I have obtained a new cell line (stably transfected) from another source and have found out that the cells should have been grown in the presence of G418. However I have ben culturing them for nearly 2 weeks without G418. Do you think the cells will have lost the gene ? Am I also right in thinking they shold be cultured in a concentration of G418 lower than that used to select stable clones ?
Any advice much appreciated

Hey,

In doubt I would start to culture your cells again in medium containing G418.
If they still grow you can be more sure that they still have their insert.
I think you have to try it.

For some analysis it might be better to culture cells without G418 prior to an experiment so it´s not uncommon to feed cells with just medium.
In general I would try to keep my cells under "constant" medium conditions.

If you are in doubt about the concentration:
-Do you have cells without the insert? In case you have you could make several dilutions for G418 and see which concentration is necessary to kill cells without insert. This is the concentration I would use for culturing my cells as you just need to provide selection pressure. It´s not necessary to use the limit as your cells are already stable clones so it´s just about not loosing the insert and not about killing untransfected cells.
Unfortunately I do not know your cell line, I just can say what I would do with the cells I know.

Hope this is some input
Good luck
Cheers

hi
normally, G418 resistant cells are cells wich have successfully intergrated the resistance gene in their genome. Hence, there are little chances you lost your gene.
By the way if you are in trouble with this lack of g418, culture your cells in 400µg/ml G418 for one month would kill cells unresistant to G418. In my lab, after establishing stable cell lines, we usually cultuvate them without G418. But every two month, we dilute them more than usual and add G418 zat the above concentration. And we never noticed a lost of gene...

fred

thanks for the advice.
I guess I was worried about the cells kicking out the plasmid if the selection pressure was not present. I have heard that some genes may not have integrated as 'Stably' as others into the genome and so if no selection pressure is present cells will loose it. Does this make sense.

hi
this partially make sense.
as you do not control the opening of your vector, you can't be sure the gene of interest is not killed. Even if you cut your vector before transfection, you're not sure that your G O I is not killled too. by selecting G418, you're only sure the G418resistance gene is integrated and intact in the cell. that's why i said it make some sense
but after integration, i don't think the cell can kick out the integrated fragment only, whithout kicking out more DNA. And generally it's lethal or damaging the cell enough that you can see a change.
I've heard about unspecific macro deleting events, but it is a process strongly nefast for he cell as it kicks out many kb, and generally cell don't survive.