Hello,

We have made several such constructs and through experience learnt that:

It is best to have reporter gene (gfp, rfp) as the first cistron followed by 2A peptide and the 2nd cistron. This also helps with minimal addition of extra amino acids (only two) to the protein of interest on its N-terminal whereas POI as first cistron will contain 8 or so amino acids on its C-terminal which may affect POI.

IRES may not solve your problem because cistron downstream IRES expresses poorly compared to cistron upstream IRES and next to the promoter.

You can however use a dual promoter vector (for instance pVITRO1-neo-mcs) in which each gene is expressed under the control of a separate promoter. 

http://www.invivogen.com/PDF/pVITRO1-neo-mcs_TDS.pdf

Thanks Syed. I agree that placing the reporter as the upstream cistron has advantages, including making the cleavage consistent. But do you know if the reporter expression can be affected by the other cistron?

Dear Tian Chi,

As you know, in this system the two cistrons should express at equimolar levels. Have you done Western blot and if yes, did you find that the two cistrons expressed differentially? If yes, did first cistron express better than the 2nd cistron? Only in this case can we say that the first cistron somehow affected the expression of the 2nd. Now if this was not the case (that both cistrons expressed at low levels) then the overall reduced expression may be due to the size of the first cistron or its possible toxicity towards the cells.

Dear Syed,

We have not checked by western blot. I agree that  the two cistron should be equally expressed. So the reduction in reporter expression must be due to the presence of the other cistron, as you said. But the other production is not toxic, and it is only 1.7 kb. So how could this reduce marker expression? BTW, have you ever seen this in your system? In other word, when your marker is co-expressed with various other proteins, is the marker expression similar or very different among these constructs?