Best cell line for expression cloning but not COS-7 c
David de Graaf
degraaf at genome.wi.mit.edu
Tue Jan 13 16:43:04 EST 1998
In article <68u1vj$e17 at panix3.panix.com>, iayork at panix.com (Ian A. York) wrote:
> In article <34B27569.5FD3 at chugaibio.com>,
> Dom Spinella <dspinella at chugaibio.com, @chugaibio.com> wrote:
> >if anyone has tried expression cloning from a system in which there is
> >chromosomal integration. There might even be an advantage to it -- you
> I see two potential problems with this: first, getting enough cells
> transfected, and second, getting the plasmid back.
If you really want to do this by transfection, I could recommend 293T
cells, since they also allow for episomal vectors. If you want a non-human
cell line, you might want to give LLC-PK1 procine kidney cells a try. You
can routinely electroporate 50% of these cells. Look up a Laurent Journot
paper in Nature on the expression cloning of PACAP receptors.
My personal bias: don't use transfection. You will have multiple
integrants in every cell, forcing you to recover your DNA and retransfect
and select multiple times. Consider doing this the right way using a
retroviral expression library. It just means you have to clone the library
into a different plasmid. A number of mouse cell lines now become
excellent targets. For example, mouse 3T3s are 100% infectable. Check Gary
Nolan's web site at Stanford for more details.
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