 fold in comparison to other protocols in use. This protocol relies on the use of a novel adenovirus 5-based helper containing the parvovirus VP transcription unit (Ad-VP).
JoVE Clinical and Translational Medicine
A Novel Surgical Approach for Intratracheal Administration of Bioactive Agents in a Fetal Mouse Model
1Molecular Virology and Gene Therapy, KU Leuven, 2Department of Woman and Child, KU Leuven, 3Neurobiology and Gene Therapy, KU Leuven, 4Division of Nuclear Medicine, KU Leuven, 5Biomedical NMR Unit/ MoSAIC, KU Leuven
We developed a novel surgical approach for intratracheal administration of bioactive agents into the mouse fetus. The delivery route is more efficient in targeting the fetal mouse lungs than the commonly used intra-amniotic injection. This procedure has to date not been described in a mouse model.
JoVE Immunology and Infection
Cost-effective Method for Microbial Source Tracking Using Specific Human and Animal Viruses
The study describes a cost-effective method for the identification of the source of fecal/urine contamination or contamination by nitrates in water using qPCR for the specific quantification of human/porcine/bovine DNA viruses, adenoviruses and polyomaviruses, proposed as MST tools.
JoVE Neuroscience
Lentivirus-mediated Genetic Manipulation and Visualization of Olfactory Sensory Neurons in vivo
Department of Cell Biology and Human Anatomy, School of Medicine, University of California, Davis
We present a lentiviral technique for genetic manipulation and visualization of single olfactory sensory neuron axon and its terminal arborization in vivo.
JoVE General
Adenovirus-mediated Genetic Removal of Signaling Molecules in Cultured Primary Mouse Embryonic Fibroblasts
Department of Molecular and Cellular Biology, University of Guelph
In this video we use an adenovirus carrying the Cre recombinase gene to infect primary mouse embryonic fibroblasts carrying a floxed Rac1 allele.
JoVE General
Genetic Modification and Recombination of Salivary Gland Organ Cultures
Department of Biological Sciences, University at Albany, SUNY
A technique to genetically manipulate epithelial cells within whole ex vivo cultured embryonic mouse submandibular glands (SMGs) using viral gene transfer is described. This method takes advantage of the innate ability of SMG epithelium and mesenchyme to spontaneously recombine after separation and infection of epithelial rudiments with adenoviral vectors.
JoVE Clinical and Translational Medicine
Neo-Islet Formation in Liver of Diabetic Mice by Helper-dependent Adenoviral Vector-Mediated Gene Transfer
1Department of Medicine, Baylor College of Medicine, 2Division of Diabetes, Endocrinology & Metabolism, Diabetes & Endocrinology Research Center, Baylor College of Medicine, 3Department of Molecular & Cellular Biology, Baylor College of Medicine
We describe hepatic neo-islet formation in STZ (streptozotocin)-induced diabetic mice by gene transfer of Neurogenin3 (Ngn3) and Betacellulin (Btc) using helper-dependent adenoviral vector (HDAd) and the reversal of hyperglycemia. Our method takes advantages of helper-dependent adenoviral vectors with their highly efficient in vivo transduction and the long lasting gene expression.
JoVE General
Protein Transfection of Mouse Lung
Department of Medicine, St. Luke's Roosevelt Medical Center
Transgenic mice or viral vectors have been used to increase protein expression within the lung. However, these techniques are time-consuming, technically challenging and have off-target effects that can confound results. Our protein transfection protocol uses a lipid based transfection reagent and an ultrafine microsprayer to uniformly deliver active protein to lung cells.
JoVE Immunology and Infection
Identifying Dysregulated Genes Induced by Kaposi's Sarcoma-associated Herpesvirus (KSHV)
Host cell factors play a critical role in the establishment and maintenance of Kaposi's sarcoma (KS). We outline methods to identify host cell factors altered in KSHV-infected DMVEC cells, and in KS tumor tissue. Cellular genes altered by virus will serve as potential target(s) for novel therapeutics.
