Hi Lena.

My name is Ben and I work in Promega Technical Services.

This thread has addressed many important issues, but there are a couple of potential points not yet mentioned: the pGL3 vector and the CMV promoter for your protein that could affect your specific promoter.

The pGL3 vector has a number of cryptic transcription factor binding sites that can often generate misleading results. Our pGL4 vector has removed many of these cryptic sites and generally results in lower basal levels of transcription in a control vector.

A second point is the use of the CMV promoter. CMV can sometimes cause cross-interaction with other promoters, such as your control promoter. This might explain some of the increase you see in your luciferase from your non-specific reporter. The other concern with the CMV promoter is that it can drain the transcription factor pool, so this can affect the expression of luciferase from your specific and non-specific promoter driven luciferase. The use of a weaker promoter, such as a TK promoter, might be worth considering.

I or my colleagues would be happy to discuss this in further detail if you would like.

Please see this page http://www.promega.com/support/customer-and-technical-support/ for contact information.

Firstly,I want to find out whether the over-express protein you mention above is a Activator of transcription；

Secondly，which the empty vector(such as pGL3) was you used:pGL3-Basic Vector/pGL3-Control Vector/pGL3-Enhancer Vector/pGL3-Promoter Vector?

Just find out the interaction between your vector backbone sequence and your protein sequence for any specific binding affinities through some bio-informatic software. Your luciferase reporter shud be under control of some minimal promoter and just find out whether your protein of interest act on that promoter. And can you tell how you overexpressed your protein of interest?

Did you try to run empty wells on your plate just with the luciferase chemistry? This can sometimes gives surprising high read-outs. What kind of plate are you using?

Did you normalized your reporter data against the protein qty or cell number? and yes, can you share with us how you overexpress the promoter's activator protein?

To Guanfan Mao

My protein is a signal transduction protein, it is connected somehow to the promoter under investigation. The connection is obvious, the question is what stands between the protein end the promoter.

I'm using pGL3 basic II vector.

About the protein over-expression. The protein-coding sequence is under CMV promoter. This plasmid is cotransfected with the luciferase reporter construct, cells are harvested 24h later.

To Christian Praetorius: I'm using Nunc white 96-well plate. The empty wells are really zero.

To Doinita Ispas: initially cells were seeded in equal amount and there is no sign of different growth speed. Protein amount is the same according WB. Any of these points cannot explain differences in the background.

I had a similar issue where my reporter plasmid was upregulated even when my overexpressed protein contained no DNA binding domain, It turned out that a p300/CBP inhibitory domain of my protein indirectly caused upregulation of p300/CBP independent promoters. One way around this would be to assay various truncated versions (al without p300/CBP inhibitory domains).

I hope this was helpful.

Maybe your protein of interest affects basal transcription rates by RNA Polymerase II? If so, the phenomenon you are observing with your negative control plasmid could be a bona fide effect. A better approach would be to use the dual luciferase system where you examine the relative luminescence ratio of two luciferase reporters (usually firefly vs. Renilla). This would allow you to better control for effects on basal transcriptional efficiency. Moreover, it will help you correct for changes in transfection efficiency that happen when you add additional plasmids (it is possible that some of your increased "2X" signal in the empty vector + GOI is due to better transfection efficiency with higher total amounts of DNA).

To check if your system is correct, did you try to over-expressed an other protein already known to control the activity of your promoter?

Dear Lena,

please describe how over-expression is induced (requested a few times before). Please also indicate which transfecting agent you are using (Lipofectamine, Mirus, Trensfectine....). There are several possible issues with the respective chemicals, but listing all is very extensive at this point, so please specify. Please also indicate which cell-line you are working with.

To Daniel Cohen: I have Renilla in the system, it's level is stable.

To Joachim Gerhold: as I said before, the protein of interest is co-transfected with the luc-reporter plasmid. The protein is under CMV promoter. Transfection was performed by JetPei reagent into H358 human lung cancer cells.

Lena- does your protein of interest upregulate or downregulate the activity of the promoter you are studying? Is this protein normally expressed and/or active in the lung cancer cells? If it is, you may need to try your assays in a heterologous system that lacks your protein of interest to see a clear effect.

To Daniel Cohen: The protein is endogenously expressed in this cell line and up-regulates expression from the promoter. I have tried over-expression in H358 cells as is and in cell line derived from H358 with shRNA to my protein. The effect is the same.

Now I'm wondering may be even 20% of the protein that we have in the sh cells is enough to produce full activation of the promoter and this is the reason for lack of over-expression effect.

Cell line that does not expressing the protein at all may be dangerous. My protein is signal transduction protein, it needs all the system around it to work. Who knows if the system exists in non-expressing cell line? But I'll try it.

Hi Lena.

My name is Ben and I work in Promega Technical Services.

