I believe my protein of interest is causing apoptosis in my cells (which is actually one of the things I want to test for once I get this assay up and running). My control plasmid works beautifully, but all of my cells transfected with my protein of interest die between 24-48 hours post transfection. I cannot currently switch to a viral vector because my lab is not BSL2 certified (we're working on that, but I need to move on this experiment now). My current plasmid contains a CMV promoter, and I think if I could find a plasmid with a weaker promoter that it might just solve my problem, but I am having real trouble finding a clear answer on whether or not a specific vector is weak or strong. Any help would be appreciated.
Hi Amanda,
If you do get an answer to your question could you please send me a message and let me know. I'd like to know that as well. Thank you.
Hope this can help you :-) ....In the past I had problems with luciferase assays because the vector had a CMV promoter and I couldn't appreciate differences between my mut and wt contructs. Then I changed CMV with the Herpes simplex virus thymidine kinase promoter (TK) and things worked very well!
Thank you, Amelia. I will look into that one. Trevor, I have a colleague trying to push me that direction, as well, but I've heard they can be very difficult to troubleshoot. What has your experience been with those systems?
If you are concerned about toxicity, then I agree with Trevor, using a Tet inducible system is the best way to go. The system is not difficult, and you can tune the expression of your gene to whatever level you want by adjusting the dox concentration in the media. If you try using a different promoter, there is no guarantee that the expression level of that other promoter will be low enough to avoid toxicity. Maybe you will get lucky, but if not, then you will be back in the same place you are right now.
Hi.
How do you know your protein is toxic?
I think you should prove it.then you can work with a vector even without promoter,you just go to pubmed,Nucleotide and searching for mammalian expression vector if you are working on mammalian cells.
I've experienced similar problems using plasmids containing CMV promoters, either the expression is excessive, or when creating stable lines the CMV promoter gets shut off altogether. As an alternative to the CMV promoter, you might find a plasmid containing the EF-1 alpha promoter better suits your needs.
In terms of promoter that are available and regarding their relative activity, this paper might be of help: Article Systematic Comparison of Constitutive Promoters and the Doxy...
I agree with Anand Patwardhan , UbC promoter is the weakest I used in my hands.
Hello,
Normally PGK promoter is also considered a weak promoter for mammalian cells.
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