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P11018An Aequorin Luminescence-Based Assay for Monitoring GPCR Function in Cryopreserved CHO Cells Transiently Expressing Either a Receptor-of-Interest or Apo-aequorin-3
Presenter Beverly Holskin, Cephalon, Inc.,USA
Additional Authors: Bruce E. Jones, Thao Ung, Sheryl L. Meyer, Sandra Flores, Emmanuel Burgeon, Beatrice Goxe, Laurent Dumortier, Mark A. Ator, and Emir Duzic
Luminescent aequorin-based functional cellular assays traditionally have been optimized for high-throughput screening (HTS) of G-protein coupled receptors (GPCRs) using stable cell lines expressing mitochondrially-targeted apo-aequorin. Outlined in this poster is a method for generating cryopreserved cells suitable for HTS using transient transfections of mammalian expression vectors expressing 1) either a Gq-coupled GPCR in a cell line stably expressing apo-aequorin-3 or 2) apo-aequorin-3 in a stable recombinant cell line expressing that same Gq-coupled GPCR. Transient transfections were performed with Lipofectamine 2000™ (Invitrogen) with rapid cyroprotection. The cryopreservation procedure used allows for long-term storage of the cells in liquid nitrogen prior to assay. The pharmacology of the receptor in these cells, as evaluated with reference compounds in the aequorin assay, was consistent with literature reports. Luminescence detection was successfully performed with both FDSS6000 (Hamamatsu Corp.) and LumiLux™ (PerkinElmer, Inc.) instrumentation. These methods were successful to produce a large signal-to-noise ratio with cell numbers as low as 1,500 cells/well with the transient apo-aequorin-3 expression. Therefore, a library screen in the range of 200,000+ compounds would be possible from a single transfection. The time and expense required for this method is compared to the building of a stable cell line. Future applications could involve transfection of the apo-aequorin-3 plasmid into a naturally-expressing cell line, increasing the utility for screening specific targets.