JoVE Clinical and Translational Medicine
The CYP2D6 Animal Model: How to Induce Autoimmune Hepatitis in Mice
Pharmazentrum Frankfurt / ZAFES, Goethe University Hospital Frankfurt
Infection of mice with an Adenovirus expressing the major human autoantigen cytochrome P450 2D6 (hCYP2D6) recognized by sera of patients suffering from type 2 autoimmune hepatitis results in a persistent form of autoimmune-mediated liver disease characterized by extensive hepatitis, fibrosis and generation of a CYP2D6-specific immune response.
JoVE Clinical and Translational Medicine
Murine Bioluminescent Hepatic Tumour Model
1Cork Cancer Research Centre, Mercy University Hospital and Leslie C. Quick Jnr. Laboratory, University College Cork, 2Department of Computer Science, University College Cork, 3South Infirmary Victoria University Hospital
This article describes a procedure for the induction of orthotopic bioluminescent liver tumours in mice, and subsequent analysis of tumour growth confined to the liver using live whole body luminescence imaging.
JoVE General
Isolation and Genetic Manipulation of Adult Cardiac Myocytes for Confocal Imaging
Institute for Molecular Cell Biology, Universty of Saarland
Adult cardiac myocytes are primary cells that can be isolated from animal hearts and cultured for several days. Within this culture period adenoviral gene transfer can be used to express genetically encoded biosensors (GEBs) or fluorescent fusion proteins. Both approaches allow cellular investigations by means of confocal microscopy.
JoVE Immunology and Infection
Induction of Experimental Autoimmune Hypophysitis in SJL Mice
Department of Pathology, The Johns Hopkins University
This video shows how to induce autoimmune hypophysitis in SJL mice and how to assess its severity by histopathology.
JoVE Immunology and Infection
Generation of Multivirus-specific T Cells to Prevent/treat Viral Infections after Allogeneic Hematopoietic Stem Cell Transplant
Center for Cell and Gene Therapy, Baylor College of Medicine
A rapid, simple and cost-effective protocol for the generation of donor-derived multivirus-specific CTLs (rCTL) for infusion to allogeneic hematopoietic stem cell transplant (HSCT) recipients at risk of developing CMV, Adv or EBV infections. This manufacturing process is GMP-compliant and should ensure the broader implementation of T-cell immunotherapy beyond specialized centers.
JoVE Immunology and Infection
Expanding Cytotoxic T Lymphocytes from Umbilical Cord Blood that Target Cytomegalovirus, Epstein-Barr Virus, and Adenovirus
1Center for Cell and Gene Therapy, Baylor College of Medicine, 2Pathology and Immunology, Baylor College of Medicine, 3Department of Stem Cell Transplantation and Cellular Therapy, University of Texas M.D. Anderson Cancer Center, 4Medicine, Baylor College of Medicine, 5Department of Pediatrics, Baylor College of Medicine
Here we describe the first good manufacturing practice (GMP)-compliant method of producing virus-specific cytotoxic T lymphocytes (CTL) from umbilical cord blood, a source of predominantly naîve T cells.
JoVE Immunology and Infection
Murine Superficial Lymph Node Surgery
1Maisonneuve-Rosemont Hospital Research Center, 2Department of Microbiology and Immunology, University of Montreal, 3Department of Medicine, University of Montreal
To follow the progression of an immune response over time within the same mouse, lymph nodes can be sequentially removed by surgery. Here, we describe how this technique can be performed.
JoVE Clinical and Translational Medicine
Mouse Models for Graft Arteriosclerosis
1Department of Surgery, Yale University School of Medicine, 2Department of Pathology, Yale University School of Medicine
We describe protocols for our mouse graft arteriosclerois (GA) models which involve interposition of a mouse vessel segment into a recipient of the same inbred strain. By backcrossing additional genetic changes into the vessel donor, the model can assess the effect of specific genes on GA.
JoVE Clinical and Translational Medicine
Ex Vivo Culture of Patient Tissue & Examination of Gene Delivery
1Cork Cancer Research Centre, Mercy University Hospital and Leslie C. Quick Jnr. Laboratory, University College Cork, 2Department of Computer Science, University College Cork
This article describes the culture of patient tissue slices for gene delivery studies and subsequent analysis of gene expression using IVIS bioluminescence imaging.
