Does the IRES-eGFP in the bicistronic construct be in-frame with the gene of interest upstream of it? I put my gene of interest upstream of EMCV-IRES-EGFP and realized that EGFP is in frame 2 while my gene is in frame 1. Will that affect the tranlation of eGFP?
Thank you.
Hi Jivan,
They may not be in frame, since both ORFs have independent ribosomal start sites for translation.
While the cassette Xprotein-IRES-GFP is downstream a proper promotor and upstream of a proper terminator signal you'll have a molecule of mRNA containing both ORF and 2 independent ribosomal entry sites. Ribosomal translation will recognize the start codon off the first ORF and another ribosome will interacting at the IRES site recognizing the start codon of the GFP ORF.
Nevertheless, IRES systems are not 100% efficient due to formation of secondary structures hindering polymerase binding among others.
Cheers!
Hi Sebastian,
Thank you for you reply.
Do you mean that the protein of my interest will not be efficiently be expressed by saying that it is not 100% efficient? Or is it just the eGFP ORF that is affected?
Hi Jivan, I just saying that is quite frequent the situation in which only one of the ORF is expressed properly or their ratio of expression is far from 1. This generally depends on the cell type and also secondaries structures present in the ORFs. Hopefully your design will work 150% and more.
Good luck!
- Badges
- Science topic