title: AAV Vectors for FRET-Based Analysis of Protein-Protein Interactions in Photoreceptor Outer Segments.
abstract: 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.
authors: Department of Pharmacy - Center for Integrated Protein Science Munich (CiPSM), Ludwig-Maximilians-Universität MünchenMunich, Germany; Department of Pharmacy - Center for Drug Research, Ludwig-Maximilians-Universität MünchenMunich, Germany.
authors: Elvir
authors: E
authors: Becirovic
authors: Department of Pharmacy - Center for Integrated Protein Science Munich (CiPSM), Ludwig-Maximilians-Universität MünchenMunich, Germany; Department of Pharmacy - Center for Drug Research, Ludwig-Maximilians-Universität MünchenMunich, Germany.
authors: Sybille
authors: S
authors: Böhm
authors: Department of Pharmacy - Center for Integrated Protein Science Munich (CiPSM), Ludwig-Maximilians-Universität MünchenMunich, Germany; Department of Pharmacy - Center for Drug Research, Ludwig-Maximilians-Universität MünchenMunich, Germany.
authors: Ong N P
authors: ON
authors: Nguyen
authors: Department of Pharmacy - Center for Integrated Protein Science Munich (CiPSM), Ludwig-Maximilians-Universität MünchenMunich, Germany; Department of Pharmacy - Center for Drug Research, Ludwig-Maximilians-Universität MünchenMunich, Germany.
authors: Lisa M
authors: LM
authors: Riedmayr
authors: Department of Pharmacy - Center for Integrated Protein Science Munich (CiPSM), Ludwig-Maximilians-Universität MünchenMunich, Germany; Department of Pharmacy - Center for Drug Research, Ludwig-Maximilians-Universität MünchenMunich, Germany.
authors: Verena
authors: V
authors: Hammelmann
authors: Department of Pharmacy - Center for Integrated Protein Science Munich (CiPSM), Ludwig-Maximilians-Universität MünchenMunich, Germany; Department of Pharmacy - Center for Drug Research, Ludwig-Maximilians-Universität MünchenMunich, Germany.
authors: Christian
authors: C
authors: Schön
authors: Department of Pharmacy - Center for Integrated Protein Science Munich (CiPSM), Ludwig-Maximilians-Universität MünchenMunich, Germany; Department of Pharmacy - Center for Drug Research, Ludwig-Maximilians-Universität MünchenMunich, Germany.
authors: Elisabeth S
authors: ES
authors: Butz
authors: Department of Pharmacy - Center for Integrated Protein Science Munich (CiPSM), Ludwig-Maximilians-Universität MünchenMunich, Germany; Department of Pharmacy - Center for Drug Research, Ludwig-Maximilians-Universität MünchenMunich, Germany.
authors: Christian
authors: C
authors: Wahl-Schott
authors: Department of Pharmacy - Center for Integrated Protein Science Munich (CiPSM), Ludwig-Maximilians-Universität MünchenMunich, Germany; Department of Pharmacy - Center for Drug Research, Ludwig-Maximilians-Universität MünchenMunich, Germany.
authors: Martin
authors: M
authors: Biel
authors: Department of Pharmacy - Center for Integrated Protein Science Munich (CiPSM), Ludwig-Maximilians-Universität MünchenMunich, Germany; Department of Pharmacy - Center for Drug Research, Ludwig-Maximilians-Universität MünchenMunich, Germany.
authors: Stylianos
authors: S
authors: Michalakis
keywords: AAV; FRET; adeno-associated viral vectors; fluorescence resonance energy transfer; outer segment; photoreceptor; protein-protein interaction