I am trying to test regulation of the promoter of some genes by luciferase assay. I cloned this promoter into pGL4.10 and transfected HEK293T cells. As a control I used empty plasmids. Why did luciferase assay with empty vector induce higher luciferase activity than a vector with my promoter? I normalise my results to renilla. BTW, I know that these cells express a lot of my gene.
Hi,
there could be a problem with the promoter stretch you cloned into the vector. As the promoter includes both responsive elements for activators and represors of transcription that act together, you may have cloned a sequence that includes more potent repressive elements than the activating ones. I would suggest to clone another sequence stretch, possibly longer than the previous one.
Thank you Lucie. In general I am happy with the luciferase in my plasmid with promoter when compared to other controls. What I am not happy is high activity of empty vector... The company state that there should be nothing above background.
Cheers
I have been working with pGL 4.10 several years ago. In my experience, the emty pGL was ative, but much less than with my promoter inside. However, I was observing a correlation between luciferase activity from empty pGL and the nuber of my cells in the culture. Therefore, there must be a regulation process occuring. However, I was working with different cell types, HepG2 and U-937. I am afraid no company could ensure no regulatory elements. Unfortunately.
I have just talked to them and they gave me some tricks to try...
Thank you Promega for fast respond :)
Sure. Here are the tricks:
1. Reduce Renilla plasmid amount.
2. Clone into empty plasmid something the same size as your tested fragment to avoid size interference.
3. Use proper control for your promoter e.g. one cell line know to not express your gen and one with high expression.
Anyway, I need to think if this model is still useful for me...
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