I am have been growing pcDNA3.1 (with insert) tranfected HEK293 cells in G418 for a month now. Although the transfected cells are alive and growing well (all the untransfected cells died), the protein of interest is not being expressed at all. Does anybody know why this might happen?

Sometimes the cells could kick out the gene of interest and retain the antibiotic resistance gene so ur cells grow in the presence of the antibiotic yet not produce the protein.

or it could b that the protein production is too low, can happen with stable cell lines.

Your gene is integrated in to cell genome. It gives various kinds of expression level in stable transfection. Like 0-100% of expression. For the your statement not see the any protein (insert), for this conditions there is only possibility is occur that is the expression level is very low.

I met the same problem. I transfected pcDNA3 with my target gene into HEK293 cell, the target gene expressed very well, I can detecte the protein, but when I transfected same gene into another cell line (HEP-2), I can not detect the protein though I can detect the gene expression by RT-PCR.

I've only cloned with pCDNA3.1 twice. It failed both times! Almost everyone I've talked to who has used the plasmid has something negative to say about it.

In my case I cloned a cDNA into it from another construct (in which it expressed well). However, once in pCDNA it no longer expressed, although the antibiotic resistance still worked. I wasted a lot of time before sending the plasmid for sequencing and found that the region coding for the C-terminal portion of the protein was not as expected. There was a section in which part of the end had been duplicated and another part was missing! Something unstable about the way the plasmid replicates????

My advice, use Bluescript / pUC etc where possible.