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

최적의 luminescence 신호 분석을 위한 유전자 전달 방법의 비교연구

  • Park, Seohyun (Department of Biomedical Technology, Sangmyung University) ;
  • Lee, Sunghou (Department of Biomedical Technology, Sangmyung University)
  • 박서현 (상명대학교 의생명공학과) ;
  • 이승호 (상명대학교 의생명공학과)
  • Received : 2016.09.26
  • Accepted : 2016.11.10
  • Published : 2016.11.30

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

Acknowledgement

Supported by : 상명대학교

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