losing protein expression after stablizing cells( please help) - (Nov/13/2007 )

hey all,
I have transfected CHO cells 9 times with GFP plasmid with Lipofectamine 2000, but I get so many clones( actually they cover the whole plate surface) that are resistant but ,say, 80% of them do not show GFP, and 20% are green, but after 20 dayes I do not see any GFP!!!!! while I used INTEGRASE gene to have my construct integrated in the genome.
what might be the reason?
tnx,

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Is this GFP only or GFP fusion protein?
After 20 days meant 20 days after transfection or 20 days after picking clones (that is, after all the selection (with antibiotic), picking clones...)?
You do not see any GFP. Did you just take a look under microscope (live cells and just look), or did you look at fixed cells, or did you do IF (that is, using anti-GFP and stain with another colour, e.g. red)? Did you induce the cells?

This is quite a common phenomenon with stable cell line. There has been a number people having the same experience. There could be many reasons for this, just for a few:
- Maybe your protein is toxic to the cells
- The resistant cells does not automatically mean that they will have your gene, they may just get the neo gene (for example) integrated into their genome, and the rest has been lost, truncated, mutated...
- Even for those that got your gene integrated, this gene still may be lost after awhile
- Stably expressed genes usually show lower expression than transient. You may not see it, but it is still there.

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you probably did not get true stable colonies from these cells. Did cells in the control group all get killed? EGFP or EGFP fused to something was used?

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after 20 days of transfection the transfected may be lost in all cells and were not integrated in the genome; or you have some stables which do only a low constitutive expression; use an inducable promoter, or do classical selection with G418

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