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Interesting gene expression in a stable transfection - (Jun/28/2005 )

sad.gif Hello, every body:
I have got a tough problem. Thing is like:
We have a stable-transfected cell line coming from other lab, which has been maintainance for several passages in our lab and confirmed to be good. Then, the -80 freezer went bad... ..
Anyway, I thawed this cell and they started to grow badly. After some generation, they seems to be recovered and survived the select pression of puromycin. But, I could not detect any functional activity of our interesting protein from these cells.
I am confused. how come these cells can survive the selection( Puromycin) , but no expression of the protein. blink.gif
Eager for the answers!!!


i assume that your resistance gene and the expression of your protein are not controlled by the same promooter and an IRES?
Hence you can not control transcription of the gene coding for your protein. Seems that cells that have survived are cells that do not express your protein. Anyway, try to make clones of these cells and after test for presence of your protein...
Last question? Does the expression of your protein confers a disadvantage to te cells that are actually expressing it? is it toxic or sthg like that?


Well, it seems not. Because the this cell line are stably transfected already. Previous people has used it fore experements. However, the -80 freezer, storing the cell went bad once. I think it must do something to the cells . they are starting very bad. so, I did not give puromycin at the first beginning and let the cell grow for several generation, then adding the puromycin to select.
And the interesting protein is dopamine receptor, which should not be toxic to the cell line (HEK293 cell).
Thanks for your answers