293 vs 293TN cells - Which would be beter control in testing against expressed proteins (Jun/29/2010 )

I work with both 293 and 293 TN cells. I know that 293 TN cells differ from that of 293 in that the 293 TN cells express SV40 T Antigen and Neomycin gene products and 293 cells do not. Otherwise, overall protein expression the same. We transfect the cells with a cDNA construct. We perform transfection based on the cDNA collection that we are working with. So one collection may code for Neomycin resistance in the cDNA, so we will use 293 since 293TN are Neomycin resistant and we can not select. Other collections have puromycin resistance, so we will use 293TN cells since they have higher transfection efficiencies and only Neomycin resistant. We would like to always just use 293TN for this reason (Higher efficiency) but again, based on the cDNA collection using, we need to select and can not if cell line has same resistance as cDNA plasmid codes for. My question is this. If you were only able to pick one cell line as a control, which would it be and why? We are trying to maximize production and need to pick one control regardless if cells were transfected in either 293 or 293TN cells. The cell lines are so similar that running 1 as a control (untransfected) should not matter (I am told). But I want to get more of an expert opinion because I was not able to find resources that would help me make the decision and back it up. We lyse the cells and run WB and would like to standardize to one control, either 293 or 293TN. Any information is greatly appreciated. Thank you.

In theory one control sounds fine... however, how do you know that the TN cells don't have genes or other regulatory mechanisms disrupted by the SV40 T antigen or the neoR gene? We are only just starting to understand that the so called "junk" DNA may actually play a role in regulation of transcripts and even protein interaction.

So... I say that you need to have both cell lines as controls unless you know absolutely that the TN does not alter expression of your gene of interest at all.