HEK293 tansfection with pcDNA-YFP - can anyone help?! - stable transfections (Aug/04/2008 )

Hi

We use this plasmid in the lab to transfect HEK293 cells in order to make stable cell lines expressing various TLR-YFP constructs. Plasmid wise all the genes of interest are in frame, correct sequence and confirmed to be present!! However - we have a slight problem!

One particular transfection line after 1month of G418 selective pressure (quite high at 1mg/ml the student tells me....) is not positive for YFP expression by FACS (analysing in FITC channel) compared to negative control. Control cells are dying at this concentration of G418 - so no 'resistant' cell line yet we hope! At 40% confluency these transfected cells are ~10% YFP positive, but at 85% or so, they are no more fluorescent than the controls.

Can anyone suggest what might be happening here? Has anyone seen this before in that cells are resistant for the selective antibiotic but no expression is observed? Is there an alternative test we can try? The student is going to extract mRNA to confirm integration of the TLR gene later this week......

Many many thanks!

I believe that some types of 293 cells are resistant to G418. You may need an additional antibiotic, depending on which antibiotic the vector that you're transfecting is resistant to (examples might include hygromycin or kanamycin). Here is also a general protocol that you can refer to on How to Produce a Stable Cell Line.

it may depend on your protein of interest rather than YFP; follow the expression of your untagged protein