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http://dx.doi.org/10.5762/KAIS.2016.17.11.640

Comparative studies of various transfection processes for the optimal luminescence signal analysis  

Park, Seohyun (Department of Biomedical Technology, Sangmyung University)
Lee, Sunghou (Department of Biomedical Technology, Sangmyung University)
Publication Information
Journal of the Korea Academia-Industrial cooperation Society / v.17, no.11, 2016 , pp. 640-647 More about this Journal
Abstract
By minimizing fluorescence interference phenomena, aequorin-based luminescence technology can provide a relatively sensitive detection platform with integration of $G{\alpha}16$ protein in order to track internal calcium mobilization by G protein-coupled receptors (GPCR). In this type of cell-based functional assay format, it is essential to optimize the transfection process of a receptor and $G{\alpha}16$ protein. For this study, corticotropin releasing factor receptor subtype 2(CRF2) was set as a model system to generate three stable cells with CRF2 and $G{\alpha}16$ in addition to transiently transfected cells under three different conditions. Agonist (sauvagine) and antagonist (K41498) responses in those cells were analyzed to develop the optimum transfection process. As a result, the effective signal ratio in the dose response experiments of sauvagine and K41498 were at least 10-fold higher (z'=0.77) in CRF2-$G{\alpha}16$ stable cells. For the transient transfection cells, stable expression of $G{\alpha}16$ prior to the CRF2 represented a two-fold higher signal (z'=0.84) than the other cases of transient transfection. In conclusion, for the utilization of transient transfection processes to develop a cell-based GPCR functional assay system, it is suggested to introduce various target receptors after stable expression of $G{\alpha}16$ protein.
Keywords
Aequorin; Cell based assay; Corticotropin releasing factor receptor; G protein coupled receptor; Transient transfection efficiency;
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