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Tanja Grau, Stylianos Michalakis, Thomas Euler; Viral transfer of the genetically-encoded chloride indicator Clomeleon into ChAT/Cre retinae to study chloride dynamics in “starburst” amacrine cells. Invest. Ophthalmol. Vis. Sci. 2013;54(15):2504.
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Starburst amacrine cells have been shown to play an important role for the retinal computation of visual motion direction (direction selectivity, DS), with their dendrites providing direction-selective input to DS ganglion cells. It was proposed that differential distribution of chloride importers and exporters along starburst cell dendrites lead to the formation of an asymmetrical distribution of chloride that contributes to the dendritic DS computation in starburst cells (Gavrikov et al., 2003, 2006). Here we aimed at generating a model that will allow investigating the physiological distribution of chloride and its potential functional role in starburst amacrine cells.
A Cre-recombinase dependent viral expression construct, pAAV-Flex-Clomeleon, was designed by insertion of PCR-amplified Clomeleon complementary DNA into pAAV-Flex-Arch-GFP vector (Plasmid #22222, Addgene) by replacement of the Arch-GFP sequence. Cloning and mutagenesis were performed by standard techniques and confirmed by DNA sequencing. Recombinant adeno-associated virus (AAV) particles were produced by triple calcium phosphate transfection of 293T cells with pAdDeltaF6, pAAV2/7Y732F and pAAV-Flex-Clomeleon plasmids followed by iodixanol-gradient purification and ion exchange chromatography on a HiTrap Q Sepharose column (Michalakis et al., 2010). Light-Cycler technology (Roche Applied Science) was used to determine genomic recombinant AAV titers. AAVs carrying a reversed and double-floxed Clomeleon were delivered into the vitreous of 21 day old ChAT/Cre mice. Three weeks post-injection ChAT/Cre retinae were removed and Clomeleon expression detected by two-photon microscopy.
Using recombinant adeno-associated viruses Clomeleon, a fluorescent chloride indicator protein (Kuner & Augustin, 2000), can be successfully delivered and expressed in starburst amacrine cells of the mouse retina. Preliminary imaging data suggests that Clomeleon is functional in the targeted cells.
The chloride biosensor Clomeleon can be functionally and selectively expressed in starburst amacrine cells using a combination of ChAT/Cre mice and recombinant AAV vectors.
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