This thread has addressed many important issues, but there are a couple of potential points not yet mentioned: the pGL3 vector and the CMV promoter for your protein that could affect your specific promoter.

The pGL3 vector has a number of cryptic transcription factor binding sites that can often generate misleading results. Our pGL4 vector has removed many of these cryptic sites and generally results in lower basal levels of transcription in a control vector.

A second point is the use of the CMV promoter. CMV can sometimes cause cross-interaction with other promoters, such as your control promoter. This might explain some of the increase you see in your luciferase from your non-specific reporter. The other concern with the CMV promoter is that it can drain the transcription factor pool, so this can affect the expression of luciferase from your specific and non-specific promoter driven luciferase. The use of a weaker promoter, such as a TK promoter, might be worth considering.

I or my colleagues would be happy to discuss this in further detail if you would like.

Please see this page http://www.promega.com/support/customer-and-technical-support/ for contact information.

To Ben Schmidt : thank you a lot, I''ll consider these points.

Since you're getting the same effects in the vectors with promoter and without promoter, its certain that some transcription factor binds upstream of luciferase sequence and activates transcription. The pGL3 to pGL4 upgradation mentioned above is a good point..!! But I would recommend to test pGL4 promoterless empty vector first before you clone your promoter of interest in pGL4. Second get the pGL3 empty vector sequence and omit luciferase sequence from it and scan it for transcription factor binding sites (using online promoter scan web site) and examine the list of transcription factors that can co-operate with the protein which you had overexpressed. This will give a clue which transcription factor makes this leaky expression of luciferase from pGL3. Once you get an idea on such binding sites, make sure that the pGL4 doesnt have such binding sites. (just computer works that could save your cloning and luciferase assay times). Good luck..!!

J.

Lena- pGL4 vectors are superior to pGL3 in my experience, also. However, because these vectors use a destabilized version of Firefly luciferase, overall signal counts are much lower than what you are used to seeing with pGL3. Absolute signal strength can matter a great deal, especially in terms of the robustness of your responses with co-transfection with your protein of interest. For example, if your Renilla counts are saturating, you might be missing a response in your co-transfection studies with your protein of interest. Conversely, if you have too little signal in your Firefly channel, the assay can get extremely noisy, and 2-fold changes become hard to interpret. I'd recommend aiming for no less than 10^4 (ideally 10^5 to 10^6 AU) in each luminescence channel. You can always check for a linear response by assaying a serial dilution of your cell lysate.

So before jumping to the pGL4 system, you should consider that you may need higher transfection efficiencies and/or higher cell #s for relatively weak promoters. Lastly, I have also seen interference by the CMV promoter; however, you can usually minimize this problem by using a relatively low amount of your CMV-GOI construct in the co-transfections (

In additon to many of the great points already made, such as promoter competiion, have you considered that your protein or your plamsid may affect the promoter which is controling your Renilla control? You say that your Renilla is stable, but what are your controls for that? In addition, we have found that some transfection reagents are not good at getting multiple plasmids into the same cell- if you have optimized your transfection efficiency with a single plasmid, it may not be the same for multiple plasmids. Again, this can lead to problems if you are using expression from a different plasmid to measure transfection efficiencies. You might consider a test where you harvest the total transfected cell DNA and do qPCR for a cellular control gene (as DNA loading control) and specific sequences in your plasmids. This definitely takes longer, but avoids (or allows you to test for) some of the above possible problems.

What is the promoter for your control vector? TK, SV40, or CMV? What is your treatment? Some hormone may increase or decrease your control expression.

This is for Laura Hanson. Hi Laura, I like your idea of qPCR for transfected cell DNA versus cellular control gene. Is the method published anywhere? Could you provide a reference? Thanks!

Hi Wen,

A couple of references you might want to look at: Heid et al. (1996) Genome Research. 6: 986-004. and Jiwaji et al. (2010) BMC Mol Biol. 11:103. The second is looking more at RT qpcr for expression levels, but still has some useful and interesting pointers.

In addition to read-through effects mentioned by the others, one possibility is that the cotransfection changes the efficiency of uptake of the plasmids. You would exclude that by checking for the effect of a mutant transcription factor (which would be a good control anyway). Also, it might be instructive to use different concentrations of the plasmids. Good luck WAS

I agree with people who have mentioned about pGL4. It reduces/ eliminates background vector effect substantially.

Http://www.promega.com/products/reporter-assays-and-transfection/reporter-assays/dual_luciferase-reporter-assay-system/

If you are using only a small segment of promoter region in pGL3 basic vector, it will not work. It is usually for full length promoters with TATA box or someother for minimum expression. If you are using promoter element, you need to use pGL3 Promoter vector. The suggestions are made assuming that you are using small promoter element in pGL3 basic vector. As suggested before by others, pGL3 promoter vector has several cryptic sites and gives high background, whereas pGL3 basic vector gives very low background expression.