stable transfected cell line in absence of G418 - (Feb/17/2008 )
Hi, this is the first time that I'm writing in this forum, I'm having a little problem in cell culture and I need Help.
It's one year that I'm working with a stable transfected cell line that usually grows in presence of G418, last time during normal split process I forgot to add G418 in the DMEM and I plated normally cells, but before than 24 hrs I remembered it and I went to add antibiotic in the Medium and I chenged it in the flask!!
My question is: Can this event affect my stable transfected cell line?? Can I use them to do membrane preparation or I waste the cells??
Thanks
In my opinion, those cells survived after your adding G418 is stable-transfect cell line.
Good luck!
I on a pretty regular base have to grow a stable transfected cell line (normally in DMEM + puromycin and G418 as it's double transfected) in absence of either antibiotic for some weeks. Expression of the "extra genes" (transfected together with puro and G418) is maintained throughout, so 24 hours won't do any harm.
If someone makes me clear , once if a stable cell line is made by drug selection (say puromycin) and a resistant clone is obtained, later what is the necessity of growing it in the presence of the drug!
Epigenetic silenceing.... presents you with a 'if you don't use it, you lose it' paradox, since genes that do not offer a selective advantage tend to be silenced by methylation and hidden in histone complexes. Once stably transfected clone is established you can lower the dose of the selective agent considerably. However it's not a good idea to remove it completely, because selective pressure favors continued expression of genes of interest that are near the near the resistance gene necessary for survival.
That said, it's probably OK to leave out the G418 for a little while in culture, but I'd certainly not advocate for total removal unless you have a compelling experimental reason to (i.e. tumor xenograft into a mouse).
Epigenetic silenceing.... presents you with a 'if you don't use it, you lose it' paradox, since genes that do not offer a selective advantage tend to be silenced by methylation and hidden in histone complexes. Once stably transfected clone is established you can lower the dose of the selective agent considerably. However it's not a good idea to remove it completely, because selective pressure favors continued expression of genes of interest that are near the near the resistance gene necessary for survival.
That said, it's probably OK to leave out the G418 for a little while in culture, but I'd certainly not advocate for total removal unless you have a compelling experimental reason to (i.e. tumor xenograft into a mouse).
Does that mean whichever celllines you have which have been immortalized/transformed by exogenous expression need inclusion of respective drugs in culture? It amuses me as the cells which I use were transformed by three vectors (puro, hygro and Blast)! but I culture them in normal DMEM+FBS.
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Does that mean whichever celllines you have which have been immortalized/transformed by exogenous expression need inclusion of respective drugs in culture? It amuses me as the cells which I use were transformed by three vectors (puro, hygro and Blast)! but I culture them in normal DMEM+FBS.
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The transformed cells will grow without the selective agents, usually with a shortened doubling time. Thus if cells that have 'lost' or 'silenced' the insert are permitted, they'll eventually overpopulate those that retain expession of the insert, so it will appear that the expression level goes decreases. If you maintain selective pressure, in my experience at least, the reporter expression is stable.