I designed the following construct: [5' PCMV_MCS1_IRES_MCS2 3']. I am only able to express genes I clone into MCS1. The genes I clone into MCS2 are not expressed. What could be the problem?
Hi Benedikt,
One thing to check is that you have the second gene cloned into the right ATG start on the IRES. The translation initiated from the IRES is positional to the correct ATG, it does not follow the standard scanning mechanism for eukaryotes.
-Richard
It can be the two possibilities for the genes that has not been expressend. Firstly the gene cloned into MCS2 might have incorporated into into the cloning site which has disturbed the it and due to that the expression was not there. Secondaly, it might be possible that the first gene that was cloned is on 5' position where which can be starting codon for the expression and second gene is on 3' position the the starting codon is not their which van initiate the gene expression
Hi Benedikt,
One thing to check is that you have the second gene cloned into the right ATG start on the IRES. The translation initiated from the IRES is positional to the correct ATG, it does not follow the standard scanning mechanism for eukaryotes.
-Richard
Hi everybody,
thank you very much for your input! I think i figured out the problem. I did not realize the IRES had it's very own start codon for the 3' gene. I thought translation would simply begin at the ATG of my 3' gene, but it is actually - as Richard said - initiated at the IRES ATG. Due to this, I accidentally inserted a stop codon in frame with the IRES ATG by adding an XbaI restriction site to MCS2 (I cloned my 3' gene via the XbaI-site and another site) . I'll clone the second gene again with regard to the new information and let you know about the outcome.
Thanks again,
Benedikt
Hi Benedikt,
Have you had the new result yet? Was the problem really due to your 3' gene used IRES ATG and hit stop codon right away?
Thanks,
Hao-Ming
Hi Hao-Ming,
yes, that really was the problem. I cloned the second gene again using different restriction sites and it worked this time. Sadly, expression of the 3' gene was always very weak, compared to the 5'gene.
Therefore I switched to using a P2A-peptide instead of the IRES. By this I got much nicer co-expression (see attached WB), but depending on your genes, you might also get some fusion protein, as I did. Anyway, I can highly recommand using P2A-peptide, as it is very small (22 AA) and allows efficient co-expression (in theory co-expression should even be equimolar). Have a look at the attached paper, if you are interested.
-Benedikt
We have used the P2A system in many constructs for expression in neurons. Yet it does not work in all neuronal types and sometimes you still have to use the IRES.
The P2A does not generate equimolar products though.
Article Analysis of the aphthovirus 2A/2B polyprotein ‘cleavage’ mec...
Hi Robert,
I found the correct ATG by looking at the vector information of pLVX-TRE3G-IRES (Clontech, see attachment). This is a commercial plasmid with two MCS. The IRES ATG was actually the last ATG in the IRES sequence, right before the MCS2. As there are slightly different IRES sequences in different plasmids you have to find the correct ATG in your vector, but usually it is at the 3' end of IRES and in frame with the 3' cloning site. Attached there's a file with the IRES sequence I used for my construct, maybe it helps you.
However, as i said earlier, expression of the 3' gene may be very weak Therefore I recommend using 2A peptides instead of IRES for co-expression.
All the best,
Ben
Hi
This thread is really full of great information. special thanks to Benedikt Kien for explanations. I am very new to histrionic vector systems, Here I am trying to insert 2 cDNAs at MCS1 and 2 of pIRES (from takara). I am just not being able to get everything in frame. I am so far just on Snapgene though. It would be great if one can have a look here,
Am I supposed to have the IRES in frame with Construct 1 and Construct 2 both? Theres no stop codon at the end of 1st ORF.
I followed the advise of putting construct 2 in frame with 3" ATG of IRES.
Please see this map from snap gene.
Hi Sneha,
your ORF1 must have a start codon (ATG) and should further have a stop codon at the end. It does not need to be in frame with the IRES, as the IRES just serves as a starting point for translation of the second CDS (A2 in your map). ORF2 (3' of IRES) must be in frame with the IRES-ATG and should also contain a stop codon at the end.
Hope this helps!
Best, Ben
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