Generation of hek-girk stable cell line as an applied model for optogenetic study

Hoda Shams najafabadi,1 Zahra-soheila soheili,2,* Shahram samiei,3 Ehsan ranaei pirmardan,4 Ali kashanian,5 Reza salmanipour,6

1. Department of Molecular Medicine, National Institute of Genetic Engineering and Biotechnology
2. Department of Molecular Medicine, National Institute of Genetic Engineering and Biotechnology
3. Ophthalmic Research Center, Shahid Beheshti University of Medical Sciences
4. Department of Molecular Genetic, Faculty of Biological Sciences, Tarbiat Modares University
5. Department of Molecular genetics, Islamic Azad University of Damghan
6. Department of Molecular Medicine, National Institute of Genetic Engineering and Biotechnology

Abstract

Introduction

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 family that activate k+ channels in neural cells stimulating pathway. g protein-coupled inwardly-rectifying potassium channels (girks) are a family of potassium ion channels which are activated via this optogenetic tools. therefore hek293 cell line that stably express girk channel can be a model for studying these principal optogenetic tools. this study aims to generate stable girk expressing hek293 cell line as a noteworthy optogenetic tool.

Methods

Pcdna3-girk1 and pcdna3-girk2 were obtained as a gift from dr. terry hebert, recovered from filter paper and were transformed to xl10 e.coli. plasmid mini preparation and subsequently sequencing experiment 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 were assessed by real time pcr.

Results

Girk1 and girk2 sequences were confirmed. 600 ug/ml were determined as an 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, the expression of girk1 and girk2 were confirmed.

Conclusion

Hek-girk stable cell line was generated as an applied model for our future optogenetic study

Keywords

Girk1 and girk2 channel, hek-girk stable cell line, optogenetic study