Loss of GFP - (Apr/16/2007 )
I transfected NIH 3T3 by calcium phospahte method. A got about 30-40% transfection efficiency.I started selecting the cells by 350ug/ml G418.But now after 10 days,few of my cells are losing the GFP-fusion gene. I could see less no. of fluorescent cells as compared to the day after transfection.
Does that mean that transfected cells are losing the gene?
What could be the reasons for that?
PLzzzz help.
-minnee-
QUOTE (minnee @ Apr 16 2007, 04:36 PM)
I transfected NIH 3T3 by calcium phospahte method. A got about 30-40% transfection efficiency.I started selecting the cells by 350ug/ml G418.But now after 10 days,few of my cells are losing the GFP-fusion gene. I could see less no. of fluorescent cells as compared to the day after transfection.
Does that mean that transfected cells are losing the gene?
What could be the reasons for that?
PLzzzz help.
Does that mean that transfected cells are losing the gene?
What could be the reasons for that?
PLzzzz help.
do you really need 10 days to kill all non-transfectants by G418 (350 µgIml)?
-The Bearer-
Most cells, even those expressing GFP will be killed, because of transient transfection nature. Only a few hundred will survive the selection. Those will grow into colonies and the colonies that carry GFP are the ones that you collect.
-genehunter-1-