We're currently using pCDNA vector to induce protein overexpression in the Ntera2 cell line and having problems with cell survival after transfection with plasmids containing the insert for our protein of interest. We see no death with either GFP-containing or mock plasmids. Cell death occurs irrespectively of the transfection medim used, suggesting that the overexpressed protein promotes death. I'm persuaded that this effect is expression level-dependent, because transfected cells survive when low amounts of DNA are used. Unfortunately, in these conditions, the transfection efficiency is low (only about 15-20% cells) and I need high transfection efficiency for biochemical approaches. I guess this could be easily solved (without the need of inducible systems which require engineering my cell line) using a promoter with considerably less efficiency than CMV (which controls recombinant gene expression in pCDNA). I've read the article by Mezzanine and colleagues (DOI: 10.1371/journal.pone.0085550) and PGK promoter seems to be a good choice. Please, let me know if this reasoning is correct. If so, does anyone know a commercial or non-commercial source of a mammalian expression plasmid with PKG upstream the cloning site?.
Thanks in advance,
G. García del Caño PhD.
Yes it sounds reasonable in principle. The relative promoter strength strongly depends on the cell line used, but it's certainly true on average that PKG should be somewhat weaker than CMV, especially immediately after transfection. But it's not a huge amount (maybe a few fold?).
If you make stable cell lines, CMV is going to be more susceptible to epigenetic silencing over time, which might make PKG come out ahead in the long run.
PKG is tiny. I would just include it in a fully synthetic sequence, along with your gene and all the other necessary features, rather than bothering with trying to clone by hand.
Some other strategies to lower the expression level: Destabilize the mRNA by removing WPRE or polyadenylation signal, if you have them. Or destabilize the protein by adding a degradation motif "PEST" sequence.
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