tet repressor info:

john shanklin shanklin at BNLUX1.BNL.GOV
Mon Aug 8 18:24:22 EST 1994


I received the following reply to our questions about the Gatz
Tet repressor system in Arabidopsis, from Peter Moddis, and
thought it might be generally of interest.

John Shanklin, Brookhaven National Lab, NY.


I read your E-mail and thought you might be interested to read about 
my experience with tet-repressor and Arabidopsis. At the time I was 
working as a post-doc with Lothar Willmitzer in Berlin where 
Christiana Gatz was also working. With Christiana I decided to try 
and set up the tet-repressor system in Arabidopsis. I found the 
plants were really difficult and slow to transform as compared to 
other constructs, however about 10 plants did come through. On a 
northern level only a couple of these showed much expression (about 
one tenth of the level shown by a good tobacco repressor line), and 
these plants turned out to be tetraploid; I dont know if this latter 
is meaningful. Using crude extracts of the plants I could show by gel-
shift that the tet-repressor protein was being produced and could 
bind the target DNA.  This incidently might be a good screen.

 In a transient expression assay I could show that CAT expression 
could be induced by tetracyclin in protoplasts of these plants. 
However when I stably super-transformed with a TX-GUS construct or 
crossed this into the repressor lines, none of the plants was cabable 
of repressing GUS activity. I think, but cannot prove, that the Tet 
repressor is toxic to arabidopsis, since all the regenerants and F2 
offspring were pretty weedy. This way one would select for low 
expressors. What the answer is I am not sure. Maybe target the 
repressor to the nucleus direct, or maybe in some way tinker with the 
protein, but if the supposed toxicity is directly connected with the 
DNA binding function, either of these options is no use. If you wish, 
I can send you seeds of the plants I made so you have a "negative 
control".

 Anyhow, I wish you good luck with your experiments. One last point, 
Christiana may have an antibody to the repressor protein. 
*******************************************************************

Peter Morris
ISV, CNRS 
Bat. 23 Avenue de la Terrasse,
91198
Gif sur Yvette
France







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