Loss of expression of EGFP-actin from MCF-7 transfected with pEGFP-actin - (Aug/29/2007 )
Please Help !!
I am facing a problem of either loss of expression of EGFP tagged to human b-actin gene or the plasmid itself from MCF-7 cells. I have respective vector control. For first 2-3 passages of the clones, % of EGFP-actin expressing cells is more than 70. but as the passages increase the expression and % cell positivity goes down to as low as 20%. the vector control cells have above 90% positivity all through out.
I am maintaining the clones in DMEM + 10% FBS + Antibiotics + 800 ug/ml G418
I have tried Sorting the cells but same thing happens to the sorted clones 
help required urgently !
Aditi
I am facing a problem of either loss of expression of EGFP tagged to human b-actin gene or the plasmid itself from MCF-7 cells. I have respective vector control. For first 2-3 passages of the clones, % of EGFP-actin expressing cells is more than 70. but as the passages increase the expression and % cell positivity goes down to as low as 20%. the vector control cells have above 90% positivity all through out.
I am maintaining the clones in DMEM + 10% FBS + Antibiotics + 800 ug/ml G418
I have tried Sorting the cells but same thing happens to the sorted clones
help required urgently !
Aditi
How is the growth of your tranfected cells compare with control?
If your starting point is about 70%, then usually the normal cells growing faster will crowd your transfected cells, push it out of the pool.
How did you do your stable transfection? This is stable clone or stable pool? A clone may give you more chance than a pool. You sure they are stable? Did you do IF or FACS and induce cells to see if maybe the protein is still there just that it is weak? Stable transfected proteins usually will express at lower level than transiently express.
Also, sometimes there is really no help for it. Then you will just have to thaw new ones every so often.
Infection may help you sometimes, too.
hey Almasy,
Thanks for the reply.
I used electroporation method for transfection. We initially checked the transfection efficiency by flow and selected 4 clones. These clones we selected by G418 and checked expression of GFP by flow. the expression was above 90 %. But after 2 weeks even after continuing G418 selection, the expression came down gradually from 90 to 30%
FACS did not help as the sorted clones also showed similar decline over time. what could be the reason ?
How is the growth of your tranfected cells compare with control?
If your starting point is about 70%, then usually the normal cells growing faster will crowd your transfected cells, push it out of the pool.
How did you do your stable transfection? This is stable clone or stable pool? A clone may give you more chance than a pool. You sure they are stable? Did you do IF or FACS and induce cells to see if maybe the protein is still there just that it is weak? Stable transfected proteins usually will express at lower level than transiently express.
Also, sometimes there is really no help for it. Then you will just have to thaw new ones every so often.
Infection may help you sometimes, too.
[/quote]
I am currently having a similar problem with my gfp transfected (also with electroporation) cell line (K562) - trying to get a stable cell line. We select using blasticidin for several weeks and have to do a couple of successive flow sorts to decrease the amount of non-green cells but it is always a battle. In addition I have cells that are green but not expressing and/ or do not contain my gene since the gene and gfp are under separate promoters. The non-green cells grow faster and out compete my green cells so I flow sorted the cells into a 96 well plate in order to get clones. Additionally the gene that I am using causes the cells to be growth suppressed which makes it even harder.
While struggling to get a stable cell line (I've been trying for over a year) I've been using transient transfected cells to get data because I have good efficiency and western blot positive data with the transient cells. If you have good efficiency this could be an option (depending on what you are trying to do) - albeit it could get expensive especially if using nucleofector but in the end we just need to get the data (and graduate in my case)!
Good luck