We have developed an improved vector for the stable expression of recombinant protein in mammalian cells. In this vector, designated pCIN, both the recombinant cDNA and the neomycin phosphotransferase selection marker are transcribed from a single promoter element. To facilitate translation of the second open reading frame, the encephalomyocarditis virus internal ribosome entry site has been inserted into the expression cassette immediately before the start codon of this sequence. We report the use of this vector to generate stable cell lines expressing the human 5-HT1Da serotonin receptor and show that following transfection and clonal selection, all ten cell lines characterized express similar and high levels of receptor (1.5-11.9 pmol receptor/mg protein). Use of pCIN should permit the rapid and efficient production of stable mammalian cell lines for the characterization of recombinant protein, as this vector appears to predispose all transfected cells to express such protein.

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Several reporter gene assays have been described where gene transcription is activated as a consequence of a specific signal transduction event, such as activation of adenylyl cyclase (1.2). Reporter genes typically consist of specific responsive elements placed upstream of a minimal promoter, which together control the expression of a readily detectable reporter protein, such as luciferase. We have developed a dual glow-signal firefly and Renilla luciferase assay, which allows the simultaneous measurement of two reporter genes in the same well of a 96-well plate. In this report we demonstrate the use of this assay for the simultaneous analysis of agonist activity at two G-protein coupled receptors which signal through activation of the G-protein alpha sub-unit, G alpha S. Chinese hamster ovary (CHO) cells stably transfected with a cAMP responsive firefly luciferase reporter were further transfected with the human Vasopressin V2 receptor. Similarly, CHO cells stably transfected with a cAMP responsive Renilla luciferase reporter were further transfected with the human beta 2-adrenoceptor. The two cell lines were mixed in individual wells of a 96-well plate and a number of compounds were screened to determine their activity at both receptors. Stimulation with vasopressin and beta 2-adrenoceptor agonists resulted in the activation of the firefly and Renilla luciferases respectively. Stimulation with forskolin, which directly stimulates adenylyl cyclase, caused the activation of both reporter genes, and stimulation with a range of further compounds with no activity at either receptor did not generate a reporter response. The dual luciferase assay allows the simultaneous screening of two receptors in a 96-well format resulting in significant time and cost savings.

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