cos stable transfection

Ian A. York iayork at panix.com
Thu Oct 10 11:57:09 EST 1996


In article <96284.100640HXC117 at psuvm.psu.edu>,
Hugo  <HXC117 at psuvm.psu.edu> wrote:
>Dose anyone have experience on stable transfection on Cos cells?  What should b
>e careful in doing this?  Is it necessary to keep the expression of foreign gen
>e low?  If yes, it would be difficult to detect its expression.  Then, it would
> be very difficult to do further studies on the protein.  Any suggestions?

Our lab has done this with no major problems.  We used plasmids which
included SV40 ori, which meant that we had to linearize the plasmids (at
least; better to cut out the ori region altogether) so that the plasmids
wouldn't replicate and kill the cell.  However, the transformation
efficiency was reasonably high, and at the end of the cloning and so forth
protein expression from the transgene (driven under CMV promoters) was and
still is good - very easily detectable.

I don't think there are any real tricks to it, other than remembering that
the plasmid must not be able to replicate under the large T control.  We
used electroporation to transfect the cells and selected with G418.

Incidentally, you should set your newsreader/mailreader/whatever you
posted with to limit your line length to less than 75 characters,
otherwise the formatting becomes ugly and your posts are difficult to read
on the majority of newsreaders.

Posted and mailed.

 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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