A problem about G418 selection - (Jun/22/2005 )
I transfection a plasmid carried GFP protein into some cell lines
and add G418 to select cells after the lipofetanmin incubate 48 hours,
After 1 month I collect the cells,
I did't detect the GFP expression by microscope , and also did't dectect
the target protein expression by western,
Why these cells keep the G418 resistance but did not express the GFP and my insert protein?
(ps: the plasmid is work and have been successed in other transfection experiment.)
Hi,
From your question I presume you didn't clone out individual colonies and therefore would have a mixed population of cells. When working with a mixed population there is going to be a subset that develop resistence or have a natural resistance to higher levels of G418, these will be selected for as the expression of GFP is actually toxic to most cells (they don't like it). Therefore over time your non-GFP cells will compete out your GFP expressing cells.
Did you notice with time a diminishing fluorescence level?
Did you check to ensure that your G418 levels were high enough to kill all cells ie. untransfected control in G418 side by side?
The other possibility is that your GFP-protein is toxic to the cells resulting in a selective pressure to 'spit out' your gene of interest, this can sometimes happen while maintaing the selective marker as it is generally a few Kb from the multiple cloning site of your vector.
To overcome this I'd try and clone out from the start using limid dilution or cloning rings. screen for positive clones early and monitor their progress over the month.
Scott
Thanks for your detail suggestions,
It's really make sense and I will try it.
From your question I presume you didn't clone out individual colonies and therefore would have a mixed population of cells. When working with a mixed population there is going to be a subset that develop resistence or have a natural resistance to higher levels of G418, these will be selected for as the expression of GFP is actually toxic to most cells (they don't like it). Therefore over time your non-GFP cells will compete out your GFP expressing cells.
Did you notice with time a diminishing fluorescence level?
Did you check to ensure that your G418 levels were high enough to kill all cells ie. untransfected control in G418 side by side?
The other possibility is that your GFP-protein is toxic to the cells resulting in a selective pressure to 'spit out' your gene of interest, this can sometimes happen while maintaing the selective marker as it is generally a few Kb from the multiple cloning site of your vector.
To overcome this I'd try and clone out from the start using limid dilution or cloning rings. screen for positive clones early and monitor their progress over the month.
Scott
Hello, Scott:
Maybe, you can help me to find out the what is going wrong in my experiments. I have some Dopamine receptor-transfected HEK293 cell, which are working well in the previous assay. I thawed the cells from frozen stcok. I noticed that the cell is not starting well, very few of tem survied, which might due to once broke-down of -80 freezer. However, these cells are growing better and better after several passages. But I could not detect any expression of the interesting protein D2 receptor.
So, we add puromycin to select the D2-expressed cell. Somehow, some of the cells survived the selection, but failed to give us signal of D2 receptor. So, how come these cells, which we know they have succed transfection, survive the puromycin selection, but no expression of our protein? Amazing???
Thanks a lot for your time!
Juan