If you want to transient transfect and select, I think it is more dependent on the cell line you use. A good selection marker (puro / G418- whichever works well in the target cell line) and not so bulky size would be 2 features necessay for the plasmid (In addition to the promoter criteria mentioned above- in case of not well tolerated proteins).
But as an exception, I have made several cell lines with CMV promoter expressing plasmids (strong promoter) and have also selected cell lines by co-transfecting resistance containing plasmids separately. At the end of the day, it depends more on the toxicity of your expressed protein and the tolerance range of the cell line..
The point regarding the promotor might get important regarding the cell lines you use. Some cell lines silece some kinds of promotor. Eg our experience is, that Hela cell silence the CMV promotor, whereas U2OS cells like it. So first you should figure out if the promotor of the plasmid you choose is fine in the cell line you use, eg by transfecting a gfp expression plasmid (or any other easy detectable reporter) and checking regularly the expression over time.
The strenght of the promor is only critical if your expressed gene is not well tolerated by the cells, but keep in mind, that potentially it might be if your expression is very high, you might some "overexpression effects" which not necessarily has something to do with the physiological role of your gene.
Best,
GAD
Agree with Guido, the promoter can be important if the expression of the protein is not tolerated by the cell line in use. Since you are trying to create a stable line, you have to make sure the plasmid contains a selection marker. Puromycin, blasticidin, G418 are some common selection markers. You can create stable lines like Manoj mentioned earlier (transient transfection followed by selection) or you can look into lentiviral transduction.
If you want expression in vitro you might get one kind of answer, like the CMV promoter to drive expression and the puro selection marker. If you are modifying ESCs or iPSCs with the intent to make a transgenic animal which expresses your protein in vivo, you will be getting different advice (CAG is a strong promoter, the viral promoters like CMV get silenced fast).
Just in case you are thinking to use Vero cells, I can tell you that, as Hela, they do like to silence the CMV promoter. I have a lot of issues trying to keep the expression my protein of interest for more than 10 passages, I might need to change to a lower activity promoter. Good luck with your experiments!
Perhaps a more basic question may be. Am I trying to mimic a disease state or a cell signaling / protein-protein interaction in cell culture. That can make the decision of which cell to choose hard or easy. Look to the literature on that problem As to plasmid expression, promoters can be ranked as CMV> LTR> SV for promoter strength. There was a report ~ 1980's I think in MCB where promoter strength was measured, You can track it down in PubMed. Please note that promoters like CMV work best in growing cells since they need, NF-kb-like transcription factors. Once stationary their expressions levels go down. If you need to have basal levels look at one's with SP1 transcription sites. These also should work in more stationary cultures. Primary cells are best transfected with HIV LTR systems. Lastly consider if your protein is toxic at high levels. In that case use a tet-on system. or a weak (SV) promoter.
Good Luck with your research
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