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Institut National de la Santé et de la Recherche Médicale

7 ARTICLES PUBLISHED IN JoVE

Medicine

Transcriptomic Analysis of Human Retinal Surgical Specimens Using jouRNAl

Marie-Noëlle Delyfer^1,2,3,4, Najate Aït-Ali^1,2,3, Hawa Camara^1,2,3, Emmanuelle Clérin^1,2,3, Jean-François Korobelnik⁴, José-Alain Sahel^1,2,3, Thierry Léveillard^1,2,3

¹U968, Institut National de la Santé et de la Recherche Médicale, ²UMR S 968, Université Pierre et Marie Curie, ³UMR 7210, Centre National de la Recherche Scientifique, ⁴Départment d'Ophtalmologie, Centre Hospitalier Universitaire de Bordeaux

We used retinal samples from retinectomy for a transcriptomic analysis of retinal detachment. We developed a procedure that allows RNA conservation between the surgical blocks and the laboratory. We standardized a protocol to purify RNA by cesium chloride ultracentrifugation to assure that the purified RNAs are suitable for microarray analysis.

Neuroscience

Vibratome Sectioning Mouse Retina to Prepare Photoreceptor Cultures

Emmanuelle Clérin^1,4,5,6, Ying Yang^1,4,5,6, Valérie Forster^2,4,5,6, Valérie Fontaine^3,4,5,6, José-Alain Sahel^4,5,6, Thierry Léveillard^1,4,5,6

¹Department of Genetics, UMR_S 968, Institut de la Vision, ²Department of Visual Information, UMR_S 968, Institut de la Vision, ³Exploratory Team, UMR_S 968, Institut de la Vision, ⁴Sorbonne Universités, Paris 06, UMR_S 968, Institut de la Vision, ⁵INSERM, U968, Institut de la Vision, ⁶CNRS, UMR_7210, Institut de la Vision

Neural retina of a mouse aged 8 days is on top of a 4% gelatin block. After isolation of the photoreceptor layer (200 µm) by vibratome, the photoreceptors are seeded after mechanical and enzymatic dissociation for culture. The photoreceptor layer can be used for molecular, biochemical analyses or transplantation.

Medicine

Using Adeno-associated Virus as a Tool to Study Retinal Barriers in Disease

Ophélie Vacca^1,2,3, Brahim El Mathari^1,2,3, Marie Darche^1,2,3, José-Alain Sahel^1,2,3, Alvaro Rendon^1,2,3, Deniz Dalkara^1,2,3

¹Department of Therapeutics, Institut de la Vision, Sorbonne Universtés, UPMC Univ Paris 06, UMR_S 968, ²INSERM, U968, ³CNRS, UMR_7210

To investigate the blood-retinal barrier permeability and the inner limiting membrane integrity in animal models of retinal disease, we used several adeno-associated virus (AAV) variants as tools to label retinal neurons and glia. Virus mediated reporter gene expression is then used as an indicator of retinal barrier permeability.

Neuroscience

Purification of Mouse Brain Vessels

Anne-Cécile Boulay^1,2,3, Bruno Saubaméa⁴, Xavier Declèves⁴, Martine Cohen-Salmon^1,2,3

¹Collège de France, Center for Interdisciplinary Research in Biology (CIRB), Centre National de la Recherche Scientifique CNRS, Unité Mixte de Recherche 7241, Institut National de la Santé et de la Recherche Médicale, ²Centre Interdisciplinaire de Recherche en Biologie, ³MEMOLIFE Laboratory of Excellence and Paris Science, Lettre Research University, ⁴Université Paris Descartes, Faculté de Pharmacie, Université Paris Diderot

We describe a protocol allowing the purification of the mouse brain's vascular compartment. Isolated brain vessels include endothelial cells linked by tight junctions and surrounded by a continuous basal lamina, pericytes, vascular smooth muscle cells, as well as perivascular astroglial membranes.

Developmental Biology

Following Endocardial Tissue Movements via Cell Photoconversion in the Zebrafish Embryo

Renee Wei-Yan Chow^*1,2,3,4, Paola Lamperti^*1,2,3,4, Emily Steed^1,2,3,4, Francesco Boselli^1,2,3,4, Julien Vermot^1,2,3,4

¹Institut de Génétique et de Biologie Moléculaire et Cellulaire, ²UMR7104, Centre National de la Recherche Scientifique, ³U964, Institut National de la Santé et de la Recherche Médicale, ⁴Université de Strasbourg

This protocol describes a method for the photoconversion of Kaede fluorescent protein in endocardial cells of the living zebrafish embryo that enables the tracking of endocardial cells during atrioventricular canal and atrioventricular heart valve development.

Developmental Biology

Defined Xeno-free and Feeder-free Culture Conditions for the Generation of Human iPSC-derived Retinal Cell Models

Amélie Slembrouck-Brec^*1, Céline Nanteau^*1, José-Alain Sahel¹, Olivier Goureau¹, Sacha Reichman¹

¹Institut de la Vision, Sorbonne Université, INSERM, CNRS, F-75012 Paris, France

The production of specialized retinal cells from pluripotent stem cells is a turning point in the development of stem cell-based therapy for retinal diseases. The present paper describes a simple method for an efficient generation of retinal organoids and retinal pigmented epithelium for basic, translational, and clinical research.

Neuroscience

Cone-Enriched Cultures from the Retina of Chicken Embryos to Study Rod to Cone Cellular Interactions

Géraldine Millet-Puel¹, Myriam Pinault¹, Marie Cordonnier¹, Valérie Fontaine¹, José-Alain Sahel¹, Thierry Léveillard¹

¹Department of Genetics - Sorbonne Université, INSERM, CNRS, Institut de la Vision

We describe a method to obtain primary cultures of cone photoreceptors from the retina of chicken embryos and its use for high content screening.