Transgenic cells lines generally have a limited number of passages for which they are usable; after that, the cells will undergo changes like the ones you mention and become useless for assays.  I have experienced that with HeLa cells expressing neuronal receptors, for instance.  The trick to working with transgenic lines (and any cell line, really) is to freeze back a large number of aliquots of the cells at a very early passage, then establish for how many passages the cells can be used before they go "bad".  You pull out a fresh vial of cells from your large early passage stock before the current batch of cells hits their passage limit.

If you used a selection agent, you may consider adding some back in your culture in order to limit transgene silencing over time.

Was the original cell line you used a normal diploid CHO line or was it already transformed (permanent) and could be grown indefinitely under normal conditions? If it was already a permanent line then you have a reasonable chance that a transgenic derivative  can also continue growing indefinitely. However, as pointed out by Charles Fernalid, there is no guarantee that the transgene will be retained and expressed since all transformed cell lines undergo genome changes. It sounds from your description that your transformed iine is under stress after 6-8 passages. I suggest you hang in there and continue a large number of cultures in parallel to see if some cells/lines eventually pull through and then assay again for transgene expression.

Thanks all for your suggestion.

@Raymond: Yes that's most preferred way to deal with.

@ Peter : That's very true, I am loosing the stable after 6-8 passages. The host cell line is CHO-K1, and transfection done using nucleofection method. I have made so many stables but few genes are really weird. In my case im using G418 and trans gene is one of the GPCR.

One more question : What course of selection pressure should be followed for stable generation  e.g .24 hr Post Transfection I dilute and seed cells in 6 well plate with different G418 right from 50 to 1000 ug/ml in different wells. Post colony or clone selection I put 600-800 ug/ml throught till cell freezing. Is it right way or gradual increasing in G418 over the passages is works better.

E.g. Passage P2- (50 ug/ml) , P3- (100), P4-(300) and so on ..

My experience with post-transfection selection is this:  With the cell line you are transfecting, you do a "kill curve" at different antibiotic concentrations on non-transfected cells to determine the minimum concentration of antibiotic that will completely kill all cells in a well/dish.  Then you use that concentration for your selection after transfection, and begin the selection 24 hours after transfecting the cells.  Once all the non-transfected cells have been killed off and you have established the resistant cells, you can either keep the antibiotic at the selection concentration, or scale it back 50% to a "maintenance" concentration.  

Yes Raymond, very true I am currently following the same proc what you mentioned. But want to try something different which may help for getting better performing clones.

Hello Nikhil, i made a dose response with G418 and CHO K1, but my cells are very resistant to G418. What is the concentration that you use?

Hi  Mathilde Poujol de Molliens,

I would suggest you to check the G418 stock, if not stored/reconstituted properly may loose its potency.  In our lab we normally use 400-800 ug/ml range as selection pressure for stable clones.

I also recommend to keep different G418 pressure ranging from 400ug/ml to 1 mg/ml in 6 well plate.

If possible prepare and store the multiple aliquots at -20 apart from one which u will keep at 4 degree(1-2 weeks).

Its better idea to run a kill curve for each lot.

Reply if you have further queries, it will be my pleasure.