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Heidelberg University

17 ARTICLES PUBLISHED IN JoVE

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Immunology and Infection

Engineering and Evolution of Synthetic Adeno-Associated Virus (AAV) Gene Therapy Vectors via DNA Family Shuffling
Eike Kienle *1, Elena Senís *1, Kathleen Börner 2, Dominik Niopek 1, Ellen Wiedtke 1, Stefanie Grosse 1, Dirk Grimm 1
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.

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Developmental Biology

Tracking Cells in GFP-transgenic Zebrafish Using the Photoconvertible PSmOrange System
Carlo A. Beretta 1,2,3, Nicolas Dross 2, Ulrike Engel 2, Matthias Carl 1
1Medical Faculty Mannheim, Department of Cell and Molecular Biology, Heidelberg University, 2COS and Nikon Imaging Center at the University of Heidelberg, Heidelberg University, 3Excellenzcluster CellNetworks, University of Heidelberg

We established the photoconvertible PSmOrange system as a powerful, straight-forward and cost inexpensive tool for in vivo cell tracking in GFP transgenic backgrounds. This protocol describes its application in the zebrafish model system.

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Medicine

A Choroid Plexus Epithelial Cell-based Model of the Human Blood-Cerebrospinal Fluid Barrier to Study Bacterial Infection from the Basolateral Side
Stefanie Dinner 1, Julia Borkowski 1, Carolin Stump-Guthier 1, Hiroshi Ishikawa 2, Tobias Tenenbaum 1, Horst Schroten 1, Christian Schwerk 1
1Department of Pediatrics, Medical Faculty Mannheim, Heidelberg University, 2Department of NDU Life Sciences, Nippon Dental University

The epithelial cells of the choroid plexus (CP) form the blood-cerebrospinal fluid barrier (BCSFB). An in vitro model of the BCSFB employs human choroid plexus papilloma (HIBCPP) cells. This article describes culturing and basolateral infection of HIBCPP cells using a cell culture filter insert system.

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Developmental Biology

In Vivo Imaging of Transgenic Gene Expression in Individual Retinal Progenitors in Chimeric Zebrafish Embryos to Study Cell Nonautonomous Influences
Stefanie Dudczig 1,2, Peter D. Currie 2, Lucia Poggi 3, Patricia R. Jusuf 1,2
1School of Biosciences, The University of Melbourne, 2Australian Regenerative Medicine Institute (ARMI), Monash University, 3The David J Apple Center for Vision Research, Department of Ophthalmology, Heidelberg University

Live tracking of individual WT retinal progenitors in distinct genetic backgrounds allows for the assessment of the contribution of cell non-autonomous signaling during neurogenesis. Here, a combination of gene knockdown, chimera generation via embryo transplantation and in vivo time-lapse confocal imaging was utilized for this purpose.

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Neuroscience

Analysis of Immune Cells in Single Sciatic Nerves and Dorsal Root Ganglion from a Single Mouse Using Flow Cytometry
Asa S. Hidmark 1, Peter P. Nawroth 1,2,3, Thomas Fleming 1,3
1Department of Medicine I and Clinical Chemistry, University Hospital of Heidelberg, 2Institute for Diabetes and Cancer IDC Helmholtz Center Munich, Germany & Joint Heidelberg-IDC Translational Diabetes Program, 3German Center for Diabetes Research (DZD)

Quantitative analysis of cell content within the murine sciatic nerve is difficult due to the scarcity of the tissue. This protocol describes a method for tissue digestion and preparation that provides sufficient cells for flow cytometry analysis of immune cell populations from nerves of individual mice.

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Medicine

Studying Diabetes Through the Eyes of a Fish: Microdissection, Visualization, and Analysis of the Adult tg(fli:EGFP) Zebrafish Retinal Vasculature
Lucas Moritz Wiggenhauser 1, Katharina Kohl 2, Nadine Dietrich 2, Hans-Peter Hammes 2, Jens Kroll 1
1Department of Vascular Biology and Tumorangiogenesis, Center for Biomedicine and Medical Technology Mannheim (CBTM), Medical Faculty Mannheim, Heidelberg University, 2V. Medical Clinic, Medical Faculty Mannheim, Heidelberg University

Here, we discuss a method protocol which will allow an easy analysis of the adult tg(fli:EGFP) zebrafish retinal vasculature as a fast read-out in settings of long-term vascular pathologies linked to neoangiogenesis and structural changes.

