In article <01bbbfd6$9e62c400$21746a8d at meb_493a.cvrc.mcw.edu> "Joe Miano" <jmiano at post.its.mcw.edu> writes:
>Question:
>>If a transgene is stably integrated in a cell line through selection with
>G418, why is continued G418 necessary with clonally-derived cells? It
>seems that once the gene is integrated and cloned, no further G418 would be
>necessary, n'est ce pas?
If the transfected gene confers a significant growth disadvantage, any
cell that managed to lose the gene would outgrow the rest of the colony
eventually. The mutation rate is not very high, but with anywhere between
1 and 10 million cells in a flask, mistakes can and will happen during
DNA replication. One alternative to constant selection is intermittent
selection, where you alternate regular medium and G418 containing medium
on a weekly basis. You might also be able to re-select on a less frequent
schedule. If a significant number of cells die during re-selection, the
transfected gene was lost in a significant % of the cells.
Annette
ah690549 at mbcr.bcm.tmc.edu
