I'm just trying to express a very small transgene by transfecting cells with a plasmid coding for the recombinant genome of AAV2. I have different constructs with different promoters, and there is only one that doesn't allow good level of expression. That same construct worked very well for expressing bigger transgenes, so here is my question: is it possible that the small distance between ITRs (2 kb) is affecting the expression of the transgene? Does anybody know about some bibliography about this? I couldn't find.
Thank you!
Hi Noelia,
It's a very interesting and particular situation. In theory there is not reason that explain a short distance between ITRs as a problem for poor performance. ITRs are prone to recombine meanwhile plasmid amplification. Were your plasmid evaluated for ITR integrity using XmaI?
I don't think the distance between the ITRs is causing your problem, I would think the problem is to do with the transgene itself or the way that it has been cloned into the vector.
How are you assessing AAV titre?
Thank you both for your reponses.
Melvin Y Rincón: Yes, I have checked the integrity of ITRs. It's very strange indeed. The funny thing is that I'm sure I read about this somewhere at some point but I can't remember where. Sometimes in our lab people had problems sequencing from recombinant AAV plasmids, apparently because of the ITRs, so I thought this could be a similar problem.
Jill Muhling: I haven't produce viral particles yet because I wanted to check the expression of the protein in eukaryotic cells first and compare different promoters that I have. So I just cloned the protein into the AAV plasmid and did transfections. I do have some expression of the protein but levels are really lower than the ones that I get with other promoters. I have checked transfection efficiency and it's similar to the other plasmids.
What really confuses me is that this promoter was the best one in our lab for expressing other proteins in vitro from AAV plasmids . The main difference is that in my case the transgene is very small, but I never had any problems expressing it before, neither from E. coli nor from eukaryotic cells.
OK, a couple more questions: which AAV system are you using, pTR/pTRUF or pAAV (or another one?)
When you say your transgene is small, how small is it? 20bp, 50bp?
If you are just doing transfections I would not think that the ITRs would affect expression that much. The plasmid will just behave like any other expression plasmid, maybe a bit less efficient due to the secondary structure of the ITRs but it should still work OK. It's just a standard AAV transgene plasmid, (ITR-promoter-transgene-ITR), right?
Jill: I’m using a standard pAAV ITR-promoter-transgene-ITR, with a strong viral promoter. The transgene is 450 bp, it’s small but not that much. I’ve cloned 2 different transgenes (both 450 bp) and I have the same problem with both.
So you don’t think it’s a problem of ITR interference? They are 2 kb away from each other
Not if you're seeing transfection issues, no. To my knowledge the ITRs should not come into play significantly if you are doing transfection only, the plasmid should behave pretty much like any non-viral expression plasmid.
You said that transfection efficiency is fine (how are you measuring that with no fluorescent protein by the way?) but transgene expression is very low. That sounds like either a protein detection issue or a promoter issue.
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