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University Hospitals of Geneva

5 ARTICLES PUBLISHED IN JoVE

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.

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 Asrih¹, Zsuzsanna Izsvák³, 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.

Medicine

Electroporation-Based Genetic Modification of Primary Human Pigment Epithelial Cells Using the Sleeping Beauty Transposon System

Sandra Johnen¹, Nina Harmening^2,3, Corinne Marie^4,5, Daniel Scherman⁴, Zsuzsanna Izsvák⁶, Zoltán Ivics⁷, Peter Walter¹, 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).

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. Wong¹, Sandra Johnen³, Zsuzsanna Izsvák⁴, 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.

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 5HT_2A-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.