loss of expression in stable transfection - (May/25/2006 )
I have established T47D cell lines stably infected w/ RTV vector that originally expressed protein of interest quite well on WB. I have continued to maintain these cells in selection media, yet all of a sudden not only is my protein of interest gone, the entire construct cannot be found by mass spec. Some knowledgeable people have indicated that this is expected to happen. Is it really and if it is what advantage then do stably transfected cell lines have over transient?
grumble, grumble...
grumble, grumble...
well not all the time but this happens, once for my tabacos even after T2 generation. one of my friends told me that this is becouse of the problem with recA protein another told me no they shutdown them self and muted fast, more friend you have more suggestion you recieve but when you have the Stable GMO they are easier to handle than transient and you could get higher protein than transient method even when transients rich higher recombinant protein / total soulouble protein ratio.
The protein expressed from the gene you transfected may have some inhibitory effect to cell growth. Try to use Tet-on or other regulated expression system for your gene see if that will help. The advantage for stable transfection is of course consistancy over transient method, your case is an anomaly. 
Thanks - I'm worried that you may be right about the inhibitory effect; our original hypothesis was that it woud promote growth...
our stable cells in 293 also have this kind of problem. however, the expression level just decline gradually. I read and agreed that once the plasmid get integrated into chromosome, the cell could selectively turn off the gene by DNA methylation at its promotor region. however, the resistant gene is still useful, therefore, that promotor is not turned off.
i do not really know how to do with this problem. stable cell line is not really stable, better freeze down more vials of low passage number cells.