Investigating methods to knock out a novel G-protein coupled receptor, GPR52, for the non-nutritive sweetener sucralose

Abstract

The effects of non-nutritive sweeteners are a disputed topic and have been linked to the disruption of signal transduction pathways, metabolic function, and physiological processes. Previous studies found that the sucralose molecule has a binding affinity for the G-coupled protein receptor, GPR52. Genetic modification using RNAi can be accomplished in relevant mammalian cells through lentiviral-shRNA delivery methods. The aim of this study was to develop and validate research tools to knockout the novel G-protein coupled receptor, GPR52. Plasmid DNA was purified and validated from transformed E. coli cultures. HEK293 T17 cells were transfected with lentivirus derived plasmid DNA and optimized accordingly, in an attempt to obtain replication incompetent virus expressing GFP. GFP expressing lentivirus was then used to infect healthy HEK293T17 cells followed by the quantification of the viral titre measuring percent transfection and transduction efficiency by counting cells. Upon facing challenges during plasmid preparation, the modifications implemented made for drastic change in both culture growth and plasmid purification and validation. Upon lentiviral transfection optimization, transfection efficiency in the pGIPZ-mGPR52-shRNA treated cells increased in not only % transfection efficiency but was found to be above 30% in the final transfection optimization trial. Similarly, we can see positive results upon optimization in both transfection efficiency and transduction progress. The validation of this genetic modification approach will allow for the development of new receptor-specific research tools and may provide insights on the function of GPR52 upon sucralose activation.