making stable cell line - (Jun/20/2008 )
hi, how important is it to have a vector that integrates into the genome for making stable cell lines? it is possible to make a stable cell line using a vector that does not integrate like ptarget? thanks.
My understanding is that any plasmid vector will integrate into the genome; the important thing is having a selection marker (neo,hygro,puro,gfp etc), to select out that rare integration event.
as cellcounter says, you need a resistance factor gene for selection, and a working promoter; but why bearing the risk to fail selecting a stable cell line with a non-integrating vector?
The reason you can't have a stable cell line without integration is that the plasmid will not be replicated at each cellular division unless it is integrated, so will eventually be lost from the cells. The loss will occur even with a selection marker, unless the plasmid is integrated.
I think EBV-ori plus EBNA based system allows one to make stable cell lines with episomal plasmid replicating in low copy numbers.
Can you clarify your objective behind why you would want to make stable line without integration?