I have two proteins and I will be preparing a vector with both genes for stable transfection. Each protein will have their own promoter and I will use piggyBac vector to insert a single cassette with both genes in it into the chromosomes. My questions are:
- If I use the same promoter (CMV, SV40 or an inducible one) for both genes how would this effect the expression levels? I read in some papers that one of the genes gets expressed lower than the other. If you have first hand experience about that I will be grateful if I can hear about it.
- If I use the same promoter for both but put them far away (say ends of the cassette) would the expression levels affected again? It sounds simple enough but I think people would have published something if they had a good result with that.
- If I use different promoters for each gene (e.g. CMV for one and Tet-inducible for the other) how would it work? I couldn't find a paper which used this and I would like to continue with this approach; therefore, if you have any experience on this I will be really grateful for your helps.
I don't want to use IRES or 2A-peptide. I would like to be able to control this experiment in transcription stage.