No, this is not typical. In fact, IRES-mediated translational efficiency is usually weaker for the second gene cassette, e.g. GFP in your plasmid. Nonetheless, the fact that you have good GFP expression suggests that your transfection was successful. If your GOI is not expressing well, it could be a problem with the cloning, Make sure you have a proper Kozak sequence present for the mRNA you are trying to express and no other potential open reading frames preceding it. Sequence verify to make sure there are no premature stop codons or missense mutations.

If the mRNA sequence checks out, it is possible that your protein is misfolding, not trafficking to the appropriate subcellular compartment, or is lacking an requisite cofactor or interacting protein for activity. You should try some Western blotting and/or immunofluorescence to investigate whether your mRNA is producing any of the expected protein. While electrophysiolgical measurements are very sensitive, they are not a direct measure of protein expression, but rather protein activity.

No, this is not typical. In fact, IRES-mediated translational efficiency is usually weaker for the second gene cassette, e.g. GFP in your plasmid. Nonetheless, the fact that you have good GFP expression suggests that your transfection was successful. If your GOI is not expressing well, it could be a problem with the cloning, Make sure you have a proper Kozak sequence present for the mRNA you are trying to express and no other potential open reading frames preceding it. Sequence verify to make sure there are no premature stop codons or missense mutations.

If the mRNA sequence checks out, it is possible that your protein is misfolding, not trafficking to the appropriate subcellular compartment, or is lacking an requisite cofactor or interacting protein for activity. You should try some Western blotting and/or immunofluorescence to investigate whether your mRNA is producing any of the expected protein. While electrophysiolgical measurements are very sensitive, they are not a direct measure of protein expression, but rather protein activity.

@Paritosh- if GFP is being made, then the mRNA is present (IRES mediates a bicistronic message, so both the GOI and GFP are present in a single mRNA). So transcription is not the problem (otherwise GFP would not be detected), rather improper translation of the gene of interest or lack of protein activity are to blame.

Hi, thanks a lot for your answers!

The biggest issue I have is the great disparity between cells! My protein of interest gives a very characteristic current response, so I can immediately tell if it is there or not. When I previously co-transfected my GOI (without IRES) with a second plasmid carrying GFP, about 95% of green cells also had my protein, even though GFP and GOI were just cotransfected. Now that they are on the same mRNA in the pIRES, I only get the response of my protein in about 50% of cells.

Of course there could be something wrong with my protein rather than the pIRES.. I just want to make sure that this is not something other people have experienced using pIRES. I hope this makes my problem more clear?

Best wishes,

Laura

That is to say, I haven't yet checked by Western blot or immunostaining if my protein is there, because there isn't really a tried-and-tested antibody for it!

There could be several issues: 1) your protein is not giving the current response you want to see 2) your protein might be less stable then the GFP 3) IRES is not working properly and you get some strange fusion which might mis-fold/not properly localise. Other mentioned reasons are less likely if you saw a response from the same construct in a different vector before. High GFP might be also toxic for the cells. Do you use the same promoter in the IRES vector and the construct before? 

The IRES-GFP cassette is designed to work in conjunction with a stop codon in your GOI. If this is absent, your GOI may not express properly. Based on what you describe, it sounds like the co-transfection experiment was a better setup. Perhaps the transfection efficiency of the IRES2-GFP plasmid is simply less good in your hands either due to increased plasmid size or due to variability in the quality of the purified DNA.

@ Dear Daniel, I have a query for you. Following your earlier argument, Should I consider adding start and stop codons to the primers for amplifying MCS-A and MCS-B inserts independently. Also, I would like to ask if it is possible to subclone the entire MCS-A+IRES+MCS-B construct into a different vector and get two independent proteins in the expression system?

Thank you....

@Ankita- I would need more context to understand your question. If by MCS you mean multiple cloning site, there is only one in pIRES2, so I don't know what you are referencing with MCS-B. Also whether you can subclone a GOI-IRES-Reporter cassette into another expression system depends on the properties of that system- eukaryotic or prokaryotic? What's the insert size limit? What's the size of your GOI? Does stoichiometry of the GOI and reporter gene matter?