I have a stably transfected cell line: CHO CRE-SEAP with GFP tagged delta opioid receptor, using dual selection antibiotics: hygromycin and G418. I did the conventional reporter gene assay (SEAP assay) with loads of optimizations but could not get the desired inhibition of forskolin mediated cAMP stimulation in my cell line. In order to make sure whether my transfection was working properly I did radiolabelled cAMP accumulation assay on these cells and found them to be working perfectly fine, giving a nice dose response curve (inhibition).
Since reporter gene assays require incubation for 4-6 hours for the expression of reporter gene product, exposure of agonist to this long time may cause GPCR desensitization, so I used different incubation time points but even then the issue could not be resolved.
I just wonder what could be the reason that I am not getting the desired response in SEAP assay, although everything seems to be working in order.
My compounds are Gi coupled (known through PTX experiments), so could it be because the SEAP works better in Gs mediated responses?
In short one assay is giving descent response (cAMP assay) while other simply failing (SEAP assay) on the same cell line?
All the standard DOR ligands in use failed to show any appreciable inhibition via SEAP. Could it be be due to downstream signalling (distal signalling) failure in this case because my ligands on other hand are showing great response in relatively proximal downstream signalling pathway (cAMP assay)?
I went for a dilution clone also but it did not improve anything except for a much better cAMP inhibition in radiolabelled assay.
Can someone throw some light on the possible reasons why SEAP system has simply failed in this case please?
I am not sure of throwing any light but what is your aim? SEAP assay or screening delta opioids?
If the latter, measure cAMP by RIA, ELISA or the Cisbio kit in the presence of blockers of PDE. This will enhance your throughput and let you forget SEAP.
I never used SEAP, but I agree with Ferenc that your cAMP assay (or any directly cAMP-measuring assay) is the method of choice. I use different luciferase reporter gene systems including CRE-luciferase and they detect Gi-effects with forskolin as the stimulant. You may consider using a Gs-coupled GPCR agonist for stimulation since forskolin is a very strong, but also tricky agent especially at high concentrations (above 1 µM).
Thanks for the reply dear Antoni and Maronde; Yes I am basically screening some delta agonists and one of the initial methods used was SEAP assay. I never exceeded 1uM limit for FSK in my experiments. I did test my compounds in other signalling pathways, showing remarkable effects except for this particular assay.
- Badges
- Science topic
- Similar topics
- Biological Science
- Microbiology
- Microorganisms