Luciferase question

Michael Andresen via methods%40net.bio.net (by jmandresen from gmail.com)
Tue Nov 20 16:40:35 EST 2007


I am trying to characterize a Purkinje cell-specific promoter using
Promega's dual luciferase system. Transfecting pRL-CMV into CHO cells
(chosen to utilize some key stably transfected lines in the lab) gives me
10x to 20x stimulation of transcription over pRL-null. The issue is that
pRL-null gives 10x stimulation over my promoter of interest. When I
transfect with transcription factors known to be active in the promoter,
transcription rises nicely, but all below the transcription level of the
"negative control" pRL-null.

I am guessing this is due to strong inhibition of the promoter by cellular
factors that help restrict promoter activity to Purkinje cells. I would like
to know if anyone knows of similar stories in the literature. I don't know
if I should abandon the assay because of its idiosyncrasy or if the results
will stand up to peer review because the luciferase assay is that sensitive.
Does anyone know any references dissecting a promoter despite overall
transcription levels below the negative control?

Thanks,

Michael Andresen


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