First of all, even though the cells do not have the antigen, 1mg/ml of G418 is not killing all wt -they survive, but their growth is really slow compared to the others'.
My real issue is: even though the cells in general are red under a microscope, when I collect protein, and do a Westerns for the viral protein, the expression levels are varied AND low. It seems like that the cells are losing the protein (or downregulating its expression).
That, of course, means that I can't do any Westerns for my proteins of interest, which we suspect might or might not be affected by the presence of this viral protein... I need to make these guys express this protein, in a relatively stable manner, otherwise the whole project is useless.
How do people go around this issue? I don't want to reinvent the wheel, and I'm running out of time for my PhD... I read a lot, but I would like to ask people as well, because I don't have a great deal of experience with creating and working with stably expressing cell lines. (While we're at it -it was a great surprise to me when I started the program, that eukaryotic cells can express plasmid encoded proteins.)
I thought about cloning the construct into lentiviral vectors, and integrating the protein into the genome, but we had experience with GST tagged versions, and even those guys got downregulated. For two months I've been doing Westerns before I realized that the antibodies work, I just have no protein...
Thank you for your time.
Edited by Andras, 05 August 2011 - 06:52 AM.