We have recently aquired plasmids with the following configuration:
EF1A>{gene of interest}:P2A:Bsd
When used for transfection (293FT cells and SH-SY5Y cells), these cells expressed the desired protein after 48 hrs (verified several times by western blot and viewing of GFP which is encoded in some of our plasmids). When the selection antibiotic is added, most cells survive, which is expected. However, after a few days, the cells no longer produce the desired protein (verified many times by western blot and viewing GFP).
To be sure, we always use a negative control for the antibiotics, cells which were not transfected, and they all died quickly (36 hrs at most).
Oddly enough, when used for lentiviral infection, there is no issue, and the cells continue expressing the protein even after a few weeks of antibiotic selection.
We have not run into this problem with other vectors acquired from other sources.
Thanks in advance
Hello, Ido!
If your cells survive selection with BSD, the problem is not in the construction but in the method used.
When we just transfected plasmids in cells the transgene is just expressed few days (about 1 week). If you desire long-term expression you must use a viral vector like Lentivirus or AAV.
Thanks for your reply!
I'd be grateful if you could clarify a point that has me stumped. Why do these cells express BSD resistance but not the desired protein? The resistance and the protein are under the regulation of the same promoter with a P2A sequence. If the cells indeed stopped expressing the desired gene, do you have an idea why they keep expressing the resistance protein?
Much obliged
Ido
How many days after transfection does BSD continue to be expressed? And when did you stop detecting your protein of interest?
Cleavage peptides such as P2A and T2A tend to be very effective for bicistronic constructions, so technically both genes would be expressed without problems.
Your problem is puzzling!
which kind of plasmid do you use ? if there is the SV40 replication origin on your plasmid then in cells expressing the T antigene they will replicate as episomes so you are not getting stable cell lines, cells will loose the plasmid progressively so the expression will lower progressively... but the resistance should also lowers. maybe the protein that gives the resistance phenotype is more stable than the gene of interrest ... or your selection is not strong enough... (it takes generally two weeks to select resistant clones ...
Attached is the plasmid structure.
Yes, it is an odd one...I retried several times because I was sure there must be some technical error, but I kept getting the same results and so did other people I gave the plasmid to.
About the gene not being stable enough, it also happens with normal gfp. Do you think it is likely that the resistance gene rna is simply that much more stable than the gene of interest?
About time for selection, I don't think that is the issue because I waited about 4 weeks and the cells are still resistant.
Ido Rosen
there is no SV40 origin on your plasmid so my hypothesis is not OK... a cryptic splicing sites in the P2A sequence and one after the eIF1 promoter ? a cryptic promoter in GFPUB ? ....
How about checking presence of your gene's DNA and mRNA presence? To know at which level the problem originates. Right now you're just wildly guessing, this would give you some starting point.
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