Hello all,
Long question, I know! In a nutshell, I have a construct that looks like:
promoterCMV::Coding1::STOPcodon::untranslated-150bp::IRES::Coding2.
The end of the translated Coding1 is 150bp away from the IRES, and I have the impression that it may be affecting the protein levels of the Coding2 (which is a puromycin resistance marker). I have that impression because when I make lentivirus with that construct, the titers are usually worse than with other constructs without that 150bp region, and by reducing the concentration of selective agent (puromycin) they improve.
The constructs I have info from don't share the same Coding1, so they are not totally comparable. The obvious experiment is to compare two constructs with the same Coding1 and with and without those 150bp of separator. But since I am not certain that the separator is the problem, I rather ask and see if someone has experience in the subject before moving into the cloning, virus production, etc.
Thanks!
Hi Ruben
I think a 150bp istance would be ok. But I strongly recommend you to use T2A or P2A instead of IRES. We found that the gene after IRES is always expressed not so good.
Genemedi is good at lentivirus and AAV production, please find more information on this website: www.genemedi.net/i/lentivirus-packaging
Hi,
in general IRES sites are not very effective. We compared and in the end decided to Use T2A based systems instead. Regarding the 150bp: From my experience the length of the insert between the LTRs plays a crucial role for transduction efficiency. This together with the non optimal efficiency of the IRES site can accumulate to the mentioned effects in my opinion.
Perhaps this helps
Sven
Hello, thanks for the answers.
Sven- not sure that LTR-insert-LTR would apply in our case, because those 150bp are about a 3.2% of the total size, and constructs without those 150bp have been working fine. Also, we chose the IRES because we did not want to aid a tail of aminoacids to our peptide... we had to make a compromise for a less efficient IRES there.
Delin- no known terminator there.
Thanks!
Hi Ruben
I think a 150bp istance would be ok. But I strongly recommend you to use T2A or P2A instead of IRES. We found that the gene after IRES is always expressed not so good.
Genemedi is good at lentivirus and AAV production, please find more information on this website: www.genemedi.net/i/lentivirus-packaging
Hi Yixiong, thanks for the input. Actually our reason to use an IRES is that we don't want to add any extra sequences to the first coding part of the construct, and the 2A peptides leave a 20-aminoacid tail when they are cut.
What I have realised is that we have a canonical polyadenylation signal in those 150bp, which could be a good reason for us to see low expression of the cassette after the IRES.
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