I have a problem with my plasmid. My plasmid design goes something like this:
I have a TurboRFP under the PGK promoter followed by SV40poly A. In the same plasmid I have a tet promoter controlling my gene of interest fused to a luciferse gene. The entire plasmid size is around 13kb.
I usually co-transfect a plasmid having a transactivator (to activate the tet promoter). The transactivator plasmid is constructed in such a way that the CMV promoter drives the transactivator and a PGK promoter drive the NGFR gene.
When I transfect these 2 vectors into my cell line NIH3T3 I can induce expression of luciferase which means that my tet promoter is active, but I do not see any TurboRFP expression. I dont know why? I dont know if there is a possible promoter interference and what I must do to change this cause I cannot change the plasmid construction.
any help will be appreciated!