Double stable transfection
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Sun Aug 13 17:56:20 EST 2006
In article <mailman.507.1155040575.20007.methods at net.bio.net>,
<arnaud.garcon at syngenta.com> wrote:
> I've developped several cell lines with two inserts and all that DK said
> makes sense.
> Invitrogen pBud vector is OK but I thought that their sectable marker
> rZeocin sucks. I know someone from this company reads this list and is
> of an obvious different opinion but there you go.
> I ended up using two linearised plasmids with two orthogonal selection
> markers(ie with different MoA). Then you need to optimised your
> conditions for each in a kind of checker board matrix then your
> selection routine. Hard work at first but works well eventually.
I maybe missing something here, but why does everyone suggest
reoptimising selection conditions for dual transfections - I don't see
the logic? I have done may dual selections using various combinations of
blasticidin, G418, zeocin and hygromycin resitance as amrkers and
although I have had to screen quite a few colonies to get what I want, I
haven't had to use a matrix of selection conditions. Selection was just
based on the original kill curves I made for the different antibiotics
for each cell line transfected. As I said perhaps I'm missing something,
but why should the kill curves shift when more than one antibiotic is
being used if each one has a different MOI?
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