JoVE Clinical and Translational Medicine
Repair of a Critical-sized Calvarial Defect Model Using Adipose-derived Stromal Cells Harvested from Lipoaspirate
1Department of Surgery, Stanford University, 2Department of Surgery, Duke University, 3Department of Surgery, Saint Joseph Mercy Hospital, 4School of Medicine, University of California, San Francisco, 5School of Dentistry, University of California, Los Angeles
This protocol describes the isolation of adipose-derived stromal cells from lipoaspirate and the creation of a 4 mm critical-sized calvarial defect to evaluate skeletal regeneration.
JoVE Immunology and Infection
Development of a Negative Selectable Marker for Entamoeba histolytica
Division of Infectious Disease and International Health, University of Virginia Health System
We report development of a negative selection system in E. histolytica based upon transgenic expression of a chimeric protein (FCU1) and selection with the prodrug 5-fluorocytosine. The FCU1 protein is a fusion of yeast cytosine deaminase and uracil phosphoribosyltransferase. Expression of FCU1 resulted in increased E. histolytica sensitivity towards 5-fluorocytosine.
JoVE General
Targeted Expression of GFP in the Hair Follicle Using Ex Vivo Viral Transduction
The hair follicle is a highly complex appendage of the skin containing a multiplicity of cell types. The follicle undergoes constant cycling through the life of the organism including growth and resorption with growth dependent on specific stem cells. The targeting of the follicle by genes and stem cells to change its properties, in particular, the nature of the hair shaft is, discussed. Hair follicle delivery systems are described, such as liposomes and viral vectors for gene therapy. The nature of the hair follicle stem cells is discussed, in particular, its pluripotency.
JoVE General
Assessment of Mitochondrial Functions and Cell Viability in Renal Cells Overexpressing Protein Kinase C Isozymes
The effects of activation of protein kinase C (PKC) isozymes on mitochondrial functions associated with respiration and oxidative phosphorylation and on cell viability are described. The approach adapts adenoviral technique to selectively overexpress PKC isozymes in primary cell culture and a variety of assays to determine mitochondrial functions and energy status of the cell.
JoVE Neuroscience
Intracranial Injection of Adeno-associated Viral Vectors
Neurobiology and Anatomy, University of Rochester
Here we present the intracranial injection of AAV vectors for fluorescent labeling of neurons and glia in the visual cortex.
JoVE Neuroscience
Stereotaxic Injection of a Viral Vector for Conditional Gene Manipulation in the Mouse Spinal Cord
1Département Nociception et Douleur, Institut des Neurosciences Cellulaires et Intégratives, Centre National de la Recherche Scientifique (CNRS), 2Departments of Anesthesiology and Pharmacology, Columbia University, 3Department of Anesthesiology, Niigata University Graduate School of Medical and Dental Sciences
Viral vectors allow for targeted gene manipulation. We demonstrate a method for conditional gene expression or ablation in the mouse spinal cord, using stereotaxic injection of a viral vector into the dorsal horn, a prominent site of synaptic contact between primary somatosensory afferents and neurons of the central nervous system.
JoVE Bioengineering
Tracking Hypoxic Signaling within Encapsulated Cell Aggregates
1Biomedical Engineering Program, University of South Carolina, 2Chemical Engineering Department, University of South Carolina
A method for photo-encapsulation of cells in a crosslinked PEG hydrogel is described. Hypoxic signaling within encapsulated murine insulinoma (MIN6) aggregates is tracked using a fluorescent marker system. This system allows serial examination of cells within a hydrogel scaffold and correlation of hypoxic signaling with changes in cell phenotype.
JoVE Immunology and Infection
Production and Titering of Recombinant Adeno-associated Viral Vectors
1School of Medical Sciences, College of Life Sciences and Medicine, University of Aberdeen, 2Translational Neuroscience Facility and Department of Physiology, School of Medical Sciences, University of New South Wales, 3Department of Biochemistry and Molecular Biophysics, Columbia University
Recombinant adeno-associated virus (rAAVs) vectors are becoming increasingly valuable for in vivo studies in animals. We describe how rAAVs can be produced in the laboratory and how these vectors can be titered to give an accurate reading of the number of infectious particles produced.
