Tet-Off system, anyone use this? Q. about No. clones to pick

G. Dellaire, Ph.D. G.Dellaire at hgu.mrc.ac.uk
Thu Aug 19 04:01:04 EST 1999


Hello All,

I have been trying to make Tet Responsive cell lines using the Tet-Off
plasmid system.  I have picked about 30 clones and of the 10 clones I
have assayed already none of them have induction levels higher than
basal level expression (measured using the pTRE-luciferase reporter).
This is pretty bad as I tried out the pTRE-luciferase with a cell line
with the tTA activator (grandfather to Tet-Off system) and I get 1000
fold induction.  It would seem that I should get some intermediate
levels of expression with the clones I picked but the 10 I picked are
all basal level!  I think maybe the plasmid we have may have a mutation
or something.

SO my question is... how many clones does one need to pick usually to
get one with high levels of induction (say 100 to 1000 fold)?  Second,
do you generally see intermediate levels of induction with different
clones rather than just basal expression as I am seeing?

Any and all suggestions, comments are welcome.

Graham Dellaire
--
********************
Graham Dellaire, Ph.D.

MRC Human Genetics Unit
Western General Hospital
Crewe Road
Edinburgh, UK
EH4 2XU

Fax: +44 (0)131 343 2620
Phone: +44 (0)131 332 2471
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