AAV vectors for FRET-based analysis of protein-protein interactions in photoreceptor outer segments

• Main Authors: Elvir Becirovic, Sybille Boehm, Ong Nam Phuong Nguyen, Lisa Maria Riedmayr, Verena Hammelmann, Christian Schön, Elisabeth Susanne Butz, Christian Wahl-Schott, Martin Biel, Stylianos Michalakis
• Format: Article
• Language: English
• Published: Frontiers Media S.A. 2016-07-01
• Series: Frontiers in Neuroscience
• Subjects: Fluorescence Resonance Energy Transfer; aav; FRET; photoreceptor; protein-protein interaction; adeno associated viral vectors
• Online Access: http://journal.frontiersin.org/Journal/10.3389/fnins.2016.00356/full

Fluorescence resonance energy transfer (FRET) is a powerful method for the detection and quantification of stationary and dynamic protein-protein interactions. Technical limitations have hampered systematic in vivo FRET experiments to study protein-protein interactions in their native environment. Here, we describe a rapid and robust protocol that combines adeno-associated virus (AAV) vector-mediated in vivo delivery of genetically encoded FRET partners with ex vivo FRET measurements. The method was established on acutely isolated outer segments of murine rod and cone photoreceptors and relies on the high co-transduction efficiency of retinal photoreceptors by co-delivered AAV vectors. The procedure can be used for the systematic analysis of protein-protein interactions of wild type or mutant outer segment proteins in their native environment. Conclusively, our protocol can help to characterize the physiological and pathophysiological relevance of photoreceptor specific proteins and, in principle, should also be transferable to other cell types.