JoVE Clinical and Translational Medicine
High-Efficiency Transduction of Liver Cancer Cells by Recombinant Adeno-Associated Virus Serotype 3 Vectors
Department of Pediatrics, Division of Cellular and Molecular Therapy, University of Florida
In this article, we describe the identification of the adeno-associated virus serotype 3 (AAV3) as the most efficient vector for targeting human liver cancer cells.
JoVE Immunology and Infection
Recurrent Herpetic Stromal Keratitis in Mice, a Model for Studying Human HSK
Department of Ophthalmology, Saint Louis University
Most studies of herpetic corneal disease use a primary infection model. However, primary infection with HSV-1 does not typically lead to human disease. Here we describe a recurrent model of herpetic corneal disease, which more closely mimics human disease.
JoVE General
Isolation and Culture of Individual Myofibers and their Satellite Cells from Adult Skeletal Muscle
1Sprott Center for Stem Cell Research, Ottawa Hospital Research Institute, 2Department of Cellular and Molecular Medicine, University of Ottawa
Isolation and culture of myofibers is the gold standard in vitro system to study the transition of satellite cells through quiescence, activation and differentiation. Importantly, the single myofiber culture system preserves the myofiber/stem cell association, which is an essential component of the muscle stem cell niche.
JoVE Clinical and Translational Medicine
A Mouse Model of in Utero Transplantation
1Department of Surgery, University of California, 2Eli and Edythe Broad Center of Regeneration Medicine and Stem Cell Research, University of California, 3Biomedical Sciences Program, University of California
The mouse model of in utero transplantation is a versatile tool that can be used to study the potential clinical applications of stem cell transplantation and gene therapy in the fetus. In this protocol, we present a general approach to performing this technique
JoVE Neuroscience
Methods for Experimental Manipulations after Optic Nerve Transection in the Mammalian CNS
Department of Surgery, University of Toronto
Optic Nerve transection is a widely used model of adult CNS injury. This model is ideal for performing a number of experimental manipulations that target the retina globally or directly target the injured neuronal population of retinal ganglion cells.
JoVE General
PAR-CliP - A Method to Identify Transcriptome-wide the Binding Sites of RNA Binding Proteins
1Howard Hughes Medical Institute, Laboratory of RNA Molecular Biology, Rockefeller University, 2Berlin Institute for Medical Systems Biology, Max-Delbrück-Center for Molecular Medicine, 3Biozentrum der Universität Basel and Swiss Institute of Bioinformatics (SIB), 4Biozentrum der Universität Basel and Swiss Institute of Bioinformatics (SIB), 5Genomics Resource Center, Rockefeller University
RNA transcripts are subject to extensive posttranscriptional regulation that is mediated by a multitude of trans-acting RNA-binding proteins (RBPs). Here we present a generalizable method to identify precisely and on a transcriptome-wide scale the RNA binding sites of RBPs.
JoVE General
Production of Replication-Defective Retrovirus by Transient Transfection of 293T cells
Molecular Oncology Research Institute, Tufts University
This technique demonstrates an efficient way to prepare replication-defective retroviral stocks encoding a human oncogene, and subsequently used for induction of myeloproliferative disease in the mouse model.
JoVE Neuroscience
Generation of an Immortalized Murine Brain Microvascular Endothelial Cell Line as an In Vitro Blood Brain Barrier Model
Klinik und Poliklinik für Anästhesiologie, University of Wurzburg
This method describes how to isolate and immortalize microvascular endothelial cells from mouse brain. We describe a step-by-step protocol starting from the homogenization of brain tissue, digestion steps, seeding and immortalization of the cells. Usually, it takes about five weeks to obtain a homogenous, immortalized microvascular endothelial cell line.
JoVE Clinical and Translational Medicine
Rat Model of Blood-brain Barrier Disruption to Allow Targeted Neurovascular Therapeutics
Department of Neurological Surgery, Vanderbilt University School of Medicine
Blood-brain barrier disruption aids the delivery of certain drugs to the brain. Mannitol delivered intra-arterially shrinks cells surrounding blood vessels in order to physically disrupt the barrier.
