I am currently working up a set of experiments to determine the signaling pathways regulating transcriptional control of our gene of interest. We already have full length and truncated promoter constructs, and would like to test them specifically in endothelial cells, to build on some previous in vivo and in vitro results. However, we can no longer access the specific cell type that was originally used, and unfortunately none of the cells we do have seem to express the gene endogenously as assessed by qRT-PCR. Admittedly we haven't yet tried just putting the luciferase constructs into cells and giving it a shot, but I have assumed that if the cells don't have endogenous promoter activity (assuming mRNA levels are a correlate of that), then we wouldn't see a luciferase signal with the transfected exogenous promoters. Is this a reasonable assumption? Or is it possible that the endogenous promoter and/or transcript levels may be suppressed (e.g. methylated, targeted by miRNA) in a way that won't affect the transfected constructs?
Dear Rea,
of course, there is the chance that these cells lack an important transcription factor for your gene.
However, there is also a fair chance that your gene is silenced on a DNA level in these cells, by methylation or chromatin structure (which would not affect your luciferase assay). So I would use cells most similar to your initial cells and give it a go, even more since you have the constructs ready.
Good luck!
best christian
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