Stably transfecting two cDNAs into the same cell !!!
mckenzie at naxos.unice.fr
Thu Apr 20 05:27:54 EST 1995
In article <3n4cdj$f6j at netnews.upenn.edu> damania,
damania_b at a1.mscf.upenn.edu writes:
>Has anybody out there tried to stably transfect two cDNAs into
>the same cell and had it work? I guess I would need to have
>two different selectable markers on my plasmid.
>Also, will both proteins be expressed to a high level?
>I am very interested in doing this myself and appreciate any
>suggestions you can give me.
>Thanks a bunch,
Good news for you - yes it does work, we routinely transfect
two three or even four cDNAs into Hamster fibroblasts. You do not need
different selectable markers although this will certainly increase the
percentage of clones which express both proteins.
To give you an idea.... I recently transfected a protein tyrosine
phosphatase, PTP1C and a G-protein linked somatostatin receptor into the
same population. The selectable marker was on the PTP1C plasmid. After
picking 20 G418 resistant clones, I found more than 50% expressing
of PTP1C and 10% co-expressing functional levels of the receptor.
You can increase the percentage of cells expressing both proteins
by modifying the relative proportion of each plasmid. For example,
1 microgram of marker plasmid (PTP1C) and 10 micrograms of non-selected
plasmid (Somato receptor) will give fewer clones, but 50 % of these clones
will express both proteins. Obviously you have to keep the total DNA
concentration at the optimal level for your particular cells
It is a good idea to use expression vectors with different
to ensure that there is no competition for available factors, but I think
that this will depend on the cells you are using.
Finally, there is no substitute for performing several
and picking lots of clones for testing.
Fergus R. McKenzie McKenzie at naxos.unice.fr
Centre de Biochimie
Universite de Nice, France.
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