expression of transfected gene - urgent (Nov/10/2005 )

I have a question about transfection and western blot.

I established cell lines stably expressing a cloned gene. I can detect the exgeneous gene has been intergrated into genome by PCR. But I cannot detect the protein by western blot. Also, I use transient transfected cells for western blot, in this experiment, I can see a very specific band from 9ug total protein which confirms the antibody works well. The difference between transient and stable transfection is that in the former I use circular DNA but in the latter I use linearized DNA. And for stable transfection, I do not select single cell clone

Thanks a lot for your suggestions.

Hi wmqiu,

How many stable cell lines have you examined??? Whenever you do a stable transfection it is a lottery as to where the gene is incorporated into the genome and hence a lottery as to the level of expression that you obtain due to repressors/enhancers etc.


Cheers,

Scott

h
in different clones level of expression can change from one to 3 or more... So in case of low all-cell-expression, selecting clones can help gfetting a good expression. For proper tests, at least 10 clones should be tested.
fred

I agree with fred33. If I may add, other problems night be poor expression due to protein toxicity, from which the cells overcome by shutting down the expression of the gene (this proccess may happen only after prolonged expression, such as stable transfection, as opposed to a transient one).
Another problem I encountered was a general promoter repression.

Finding your transfected gene by PCR does not essentially mean that it is integrated and getting expressed. I know not of your PCR protocol, but it is a technique with a potential to amplify one single copy of a DNA fragment (even half of it might be sufficient, come to that ) which might be present in your medium even after 21 days.

As far as I know, transfecting linear DNA is not a common practice as the efficiency is much lower (it is usually electroporated). For stable lines, I usually transfect circular plasmids and seed the cells quite dilute. Then I apply about 500ug/ml geneticin for selection. And I do pick clones.
Good luck!

Diril