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Cell lines expressing recombinant aequorin and a G-protein coupled receptor for functional screening

THE CELL LINES

Cell lines (usually CHO-K1, but other ones in some cases) were stably transfected with plasmids intended for expression of apoaequorin and of a GPCR. After selection with antibiotics, recombinant cells were subjected to a limit dilution, and clones expressing the G-protein coupled receptor and apoaequorin at a high level were selected. If the G-protein coupled receptor is not naturally coupled to a calcium signalling pathway, a universal coupling effector is coexpressed in order to redirect the coupling towards intracellular calcium release. This universal coupling effector is usually the Gα16 protein (Milligan et al., 1996), that was shown to be able to couple many GPCRs to the calcium pathway.

HOW TO PERFORM THE ASSAY

As explained above, detection of putative agonists by means of mammalian cell lines expressing apoaequorin and a GPCR requires the measurement of the emitted light to be performed just after placing the cells in contact with the supposed agonist. This can easily be measured at low throughput using a single-tube luminometer but until recently could not be considered for HTS because luminometer dispenser designs make it impossible to inject a different drug into each well.

Euroscreen has developed a method1 for performing highthroughput screening of drugs binding to GPCR by the use of mammalian cell lines expressing apoaequorin and a GPCR and by the use of a conventional luminometer. Following this method, the solutions to be tested for agonistic activities are placed in the wells of a 96-well plate. Cells expressing apoaequorin and a GPCR are detached from the culture plate (if they are adherent) and are incubated with coelenterazine to reconstitute active aequorin. These are then maintained in suspension with a stirrer and the cell suspension is injected, well by well, on the solutions of tested compounds. Light emissio
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