I'm trying to do lipofectamine transfection for the expression of DsRed fluorescent protein under EF1a promoter. The structure of the expression is bicistronic since it expresses TetR and DsRed (divided by IRES sequence). The vector itself is a pLV vector. The structure of the expression sequence is EF1a-cPPT-TetR-SV40NLS-IRES-DsRed. I thought the structure should drive the expression properly, but it didn't. I tried with other school vectors with GFP and it worked just fine. Do you have any idea why?
Maybe try P2A instead of IRES? can alrso add WPRE to stabilize expression.
Is there another upstream ATG that could be utilized such that you don't get protein in the correct frame (depending on the IRES, there's usually a preferred ATG)? I would try making sure that the ATG of the DsRed is exactly the ATG of the GFP in the construct that does work.
Would you like to tell us about your IRES sequence.Because, there are some IRESES that express only under stress conditions.is it cellular or viral.
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