GFP as a reporter gene? - (May/15/2006 )
I am studying a promoter region. I want to measure its transcriptional activity through a reporter gene. I know that luciferase is more used as a reporter but I wonder if GFP could be appropiate for that. I think that GFP could help me to determine the porcentaje of transfection (I don't see how could I do that with luciferase). Does it exist a plasmid with GFP without a promoter to can do it? (some plasmid equivalent to pGL3-basic for luciferase).
Thanks a lot.
GFP could help you determine the percentage of transfected cells, but if you want to measure the activaty of GFP in your cells you would have to quantify per cell (as most fluorometers aren't as sensitive as luminometers for luciferase). Alternatively you could try to analyse your cells by flow cytometry and compare plots with GFP driven by oterh promoters.
Maybe give us more detail on what you're exactly about to do (i.e. transient transfection for promoter tagging, transient transfection of a vector with GFP and your promoter, or stable transfection with a vector with your promoter and GFP).
Clontech offers most vectors with GFP and other fluorescent proteins, www.clontech.com
let me tell you what i want i'm up to:
I've cloned a new promoter of a gene that shows an upregulation under a experimental condition. i want to subclone the promoter controlling the expression of a reporter (luciferase, GFP or both) and transfect the construct (transient). Then i want to compare the transcriptional activity of the promoter under control and experimental condiction.
I've always considered to subclone my promoter in pGL3 basic which has luciferase as reporter. But my boss told me that wanted to use GFP as reporter. His most understandable reason for that is he wants to determine percentage of transfection (because my cells are difficult to transfect -caco cells-).
We have used GFP as a reporter for comparing different promoter but if u use luciferase, u can have actual numbers when comparing the promoter expression. Later we used both (GFP and luciferase) to assess the different ptomoters.
As you want to determine the percentage of transfection, better GFP as u can just look at the cell and have a rough estimate. Transfection do vary everytime so u r better off with GFP than with luciferase.
For promoter studies I use Firefly luciferase as a reporter (pGL3) and then normalize using Renilla luciferase (phRL-tk from Promega). When you transfect, both plasmids tend to enter the same cells and you'll be able to quantify this data using something like the Dual Luciferase Reporter system (also Promega).
Try this paper, it used gfp and unstable variants to measure promoter activity. Unstable variants have the added advantage of being degraded so you can see multiple upregulation events over a growth cycle.
Andersen JB, Sternberg C, Poulsen LK, Bjorn SP, Givskov M, Molin S.
New unstable variants of green fluorescent protein for studies of transient gene expression in bacteria.
Appl Environ Microbiol. 1998 Jun;64(6):2240-6.