About the choice of 2A sequence

http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0018556

I have a lentivirus that contains 6 ORFs separated by 5 different 2A-peptide sequences and after transduction they all express. Theorectically they should all be expressed equally given they are being translated from one polycistronic message. Of course expression of all of them is dependent on the transduction efficiency/number of integrations of the lentiviral DNA into the host genome.

Either promoter will work. Both are constitutively active. CMV is stronger than EF-1 alpha but can sometimes be down-regulated/inactivated in the cell. If you can, make constructs with both promoters and see which works best in your situation.

Good luck!

The expression level of each individual protein can be different If you use IRES to separate the cystrons. However, if you use 2A-peptide, it should not be any different. There is report that using T2A-peptide gives better expression efficiency compared to using F2A or E2A-peptide. CMV promoter does not give consistent expression efficiency in stem cells. CAG promoter is better than CMV. In our hand, EF-1a promoter sometimes is silenced in stem/progenitor cells. Here is the link of a paper about that.

Hey,

we used EF1 promoter, however only with one T2A peptide. I would also assume that all orf will be translated but I could imagine that the efficience of T2A site is not 100% so the latter ones could be translated wiith less efficiency. Howevver tis is just a theory based opinion.

Regards

Sven

 Great advice.Thanks a lot!!

CMV is not a good promoter for IPCS

I advise you to use EF1, PGK, or ubiquitin

The 2A system is not a guarantee to have exactly the same level of expression of your two sequences of interest but, at the moment, I think it is the best design for multicistronique vectors  (forget IRES, some people become crazy with this system ;)

Nicolas

I'll take that into consideration, thanks a lot!!

I think quite a few iPSC vectors use multiple 2A sequences to express multiple genes so at least it's a fairly common technology. They are different 2A sequences though as repeat sequences are generally not a good idea when trying to make LV. Off the top of my head I believe that different 2A peptides have different skipping efficiencies - that is, protein separation doesn't always occur and sometimes you get a fusion protein. P2A has the highest efficiency and I think F2A might be the worst. It's also cell type dependent.

About the choice of 2A sequence

http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0018556