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University Hospitals of Geneva

5 ARTICLES PUBLISHED IN JoVE

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Biochemistry

Spectrophotometric Screening for Potential Inhibitors of Cytosolic Glutathione S-Transferases
Shannon K. D. Robin 1,2, Marc Ansari *1,3, Chakradhara Rao S. Uppugunduri *1,3
1Research platform of Pediatric Onco-Hematology, Department of Paediatrics, Gynaecology and Obstetrics, Faculty of Medicine, University of Geneva, 2Section of Biology, Faculty of Science, University of Geneva, 3Onco-Hematology Unit, Department of Women, Children-Adolescents, University Hospitals of Geneva

Glutathione S-transferases (GSTs) are detoxification enzymes involved in the metabolism of numerous chemotherapeutic drugs. Overexpression of GSTs is correlated with cancer chemotherapy resistance. One way to counter this phenotype is to use inhibitors. This protocol describes a method using a spectrophotometric assay to screen for potential GST inhibitors.

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Medicine

Induction and Analysis of Oxidative Stress in Sleeping Beauty Transposon-Transfected Human Retinal Pigment Epithelial Cells
Thais Bascuas 1,2, Martina Kropp 1,2, Nina Harmening 1,2, Mohammed Asrih 1, Zsuzsanna Izsvák 3, Gabriele Thumann 1,2
1Experimental Ophthalmology, University of Geneva, 2Department of Ophthalmology, University Hospitals of Geneva, 3Max Delbrück Center for Molecular Medicine

We present a protocol for the development and use ofan oxidative stress-model by treating retinal pigment epithelial cells with H2O2, analyzing cell morphology, viability, density, glutathione, and UCP-2 level. It is a useful model to investigate the antioxidant effect of proteins secreted by transposon-transfected cells to treat neuroretinal degeneration.

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Medicine

Electroporation-Based Genetic Modification of Primary Human Pigment Epithelial Cells Using the Sleeping Beauty Transposon System
Sandra Johnen 1, Nina Harmening 2,3, Corinne Marie 4,5, Daniel Scherman 4, Zsuzsanna Izsvák 6, Zoltán Ivics 7, Peter Walter 1, Gabriele Thumann 2,3
1Department of Ophthalmology, University Hospital RWTH Aachen, 2Experimental Ophthalmology, University of Geneva, 3Department of Ophthalmology, University Hospitals of Geneva, 4Université de Paris, CNRS, INSERM, UTCBS, Unité des technologies Chimiques et Biologiques pour la Santé, 5Chimie ParisTech, PSL Research University, 6Max Delbrück Center for Molecular Medicine in the Helmholtz Association, 7Division of Medical Biotechnology, Paul-Ehrlich-Institute

We have developed a protocol to transfect primary human pigment epithelial cells by electroporation with the gene encoding pigment epithelium-derived factor (PEDF) using the Sleeping Beauty (SB) transposon system. Successful transfection was demonstrated by quantitative polymerase chain reaction (qPCR), immunoblotting, and enzyme-linked immunosorbent assay (ELISA).

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Biology

Isolation, Culture, and Genetic Engineering of Mammalian Primary Pigment Epithelial Cells for Non-Viral Gene Therapy
Thais Bascuas *1,2, Martina Kropp *1,2, Nina Harmening 1,2, Brittany M. Wong 1, Sandra Johnen 3, Zsuzsanna Izsvák 4, Gabriele Thumann 1,2
1Experimental Ophthalmology, University of Geneva, 2Department of Ophthalmology, University Hospitals of Geneva, 3Department of Ophthalmology, University Hospital RWTH Aachen, 4Max Delbrück Center for Molecular Medicine

Here, a protocol to isolate and transfect primary iris and retinal pigment epithelial cells from various mammals (mice, rat, rabbit, pig, and bovine) is presented. The method is ideally suited to study ocular gene therapy approaches in various set-ups for ex vivo analyses and in vivo studies transferable to humans.

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Neuroscience

Fluorescence-Activated Cell Sorting-Radioligand Treated Tissue (FACS-RTT) to Determine the Cellular Origin of Radioactive Signal
Quentin Amossé 1,2, Kelly Ceyzériat 1,3,4, Stergios Tsartsalis 1,5, Benjamin B. Tournier 1,2, Philippe Millet 1,2
1Division of Adult Psychiatry, Department of Psychiatry, University Hospitals of Geneva, 2Department of Psychiatry, University of Geneva, 3Division of Nuclear medicine and Molecular Imaging, University Hospitals of Geneva, 4Division of Radiation Oncology, Department of Oncology, University Hospitals of Geneva, 5Department of Brain Sciences, Faculty of Medicine, Imperial College London

Fluorescence-Activated Cell Sorting-Radioligand Treated Tissue (FACS-RTT) is a powerful tool to study the role of the 18 kDa translocator protein or Serotonin 5HT2A-receptor expression in Alzheimer's Disease at a cellular scale. This protocol describes the ex-vivo application of FACS-RTT in the TgF344-AD rat model.

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