Best cell line for expression cloning but not COS-7 ?
mantei at neuro.biol.ethz.ch
Wed Jan 7 01:17:49 EST 1998
In article <grund-0601981652490001 at 220.127.116.11>, grund at cmu.unige.ch
(Christophe Grundschober) wrote:
> Hi everybody,
> I'm trying to clone a polymorphic gene present in 60% of humans by
> expression cloning (i.e. transfection of pools of a cDNA library in a
> eukaryotic cell line and then screening of the transfected cells for the
> function of the unknown gene).
> I tested COS-7 cells which are easely transfected but unfortunately they
> already express the gene I'm looking for. I'm now looking for other cell
> lines which could be used. They have to be easely transfected (by
> electroporation or other) and grow well. Does anybody have a suggestion?
We recently did expression cloning using a CHO derivative, CHOP2 (our work
is described in Bakker, et al., JBC 272:29942-6). We could transfect up to
50% of the cells using a DEAE-dextran method, which is cheap and easy.
Someone else in the lab had much lower efficiencies with "regular" CHO
cells--I'm not sure whether this represents differences in the cell line
or in the operator. Note that CHOP2 cells have a polyoma, not an SV40
large T antigen gene. You need to either use a vector with a polyoma
origin of replication, or be satisfied not to amplify the transfected DNA
(we did the latter).
We transfected pools of clones, but had saved the pools as filter replicas
of the colonies at -80 C. Expression was detected with immunocytochemical
staining. In the first round, with 5-10,000 clones per pool, only about 10
cells in a well (6-well plate) were positive, but this is enough to see.
In the last round about 10% are positive. The pools could be subdivided by
cutting the filters into pieces (two cycles of subdividing into about 12
subpools). With this method we never had to isolate DNA from the
transfected CHOP2 cells. This made everything very quick and easy, and the
whole screening was done in a few weeks.
Dept. of Neurobiology, Swiss Federal Institute of Technology
CH-8093 Zurich, Switzerland
mantei at neuro.biol.ethz.ch Fax: +41-1-633-1046
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