pCDNAI and pCDM8 problems in stable transfection ?

Ian A. York york at mbcrr.dfci.harvard.edu
Wed Jul 5 09:45:57 EST 1995


In article <marcvdc.130.2FF9500F at lmb1.rug.ac.be> marcvdc at lmb1.rug.ac.be writes:
>
>It seems that there are problems with the stable transfections of genes 
>cloned in pCDM8.  Although the transient transfections of the gene gave 
>high expression levels, no protein was detected in the stable transfectans.  
>The same was done with an biological inactive form of the protein and this 
>gave the same results. So, it is not because of the toxicity of the gene 
>expressed.

Are you transfecting COS with this plasmid?  If so, the plasmid will 
replicate and kill the cells whether or not theprotein is expressed.  
Linearizing plasmids with the SfiI site in the SV40 promoter may help 
with this, but I suspect that enough plasmid will re-anneal that it will 
be a continual problem.  If you use an SV40 origin plasmid in COS, you 
should probably cut out the SV40 origin altogether.  However, it isn't 
absolutely essential; people in my lab have made stable COS transfectants 
with pCDM8-based plasmids.

If you aren't using COS, it probably should work.  Others in my lab have 
made several stable cell lines using pCDM8 derivatives.  However, stables 
are much less predictable than are transients, and you can't guarentee 
anything.  I've had a lot of difficulty making stable transfectants, 
using pCDM8 and also several other plasmids.

Ian

-- 
Ian York   (york at mbcrr.harvard.edu)
Dana-Farber Cancer Institute, 44 Binney St., Boston MA 02115
Phone (617)-632-3921     Fax  (617)-632-2627




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