Colleagues,

I tried to express two proteins A and GFP in one construct using IRES/P2A. However, GFP expression is too weak, and barely detectable. Then I thought this low expression might be caused by the big size of A (2.4kb) and/or unstable mRNAs. Then I switched to AdTrack-MYC which has two CMV promoters (two transcriptional units with their own polyA tails), one for A and one for GFP. Without any gene insertion, GFP signal is very strong in neurons. I saw the dendrite labeled by GFP nicely. With A inserted into the same construct, GFP signal became way too weak, and barely detectable. I really appreciate your thoughts and help here.