JoVE Immunology and Infection
Ex Vivo Red Blood Cell Hemolysis Assay for the Evaluation of pH-responsive Endosomolytic Agents for Cytosolic Delivery of Biomacromolecular Drugs
1Department of Biomedical Engineering, Vanderbilt University, 2Vanderbilt Institute for Nanoscale Science & Engineering, Vanderbilt University, 3Interdisciplinary Materials Science Program, Vanderbilt University, 4Monroe Carell Jr. Children's Hospital, Vanderbilt University Medical Center, 5Department of Chemical & Biomolecular Engineering, Vanderbilt University, 6Department of Cancer Biology, Vanderbilt University
A hemolysis assay can be used as a rapid, high-throughput screen of drug delivery systems' cytocompatibility and endosomolytic activity for intracellular cargo delivery. The assay measures the disruption of erythrocyte membranes as a function of environmental pH.
JoVE Clinical and Translational Medicine
Labeling Stem Cells with Ferumoxytol, an FDA-Approved Iron Oxide Nanoparticle
1Department of Radiology, Molecular Imaging Program at Stanford (MIPS), 2Stanford School of Medicine, Stanford University
We describe a technique for labeling and tracking stem cells with FDA-approved, superparamagnetic iron oxide (SPIO), ferumoxytol (Feraheme). This cellular imaging technique that utilizes magnetic resonance (MR) imaging for visualization, is readily accessible for long-term monitoring and diagnosis of successful or unsuccessful stem cell engraftments in patients.
JoVE General
Evaluation of Respiratory System Mechanics in Mice using the Forced Oscillation Technique
1Meakins-Christie Laboratories, Department of Medicine, McGill University, 2SCIREQ Scientific Respiratory Equipment Inc.
The present protocol provides a detailed step-by-step description of the procedures required to execute measurements of respiratory system mechanics as well as the assessment of airway responsiveness to inhaled methacholine in mice using the forced oscillation technique (flexiVent; SCIREQ Inc, Montreal, Qc, Canada).
JoVE Immunology and Infection
Glass Wool Filters for Concentrating Waterborne Viruses and Agricultural Zoonotic Pathogens
1Wisconsin Water Science Center, United States Geological Survey, 2University of Wisconsin – Madison, 3Agricultural Research Service, United States Department of Agriculture, 4Alaska Science Center, United States Geological Survey
Glass wool filters have been used to concentrate waterborne viruses by a number of research groups around the world. Here we show a simple approach for constructing glass wool filters and demonstrate the filters are also effective in concentrating waterborne viral, bacterial and protozoan pathogens.
JoVE Clinical and Translational Medicine
Ex Vivo Infection of Live Tissue with Oncolytic Viruses
Center for Innovative Cancer Research, Ottawa Hospital Research Institute (OHRI)
Oncolytic viruses are promising for cancer therapeutics. The ability to ascertain the infectability of live tissue specimens obtained from patients prior to treatment is a unique advantage of this therapeutic approach. This protocol describes how to process tissues for ex vivo infection with oncolytic virus and subsequent viral quantification.
JoVE Immunology and Infection
Generation of Organotypic Raft Cultures from Primary Human Keratinocytes
1Department of Microbiology & Immunology, University of North Carolina-Chapel Hill, 2Lineberger Cancer Center, University of North Carolina-Chapel Hill
An in vitro method to mimic in vivo epithelial differentiation is described. Many viruses target epithelial cells as part of their viral life cycle, and this method provides a means of examining virus:host interactions that more closely resembles that which occurs in vivo. This technique can be used with primary keratinocytes, established cell lines, as well as normal or diseased biopsy tissue.
JoVE General
A Convenient and General Expression Platform for the Production of Secreted Proteins from Human Cells
Department of Laboratory Medicine and Pathobiology, University of Toronto
In the post-human genomics era, the availability of recombinant proteins in native conformations is crucial to structural, functional and therapeutic research and development. Here, we describe a test- and large-scale protein expression system in human embryonic kidney 293T cells that can be used to produce a variety of recombinant proteins.
JoVE Immunology and Infection
Preparation of Viral DNA from Nucleocapsids
Department of Molecular Biology, Princeton University
We describe the process of isolating high purity herpesvirus nucleocapsid DNA from infected cells. The final DNA captured from solution is of high concentration and purity, making it ideally suited for high-throughput sequencing, high fidelity PCR reactions, and transfections to produce new viral recombinants.
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