For promoter assay, which vector I should use? - (Jul/22/2013 )

Hi all,
I would like to study the function of some SNPs in promoter activity. I clone 2 inserts containing 2 alleles of a SNP into pGL3-basic vector, but they all showed no promoter activity.
I need to compare the function of the 2 alleles in promoter activity, thus can I clone the inserts into pGL3-enhancer vector, which contains SV40 enhancer but not SV40 promoter?
Thank you so much for your help...

Hello DrLeo,

I've had success with the pGL3 vectors in the past. First you have to understand that the SNPs that you are using may not cause any depreciable change in reporter activity since the promoter system does not completely mimic what is occuring in the body, i.e. the impact of other transcription factors or enhancers acting on transcription. With that in mind, I may suggest checking your protocols to make sure you optimized for transfection efficiency, toxicity of your insert, and any contaminants in your cells. In the past, I've had trouble with a promoter assay only to realize that my insert was actually toxic to my E.coli culture leading to low yields or poor transfection-ready DNA. The pGL3-enhancer vector would be a great next step to assay for promoter capabilities of your SNPs, but keep in mind that the cells that you transfect with your promoter system should be compatible with the SV40 enhancer.
Good Luck!

Thanks HawkeyeGrad.
I transfected my construct (insert-pGL3 basic vector) into A549 cells (human airway epithelial cells) and human mast cells (HMC-1), it did not cause any toxic effect to these cells. The transfection efficiency was good. I used dual luciferase assay to confirmed the promoter activity of my constructs.
As I know, the promoter activity function of insert depends on which sequence we design. I think my insert lacks of some transcription elements. Thus I would like to clone it into pGL3-enhancer and hope the enhancer can help.
Does it make sense?
Thank you again!

DrLeo on Mon Jul 22 09:15:54 2013 said:

Sure, Jason. I will share it here if i can find out the reason and solution...

I think your protocols are fine based on what you wrote so I would suggest any of 4 options:
1. Clone into the pGL3-enhancer vector. Cloning it into the pGL3-enhancer vector would be my next step since this vector should enhance or increase the level of transcription of the reporter gene.
2. Use the alternate MCS in the pGL3-basic vector. I've read some studies that show differences in expression level based on the MCS used for the insert. This hasn't been the case in the studies I have done but you could give it a try and see if your signal changes.
3. Another option would be to try one of the pGL4 vectors since those are optimized for greater expression and improved sensitivity.
4. Use a very easy to transfect cell line like HEK293 that grows like weeds and also contains the SV40 T antigen that promotes replication and cause greater increase in expression.

And as an added note, keep in mind that your insert may just have no promoter activity. How large is your insert? Sometimes large inserts have cryptic silencer elements that decrease transcription and further subcloning may reveal the functional domain.

Hope that helps.