Hi guys,
Recently, I have been getting some very strange results when I co-transfect my two luciferase reporter vectors into my HEK cells. Twenty-four hours post-transfection, the cells begin to show fluorescence in both my mCherry and GFP (especially GFP) channels of my Leica confocal. When singly transfected neither plasmid shows this high background fluorescence. I have thawed out a younger batch of HEKs and still observe the same phenomenon. I can do a dose-response curve with forskolin and the plasmids seem to be functional. I have no explanation for the artificial fluorescence. Should I prepare new glycerol stocks of the plasmids?
I have no explanation but I observed in different human and mouse cell lines that background in stably transfected single cell clones is very different. Background in CRE-luciferase-transfected cell lines is also different because of high basic PKA activity caused by high basal cAMP levels and other non-cAMP-dependent factors dissociating PKA or even making the partially dissociated PKA holoenzyme active. The latter has been described in detail by Stein-Ove Doeskeland`s group in Bergen, Norway.
Hi Hui,
Even in the absence of my split GFP constructs I still see the abnormal fluorescence. In fact, recently I cotransfected a non-GFP containing plasmid, my Cre luciferase, and Renilla luciferase and still observed the fluorescence. I am beginning to think that it is the dose of Renilla that I am using that might somehow be toxic. For my studies I am using 80 ng of Renilla transfected into a confluent 6 well plate. Perhaps I need to tone down the dose? The renilla is being driven by a Tk promoter.
Hi Sushanth,
I have used 10ng of Tk promoter-driven renilla per well and it had worked efficiently for me. So you can definitely try lowering down your renilla plasmid concentration.
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