COS-7 transfection

Ian A. York iayork at panix.com
Wed Aug 6 15:57:50 EST 1997


In article <Pine.GSO.3.96.970806161225.24827A-100000 at xena.acsu.buffalo.edu>,
Manchao Zhang <mczhang at ACSU.BUFFALO.EDU> wrote:
>
>    We are transfecting COS-7 cells with pCDNA3 expression vectors to get
>stable expression of several cell receptors, but the transfected cells
>grow so slowly in G418 selection medium we can not do some analysis.What's
>the problem? any suggestion? Thank you!  M.Zhang

Are you linearizing the pCDNA3 before transfection?  If not, then the
plasmid will replicate (because it has the SV40 origin of replication) and
kill off the transfected cells fairly quickly.  You may not be getting any
stably transfected cells because of this.

We've been able to make stable COS transfectants using plasmids in that
series (though I don't know if we've ever actually used pcDNA3 itself; 
we've used pcDNA1Neo, and co-transfected Neo resistance with pCDM8 and
pcDNA1Amp).  We always linearize the plasmid before transfection (which
also supposedly improves integration, too).  We find that COS7 are very
resistant to G418 to start with and need to go to 1 mg/ml of G418 to get
selection. 

Good luck.

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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