stable cell line works, but transient transfections wont - why?

Ozan Aygun via methods%40net.bio.net (by metugenetics from yahoo.com)
Wed Jul 25 13:23:42 EST 2007


Hi,

This weard situation might happen sometimes if the
overexpression of your gene is very toxic to the
cells. I had a similar experience with a protein that
is a stichiometric component of a large complex. When
I was trying to transiently express it, I couldn't. 

I was also trying 24 and 48h points after
transfection. However, no signal was there in WB. On
the other hand, when I started checking in 2h
intervals immediately after the transfection (after
changing the media) I realized that my protein is
rapidly degraded within the first 6 hours as I have
seen the cleavage products that are eventually lost
after the 3rd time point. 

For your case, the same protein may be expressed in
the stable cell line established since its expression
is in tolerable levels for the cell (you also mention
that your casette can be regulated). Moreover, we also
experienced that some really wild degredation can
occur in some of the transiently overexpressed
proteins (especially those expressed from the CMV
promoter), whereas in their stable expression such is
not observed as the expression level drops
considerably after the integration.

Hope this may be any help.

Good luck!

Ozan


       
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