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Biochemistry

Split-BioID — Proteomic Analysis of Context-specific Protein Complexes in Their Native Cellular Environment
Isabel M. Schopp 1,2, Julien Béthune 1,2
1Cluster of excellence CellNetworks, Heidelberg University, 2Heidelberg University Biochemistry Center (BZH)

We provide a step-by-step protocol for split-BioID, a protein fragments-complementation assay based on the proximity-labeling technique BioID. Activated on the interaction of two given proteins, it allows the proteomics analysis of context-dependent protein complexes in their native cellular environment. The method is simple, cost-effective and only requires standard laboratory equipment.

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Biochemistry

Plate-based Large-scale Cultivation of Caenorhabditis elegans: Sample Preparation for the Study of Metabolic Alterations in Diabetes
Katharina Kohl 1, Thomas Fleming 2,3, Kübra Acunman 1, Hans-Peter Hammes 1,4, Michael Morcos *1, Andrea Schlotterer *1
15th Medical Department, Medical Faculty Mannheim, Heidelberg University, 2Department of Internal Medicine, Heidelberg University, 3German Center for Diabetes Research (DZD), 4European Center for Angioscience, Medical Faculty Mannheim, Heidelberg University

This protocol describes a method for the large-scale cultivation of Caenorhabditis elegans on solid media. As an alternative to liquid culture, this protocol allows obtaining parameters of different scales under plate-based cultivation. This increases the comparability of results by omitting the morphological and metabolic differences between liquid and solid media culture.

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Bioengineering

Correlative Light Electron Microscopy (CLEM) for Tracking and Imaging Viral Protein Associated Structures in Cryo-immobilized Cells
Rachel Santarella-Mellwig 1, Uta Haselmann 2, Nicole L. Schieber 1, Paul Walther 3, Yannick Schwab 1, Claude Antony 1, Ralf Bartenschlager 2,4, Inés Romero-Brey 2
1European Molecular Biology Laboratory, 2Department of Infectious Diseases, Molecular Virology, Heidelberg University, 3Central Facility for Electron Microscopy, Ulm University, 4Heidelberg Partner Site, German Center for Infection Research

A correlative light electron microscopy (CLEM) method is applied to image virus-induced intracellular structures via electron microscopy (EM) in cells that are previously selected by light microscopy (LM). LM and EM are combined as a hybrid imaging approach to achieve an integrated view of virus-host interactions.

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Cancer Research

Paramyxoviruses for Tumor-targeted Immunomodulation: Design and Evaluation Ex Vivo
Johannes P.W. Heidbuechel 1,2, Christine E. Engeland 1,3
1Department of Translational Oncology, German Cancer Research Center (DKFZ) and National Center for Tumor Diseases (NCT), 2Faculty of Biosciences, Heidelberg University, 3Department of Medical Oncology, NCT and Heidelberg University Hospital

This protocol describes a detailed workflow for the generation and ex vivo characterization of oncolytic viruses for expression of immunomodulators, using measles viruses encoding bispecific T cell engagers as an example. Application and adaptation to other vector platforms and transgenes will accelerate the development of novel immunovirotherapeutics for clinical translation.

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Education

A Simple Approach to Perform TEER Measurements Using a Self-Made Volt-Amperemeter with Programmable Output Frequency
Marianne Theile 1, Linus Wiora 1, Dominik Russ 1, Jonas Reuter 1, Hiroshi Ishikawa 2, Christian Schwerk 3, Horst Schroten 3, Stefan Mogk 1
1Interfaculty Institute of Biochemistry, University of Tübingen, 2Laboratory of Clinical Regenerative Medicine, Department of Neurosurgery, Faculty of Medicine, University of Tsukuba, 3Department of Pediatrics, Medical Faculty Mannheim, Heidelberg University

Here, we demonstrate how to set up an inexpensive volt-amperemeter with programmable output frequency that can be used with commercially available chopstick electrodes for transepithelial/endothelial electrical resistance measurements.

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Biology

A High Output Method to Isolate Cerebral Pericytes from Mouse
Anupriya Mehra 1, Lucie Dehouck 1, Elodie Vandenhaute 1, Marc Fatar 2, Laurence Fenart 1, Fabien Gosselet 1
1Laboratory of the Blood Brain Barrier, University of Artois, 2Department of Neurology, Universitätsmedizin Mannheim, Heidelberg University

We present a protocol for the extraction of murine cerebral pericytes. Based on an antibiotic-free enrichment oriented pericyte extraction, this protocol is a valuable tool for in vitro studies providing high purity and high yield, thus decreasing the number of experimental animals used.

