Stable transfection of COS cells?

Ian A. York iayork at panix.com
Fri Jun 16 07:44:24 EST 2000


In article <394A270B.E3303640 at icrf.icnet.uk>,
Ian Mc <i.mcfarlane at icrf.icnet.uk> wrote:
>I have been told by several people that it is not possible to make stable
>transfections in COS cells. I cannot understand this. Can anyone tell me
>whether it is possible to stably express proteins using Invitrogen expression
>vectors such as pcDNA3 or pCEP4?

If not, we've been living a lie for the past ten years.

COS cells can certainly be stably transfected.  However, there are some
points to keep in mind.  Because of the TAg in COS, they'll replicate
plasmids containing the SV40 origin of replication, such as pcDNA3.  (I
don't know about pCEP4.)  For plasmids that don't have an SV40 ori, you
don't need to do anything unusual to get stables, but when there's an ori
present, the plasmid will be amplified a hundred-thousand-fold, and kill
the cell, usually within a few days.  What you need to do, then, is
linearize the plasmid (always a good idea for stables anyway, because it
enhances integration) before you transfect.

Even if you don't linearize, SV40-ori plasmids can still yield stable
transfectants.  I'd recommend not using these for anything; presumably,
they have been selected for (as well as the drug resistance)
low-efficiency replication of SV40 plasmids, or they're odd in some other
way.

Linearizing is pretty simple, and I find decent efficiency that
way.  We've made quite a few stables in COS with no real
problem.  Occasionally I find that they're unstable--I find a positive
clone, but it continuously loses expression of the protein, even under
selection.  When this has happened to me, I've always lost the clone, but
one of our neighbouring labs ran into that and managed to pull through by
repeated subcloning and re-selection, eventually ending up with a stable
stable.

Hope this helps.

Ian 
-- 
    Ian York   (iayork at panix.com)  <http://www.panix.com/~iayork/>
    "-but as he was a York, I am rather inclined to suppose him a
     very respectable Man." -Jane Austen, The History of England






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