Optogenetic is a biological technique that uses light to restore neurons’ function in neurological disorders or to fix vision in visual impairment. Novel optogenetic tools belong to the GPCR protein (Opto-mGluR6) family that activates K+ channels in neural cells’ excitation. G protein-coupled inwardly-rectifying potassium channels (GIRKs) are a family of potassium ion channels which are activated via Opto-mGluR6. Therefore HEK293 cell line that stably express GIRK channels stands as a validated model for studying the principal optogenetic chimeric proteins. This study aims to generate stable GIRK expressing HEK293 cell line as a noteworthy model cell line.

PcDNA3-GIRK1 and pcDNA3-GIRK2 were obtained as a gift from Dr. Terry Hebert, recovered transformed to XL10 E.coli. Plasmid mini preparation and subsequently sequencing experiments were performed, killing curve assay developed and optimal dose of G418 for HEK293 cell line was determined. HEK 293 cells were cotransfected with pcDNA3-GIRK1 and pcDNA3-GIRK2, maintained under G418 selection, and G418-resistant single cells were isolated. GIRKs expression in HEK-GIRK stable cell lines was assessed by RT-PCR.

GIRK1 and GIRK2 sequences were confirmed. 600 ug/ml was determined as optimal dose for G418 treatment. GIRK1 and GIRK2 plasmids efficiently co-transfected to HEK 293 cell line and resistant cells were detected. Through single cell isolation and expansion, expression of GIRK1 and GIRK2 were confirmed.

HEK-GIRK stable cell line was generated as an applied model for future optogenetic study.