Is there a quicker way of making stable cell line? - (May/25/2006 )
Transient transfect is too variable while stable transfection and selecting for clones are too slow.
Is there a proper scientific methods to do sth in between. For example, use linearized plasmid DNA in 'transient transfection', and then use selection media to select for a polyclonal stable population. Is it a method in literatures, or is it just my day-dreaming?
Or any other way to do a quicker stable transfection, if I don't want to wait ages for singal colony to come up and risking the fact that the months effort may not work at all?
do a transfection with puro marker. Will do the selection within a week
Puro marker? What's that?
Co-transfect EGFP, either with an IRES based vector or mix in a pEGFP vector at a low ratio and use cell sorting to sort for GFP positive cells.
What's IRES based vector? Is IRES the name of a promotor, like CMV?
And is it OK to grow a pooled GFP positive cells as experiment material? Or I still need to put single cell onto single well and wait ages for one cell to form a colony, to get a monoclonal population?
IRES: Internal Ribosome Entry Site. Small viral sequence, making sure your ribosomes can translate a gene that's not near the capped site of your RNA. Vector would look like this: promoter followed by your gene of interest followed by IRES followed by EGFP.
I would still go for the colony. Not sure what you will do with the cell line, but you never know the side effects of different locations of insertions of your genes in the chromosome.
Apart from this: puro stands for puromycin, an antibiotic that enables quicker selection that G418 and others.
Thanks guys, you've all been very helpful. I'm doing functional study of a protein in a signaling pathway. Therefore I overexpress the protein by transfection, and see what happens. I'll go for single conoly, if this is better than a pool of transfected cells.
well puro marker is a short way to mean that the vector used in transfection holds a promoter/ORF couple. This is a derivative of the pac gene which encodes a puromycin N-acetyl-tranferase (PAC), that allow the expression of which confers to eukaryotic cells the resistance to puromycin.
It's the quickest selection marker to establish cell lines.