Stable transfection using plasmid with no selection gene - (May/10/2007 )
I've read you can get stable transfections using a plasmid that doesn't have an appropriate antibiotic resistance gene for eukaryotes if you co-transfect it with a plasmid that contains it. I can see how you can select transfected cells using this strategy BUT can you propagate these positive cells? Would not the plasmid of interest be segregated only into one daughter cell since it won't be integrated into the genome. I mean, for a stable transfectin, in addition to a resistance gene, you also need genes that allow recombination of that plasmid into the genome, don't you?
Input appreciated. Cheers.
woww woww... you are doing a addition of recombinason that occurs in yeast relatively easily and integration in genome of the cell.
When you cotransfect 2 genes you need to transfect 5moles of plasmid of interest (poi) for 1mol of plasmid carrying the resistance gene (pcrg).
Once you get resistant cells, the pcrg is integrated. You have to establish cell clones to then study for your poi to be there. But these plasmids are integrated in the genome as they can't propagate. So if they are integrated, they replicate with the standard genome replication.
if you have fluorescence protein in the poi, selecting tranfectants with FACS is an other good way.
As Fred says, it's possible, but you would indeed need to transfect using more plasmid that doesn't have the selection marker (don't know the ratio, but 5:1 might be a good start), then select resistant clones after which you can sort them with FACS (using a fluorescent reporter or anti-body labelling if you are lucky enough to work with a membrane-bound molecule and fluorescently labelled antibody's are available), or study multiple clonal population with real-time PCR or western blotting for your gene/protein of interest.