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

Zhonglin Chai Zhonglin.Chai at med.monash.edu.au
Thu Aug 19 19:05:29 EST 1999


Hello Graham,
In ClonTech's Tet-off/on system manual, they suggest to test at least 30
clones, and they have screened as many as 100 clonal cell lines to obtain
one line that exhibits suitably high induction and low background. Hope
this of help.

Zhonglin Chai

>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
>********************


ZhongLin Chai, PhD
__________________________________________________________
Department of Pathology and Immunology
Monash University Medical School
Alfred Hospital
Commercial Rd, Prahran, VIC 3181, AUSTRALIA
Telephone: (61 3) 9903 0698 (lab)
           (61 3) 9903 0696 (office)
Mobile:	   0413 58 1940 or International: +61 413 58 1940
Fax:       (61 3) 9903 0731
email:     zhonglin.chai at med.monash.edu.au
___________________________________________________________





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