CMV promotor upregulation? - (Nov/08/2005 )

I have made a stable eukaryotic cell line using PCDNA3.1, and zeo as the selection marker. The amount of fluorescence in this particular colony has gone down over time (its still on Zeo). The promotor is hCMV promotor.

Anyways, I'd like to get a bit more of my product if possible without making a whole new cell line. Will serum starvation work? Or is there maybe something else?

This has been know to happen to genes cloned into pcDNA3. They lose activity over time. You can try to compensate loss by adding Sodium Butyrate to your cells at a final concentration of 10mM. Make up a 1M stock in water and then add to cells directly or as a media supplement.

I'm not sure what the mechanism of action is.

Thanks....I'll give it a try and see how it goes. I'll report back in a week or so....Anyone else with other tricks?

CMV is transactivated by Sp1 (and other transcription factors) Sp1 transcriptional activity is upregulated by sodium butyrate, a histone deacetylase inhibitor.

Sp1 activity can also be increased by signalling through the Ras-MAPK pathway induced by PMA or by co-transfecting Sp1 expression plasmids.

You could also look at mechanisms for upregulating the other TFs that bind to the CMV promoter, I am not sure what they all are of the top of my head...

HTH