reporter assay with ligand activated TF - (Feb/24/2009 )

Hi,

I'm trying to set up a reporter assay for testing ligands to a receptor that work as a transcription factor. I'm expressing the receptor in pCMV script vector and cotransfect with a reporter vector with luc expression. This is done in HepG2 cells. My interest is in fish, but for now I'm working with the human receptor with known ligands to get a good protocol.
My problem is......
If I compare cells with pCMV including my receptor with empty pCMV, the first one gets high luc activity, without ligand added. My guess is that a functional ligand must be present 'by accident'...
We have tried to change solvent (from DMSO to MeOH) and use serum free media, but the activity is still high.
Something in the media? (MEM) or endogenous from HepG2??

If we compare the fold change, the difference between ctrl and overexpressing receptor but no ligand added is app. 20 times, and the diff. between solvent and a strong ligand exposure with overexpressed receptor is only 3 times.
It feels like theres is A LOT of 'noice' that will make it hard to see effects of weaker ligands.

If You have any piece of advice for me about what to do, I'll be so HAPPY!!!

quipu on Feb 24 2009, 02:25 PM said:

There may not be a ligand present in the media. High constitutive activity is common with transient transfection of reporters and transcription factors because transient transfections typically result in >1000 fold over-expression of your proteins of interest, which alters the intracellular dynamics, localization, and protein-protein interactions of your transcription factors. Note that in your experiment, the activity is high compared to empty vector, but I doubt it will be high compared to your activated control. Have you tried performing an experiment yet with factor - ligand compared to factor + ligand. In this case, I would expect that you may see several 100 fold greater activity in your + ligand treatment group over the - ligand. For your studies, this fold activation is all that matters.

Hi,
I really appreciate your comments!
If I understand you right, I have already done that +/- ligand test with factor present and it was only a 3-fold change. It seems really 'bad' right. That ligand is supposed to be strong, but the 'true' endogenous ligand is still not known for this receptor (PXR).

From what I know of PXR, depending on the cell type, 2-3 fold increase is about right.

How much DNA are you transfecting? May want to bring it down a bit so that your signal is lower...then maybe the difference +/- will be greater.

genehunter on Feb 27 2009, 01:26 PM said:

OK, good to know, feel that I have to learn more about this stuff..... do you know why this is the case..
Thanks for your comment!

NemomeN on Feb 27 2009, 04:17 PM said:

Actually I'm not sure right know.. (I'm on maternity leave right know, but my colleague is keeping up the work).
I have tried different amounts of DNA, and different ratios (expression vector: reporter), but there is of course more testing to do!
I'm still not sure what is a good ratio, 50/50 or else...
Thanks :-)

Low levels of receptor with higher levels of reporters tend to work best. Reason being, high levels of receptor resulting from transient transfection may lead to dissociation from chaperone proteins and non-ligand initiated activation. Very low levels of receptor tend to give more physiological response and decreased constitutive reporter activity. High levels of reporter ensures that the highest amount of activity possible (when all reporter promoters are maximally active) results from the activation of many transcription complexes rather than a few.

Dr Teeth on Mar 6 2009, 02:24 PM said:

Thanks for your reply and the clear explanation!! , I will keep that in mind, and make sure that we test really low levels of receptor, and hopefully reduce the high constitutive activity