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Biology

A Modified Surgical Model of Hind Limb Ischemia in ApoE-/- Mice using a Miniature Incision
Kaixuan Yan 1,2, Jiaxing Zheng 1,2, Frank G. Zöllner 3,4, Kay Schwenke 1, Prama Pallavi 1,2, Michael Keese 1,2
1Department of Surgery, Medical Faculty Manheim, Heidelberg University, 2European Center of Angioscience ECAS, Medical Faculty Manheim, Heidelberg University, 3Computer-Assisted Clinical Medicine, Mannheim Institute for Intelligent Systems in Medicine, Medical Faculty Mannheim, Heidelberg University, 4Cooperative Core Facility Animal Scanner ZI, Medical Faculty Mannheim, Heidelberg University

This article demonstrates an efficient surgical approach to establish acute ischemia in mice with a small incision. This approach can be applied by most research groups without any laboratory upgrades.

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Neuroscience

Live Imaging of the Mitochondrial Glutathione Redox State in Primary Neurons using a Ratiometric Indicator
Athanasios Katsalifis 1, Angela Maria Casaril 1, Constanze Depp 2, Carlos Bas-Orth 1
1Department of Medical Cell Biology, Institute for Anatomy and Cell Biology, Heidelberg University, 2Department of Neurogenetics, Max-Planck-Institute for Experimental Medicine

This article describes a protocol to determine differences in basal redox state and redox responses to acute perturbations in primary hippocampal and cortical neurons using confocal live microscopy. The protocol can be applied to other cell types and microscopes with minimal modifications.

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Bioengineering

Control of Cell Adhesion using Hydrogel Patterning Techniques for Applications in Traction Force Microscopy
Joel Christian 1, Johannes W. Blumberg 2, Dimitri Probst 2, Cristina Lo Giudice 1, Sandra Sindt 3, Christine Selhuber-Unkel 3, Ulrich S. Schwarz 2, Elisabetta Ada Cavalcanti-Adam 1
1Department of Cellular Biophysics, Max Planck Institute for Medical Research, 2Institute for Theoretical Physics and BioQuant, Heidelberg University, 3Institute for Molecular Systems Engineering (IMSE), Heidelberg University

Near-UV lithography and traction force microscopy are combined to measure cellular forces on micropatterned hydrogels. The targeted light-induced release of single cells enables a high number of measurements on the same sample.

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Biology

Evaluating Toxicity of Chemicals using a Zebrafish Vibration Startle Response Screening System
Gaëlle Hayot 1, Daniel Marcato 1,2, Christina A. Cramer von Clausbruch 1, Giuseppina Pace 1, Uwe Strähle 1,3, John K. Colbourne 4, Christian Pylatiuk 5, Ravindra Peravali 1, Carsten Weiss 1, Stefan Scholz 6, Thomas Dickmeis 1
1Institute of Biological and Chemical Systems - Biological Information Processing, Karlsruhe Institute of Technology - Campus Nord, 2DITABIS AG - Digital Biomedical Imaging Systems AG, 3Centre for Organismal Studies, Heidelberg University, 4School of Biosciences, University of Birmingham, 5Institute for Automation and Applied Informatics, Karlsruhe Institute of Technology - Campus Nord, 6Department of Bioanalytical Ecotoxicology, Helmholtz-Centre for Environmental Research - UFZ

We describe a screening system's workflow and data analysis for evaluating chemical compound toxicity based on the zebrafish embryo vibration startle response. The system records the movements of zebrafish embryos upon exposure to a vibration stimulus and allows for an integrated evaluation of general toxicity/lethality and neuromuscular toxicity.

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Developmental Biology

Differentiation and Characterization of Osteoclasts from Human Induced Pluripotent Stem Cells
Alexander Blümke 1,2, Jessica Simon 1, Elizabeth Leber 1, Marta Scatena 1, Cecilia M. Giachelli 1
1Department of Bioengineering, Department of Medicine, University of Washington, 2Department of Orthopedics and Trauma Surgery, Medical Faculty Mannheim, Heidelberg University

This protocol presents the differentiation of human osteoclasts from induced pluripotent stem cells (iPSCs) and describes methods for the characterization of osteoclasts and osteoclast precursors.

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