Tet Inducible Gene Expression

ege ege at mit.edu
Wed Nov 29 15:00:19 EST 1995


In article <49i4eu$lt0 at usenet.srv.cis.pitt.edu>, iddavis at vms.cis.pitt.edu wrote:

> 
> Are you using lines cloned from a single transfected cell, or bulk
> cultures?  If bulk, it's not very surprising.  What cells are you using?  
> Some cells like to spit out genes more rapidly than others.  What gene are 
> you expressing?  If it's toxic to the cells, even a very low level of
> baseline expression will select in favor of cells that do not express the
> gene.  Are you using the inducible or repressible transactivator?  If it's
> the inducible, remember tetracycline is not particularly stable in aqueous
> media so your loss of expression might be due to insufficient tet in the
> culture medium.


I apologize for being so vague.  I'm refering to clonal lines derived from
Rat 1 fibroblasts.  I am expressing E2F family members so there may be
selection against leaky expressors via apoptosis.  However I'm inclined to
believe these cells are mutant for p53 so transformation due to leaky
expression is more likely than apoptosis.  I am utilizing the original
system developed by Bujard so expression is repressed by tet.  Your point
of tet stability is good.  Perhaps my tet stock is degraded to the point
where I'm no longer blocking expression in the uninduced.  Can you tell me
how long tet is stable in media at 4 degrees?  How about in H20 at -20
degrees.  Thanks

Scott



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