Enhancement of Glycoprotein Hormone Alpha Subunit Promoter Reporter Gene Activity in Co-transfection Studies – A Cautionary Reminder

Abstract

Reporter constructs have a wide range of application in determining eucaryotic gene regulation and expression. In vitro models frequently necessitate co-transfection and there have been reports of interference, predominantly inhibition, between promoters. Studies investigating the biological activity of a mutant thyrotropin receptor involved co-transfection with receptor constructs and a cAMP responsive luciferase reporter driven by the glycoprotein hormone alpha subunit promoter. We observed considerable enhancement of basal luciferase activity by co-transfecting the empty expression vector, which contained the SV40 late promoter. We have investigated the mechanism using different concentrations and several viral promoters in COS cells, following transient co-transfection. The increase was dose dependent but plateaued at 50 ng of vector DNA. This was not due to an adjuvant effect since luciferase activity was unchanged by adding increasing amounts of a promoterless plasmid. Enhancement was maintained when truncating the promoter to −346, but eliminated in the promoter truncated upstream of −244, indicating a binding site for a putative repressor of glycoprotein hormone alpha expression between −244 and −346. Enhancement was maintained with the addition of a second constitutive reporter (bos β-gal) to correct for transfection efficiency, although this was the consequence of enhanced luciferase activity by the bos β-gal, which in itself was inhibited by the SV40 promoter. Artefactual enhancement of reporter activity can occur and highlights the need for careful choice of controls when performing transient co-transfection experiments. In silico analysis of the promoters identified a possible shared forkhead transcription factor binding site.

reporter assays - transcription - squelching

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