Sounds interesting, are you trying to compare different cell lines? Have you used any reporter gene as a positive control?

Maybe using another transfection reagent could rule out the possibility that the main reason behind failed transfection was PEI.

Expressions have been successful using CHO cell lines and PEI in the past, here is an example

Article Transient transfection of CHO cells using linear polyethylen...

CHO-S cells tend to be more difficult to transfect transiently (it is a common issue when expressing antibodies) than other cell lines like HEK which are much more efficient. You may want to consider stable transfection with CHO, or using an engineered CHO cell line. There are some commercial CHO cell lines that produce higher yields (but are more expensive) such as ExpiCHO you can try for transient transfection. You may look into flow electroporation as an alternative transfection approach. You may also try other transfection reagents, but that was not helpful for us.

It would be really useful to use a +ve control plasmid such as one expressing SEAP since it is easy to assay and very low levels of SEAP can be detected. The optimal transfection conditions with PEI are very process specific.

Article Cell line specific control of polyethylenimine-mediated tran...

A few simple optimization experiments will go a long way and can give massive increases in yield.

CHO-S cells have been used in many PEI expression experiements so it is unlikely that the cell line is the problem if it is standard CHO_S you are using.

Check the quality of the DNA. Could there be high levels of endotoxin?

Additionally media components can massively decrease tfx efficiency.

Article Iron (III) citrate inhibits polyethylenimine-mediated transi...

Could there be something in the media causing problems?

Hope this is useful.

Good luck.

Ben

Hello,

Thank you all for suggestions. Gave me some new ideas.

The CHO-S cell line is a standard one and been used in our lab already so I do not think this is a problem, however other CHO cell lines might be better, definitely something to think for the future.

DNA is endotoxin free (checked), PEI freshly prepared; I chose PEI as its pretty straight forward and also, would give comparable data to other cell lines we used. I will try to optimise PEI:DNA and DNA:cell ratios with reporter gene first - as you said, it will be easy to see if I get anything or not. If it doesn't work, might use another transfection system.

I was also wondering - since CHOs are quite resistant to transient transfections - is it possible that I get lots of host proteins (high abundance) and only a little of protein of interest (low efficiency, low abundance) and I simply cannot see it on a gel? Maybe it is worth doing WB against the protein (would see small amounts)?

@Alex Kashkin

thanks Alex for your suggestions; I am now finished with the study - have done GFP transfections and they worked however transfections with our protein did not work; it's hard to express protein and further investigation/experiments/different cell lines/transfection protocols will need to be performed