Problems obtaining stable transfected 293 cells - (Aug/02/2007 )

Hi!

I transfected 293 cells using pCI-neo. The inserted gene is quite huge - 4,1 kb. In transient transfection I get a full-length protein, which I can easily detect by Western. But although cells grow in the presence of 600 µg/ml G418 (controll cells die) I cannot detect any recombinant protein in growing cells.
What can be the reasons? Should I use a different vector?

I also have some very basic questions regarding the selection procedure:
When should I start selection? How often should I change selection medium? What should be the suited cell density? Should I split cells every time, when I add new selection medium? Or should I just remove the old medium and add new one? How do I obtain single clones?

Regarding the vector system: Do you think, using a bicistronic vector could help? Or do you know any vector system which carries two different selection markers flanking my inserted gene of interest?

As you can imagine from my questions I do not have experience in this area. So any reply would be of great help for me - even if you answer only a single question. Thanks!!

I have had the same results trying to make a stable cell line using the FlpIn-Trex system with Invitrogen. In fact, after selecting and isolating colonies, I would be able to detect expression when induced (my line was supposed to be Tet-On) for about one week. Then it just disappeared, ie: no expression when induced, in the same population of cells! (My construct was GFP-tagged.) I tried to make the line three times before giving up. Meanwhile, I was able to successfully make four other lines in the same system. I know that what I was trying to express is extremely toxic to cells and I assumed that the small amount of leakiness in the system was killing some cells while others had found a way to silence my insert. I never tested by Northern blot to see if my construct was still integrated..seemed like a lot of work when all I knew was the cell line wasn't going to happen. Perhaps your protein is toxic as well? Are your cells healthy after transient expression or do they die after awhile? As for the selection procedure, I follow the instructions in the FlpIn manual. It hand walks you through each step and as I mentioned, I was able to easily produce four other stable cell lines, which express (under tet-control) non-toxic proteins. At least it would give you an idea to the timing and neccessary steps to create a stable line.