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T357:lyons:TheUseofBacMamTechnologytoProduceAequorinCellLinesforHTS

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Author(s) name:

Jacquelyn A. Lyons, Thao Ung, Kristen Murray, Beverly P. Holskin, Rita Raddatz, Sheryl L. Meyer, Emir Duzic, Bruce E. Jones


Affiliation: Worldwide Discovery Research, Cephalon, Inc., West Chester, PA 19380

Abstract :

G-protein coupled receptors have typically been screened using calcium-coupled assays. Initially these assays used cell-permeable fluorescent dyes, but use of the recombinant photoprotein aequorin (AEQ) has become a popular alternative. One hurdle to full scale adoption of this technology is efficient generation of cell lines that express both the AEQ apoprotein and the target of interest. This can be accomplished by creating stable lines that express the protein and receptor, but this is a labor-intensive process. Reports indicate that transient transfections may be used, however this technique is limited to specific cell types. The use of BacMam viral delivery is reported to be more applicable to a variety of cell types while accommodating larger and more diverse genetic material. Herein we describe the use of the BacMam system to deliver oxytocin receptor cDNA to cells stably expressing AEQ photoprotein and use of these cells in subsequent screening.

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