stable transfections with Cos-7 cells
Ian A. York
iayork at panix.com
Fri May 4 07:22:51 EST 2001
In article <fs45ftgahogvdrs1or40iieq1767fp6lpu at 4ax.com>,
Tom Vink <eikel at xs4all.nl> wrote:
>What vector do you use ? If you use a vector with a SV40 origin you
>will never get stable transfectants in Cos cells unless you inactivate
>the ori by cutting with a RE. Anyhow for stables better use CHO or BHK
>in my opinion.
You can certainly get stables with COS--we have a half-dozen of them.
We've used vectors with SV40 origins, though we've linearized them first.
We haven't always cut in the middle of the SV40 ori, though I agree that's
the best choice; but a linearized plasmid shouldn't replicate. (OF
course, there are always un-linearized plasmids in the mix, and they'll
amplify and kill the cell they're in; you need to minimize that.
> On 3 May 2001 23:10:17 -0000, Nadia Ehtesham
><Nadia.Ehtesham at cshs.org> wrote:
>>I am doing transfections using Cos-7 cells and Superfect reagent (Qiagen).
I find Fugene6 (Boerhinger; no affiliation) slightly better than
Superfect, for efficiency and for viability.
>>My transfection rate was about 50% (48 hours post-transfection). At that
>>time I passed my cells into selective media (600 microgram/ml G418) but none
I select my stable COS with 1000 ug/ml, but clones differ.
>>I don't have much experience with using cell scrapers but I figured that
>>would be less damaging than trypsin, could there be a problem due to that?
Scrapers are definitely *more* damaging than trypsin; do a trypan blue
stain after each procedure and you'll see the dead cells with the scraper.
>>Also, is there an easy way of trypsinizing cells in 60 or 100mm dishes. In
Wait a little longer after adding the trypsin, say 2-3 minutes, add
medium, and then wash vigorously with the stream from your pipette. If
they don't all come off, you haven't waited long enough. With a little
practice you can see them under the scope rounding up and getting just
ready to come off.
Double-check your plasmids to be sure you've eliminated the circular form;
SVori-free plasmids are the best choice, though as I say above the ones
with the ori can work. (Also, of course, double-check to be sure you've
got the right selectable marker.) Run a titration of your selectable
marker; use it at the lowest concentration that gives 100% killing of
untransfected cells. Wait two or three days after transfection, then pass
your cells into selection. At this time, run a dilution series of the
cells, in 96-well plates, in blocks of 48 wells each, starting from say
10^5 cells per well down to about 10 cells per well; the aim is to get a
block in which 1 in three of the wells have live cells, which means
they're almost certainly single clones. Take the remaining cells after
this and put them all in selection in a flask.
Wait. Usually it takes a couple of weeks before you can be very sure about
your individual clones; it takes a week or more to be comfortable about
your pooled flask.
After you get your clones, remember that COS are very unstable cells,
chromosome-wise; they'll spit out your gene of interest given half a
chance (this is common though not inevitable) so keep them in selection
most of hte time--generally alternate passages.
Ian York (iayork at panix.com) <http://www.panix.com/~iayork/>
"-but as he was a York, I am rather inclined to suppose him a
very respectable Man." -Jane Austen, The History of England
More information about the Cellbiol