Hereditary retinal degenerative disorders such as retinitis pigmentosa (RP) are among the leading causes of blindness worldwide. Optogenetic gene therapy, introduces light-sensitive proteins into surviving retinal cells. Opto-mGluR6 is a chimeric protein consisting of the intracellular domain of the ON-bipolar cell specific receptor, mGluR6, and the light-sensing domain of melanopsin. Melanopsin, is a blue-light-sensitive retinal photopigment (mouse λ = 467 nm). Opto-mGluR6 activate K+ channels, therefore HEK293 cell line that stably express GIRK channel can be a model for studying the aforesaid optogenetic tool. This study aims to express Opto-mGluR6 in HEK-GIRK cell line.

Opto-mGluR6 gene was cloned into pAAV-MCS-IRES-EGFP plasmid. To check the accuracy of cloning, the resultant constructs were subjected to digestion and sequencing experiments. HEK-GIRK stable cells were transfected with the constructs using calcium phosphate precipitation method. After 48 hours, expression of constructs was examined. Expression of Opto-mGluR6 in HEK-GIRK stable cell line was assessed by RT-PCR.

pAAV-MCS-IRES-EGFP-Opto-mGluR6 plasmid was constructed. Digestion and sequence analysis revealed the true identity of the construct. Gene expression for Opto-mGluR6 was confirmed in transfected HEK-GIRK cell line.

Expression of Opto-mGluR6 in HEK-GIRK cell culture was successfully confirmed.