COS-7 transfection

Dom Spinella dspinella at chugaibio.com
Thu Aug 7 09:41:27 EST 1997


Manchao Zhang writes:

> 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

Its not clear to me why you are using COS-7 for stable integrants.  COS
is a good choice for transient expression due to the presence of the
SV40 T antigen and its ability to permit episomal replication of
plasmids (such as pCDNA3) that have an SV40 ORI.  However, I don't think
its the best choice for producing stable transfectants.  My lab
routinely performs transfection with similar constructs to yours for
both stable and transient expression.  In our hands, cells like CHO or
293 give better results than COS for stable integration -- they are less
resistant to G418 rendering them easier to select for stable integrants
because you don't have to use such a high dose which, as you observed,
causes even the resistant cells to grow very slowly.  Moreover, the
selected clones from CHO or 293 produce higher levels of expressed
protein than homologues in COS.   

In the COS system, you won't know if you have a stable integrant or are
looking at expression derived from episomes unless you do a thorough
analysis of a Hirt supernatant.  Transient expression in COS can last
for up to 8 weeks or more (and will increase under G418 selection) until
the cells finally die off -- perhaps much to your chagrin because you
thought you were looking at a stable integrant. One way to avoid this is
to linearize the plasmid as recommended by a previous respondent -- but
you must make sure it is completely cut. Finally, if you are dead set on
using COS, it may help to increase your transfection effciency.  We have
looked at a lot of methods and have found Lipofectamine transfections to
be the best for COS.



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