I tried to produce Lentiviruses using the plasmids pRSV-Rev, pCMV-VSVg and pMDLg-pRRE, as well as a plasmid containing TetOn-GFP. After transfection into HEK293T cells a lot of cells were greenfluorescent (without adding the transactivator or Doxycycline). Is it possible, that TetOn-GFP can somehow be expressed without the transactivator/Doxycycline or did I probably receive a wrong plasmid? Thank you in advance.
Most likely culprit is your FBS containing Tet - quite well established problem. You should use Tet-free FBS.
Secondly, TETon promoters are notoriously leaky.
How bright was your green fluorescence? Because thirdly, this may not be GFP fluorescence but autofluorescence from cell death...
Last point: I'd ALWAYS check plasmids received from colleagues/other labs by at least restriction digest, but preferably sequencing. It's well worth doing that to avoid wasting time trying to clone something that's not there... if from Addgene, it should be fine.
Most likely culprit is your FBS containing Tet - quite well established problem. You should use Tet-free FBS.
Secondly, TETon promoters are notoriously leaky.
How bright was your green fluorescence? Because thirdly, this may not be GFP fluorescence but autofluorescence from cell death...
Last point: I'd ALWAYS check plasmids received from colleagues/other labs by at least restriction digest, but preferably sequencing. It's well worth doing that to avoid wasting time trying to clone something that's not there... if from Addgene, it should be fine.
Hi Dominik,
I believe Petra already gave you the right answer, Tet-On promoters are leaky, sometimes less, sometimes more, sometimes unacceptably highly... I guess you end up in the last option...
One solution has been the co-expression of a Tet-controlled transcriptional silencer (tTS or TetR-KRAB), which binds to the same tetO regulatory sequences as the rtTA, but in the absence of Tet/Dox. Only when you provide the rtTA (possibly the advanced versions) and Dox you should activate your regulated gene expression by relieving the tTS repression and promoting the rtTA binding to the promoter... Never tried it personally! But I was reading a bit about it...
Good luck!
Hello,
There is Tet-ON and Tet-OFF. Check which plasmid you have ?
Tet-ON is Green without drug. Adding drug shuts if off....
Small contamination from Serum can turn on the gene.
Good luck
Late comment on this question, depends on if you are using 3rd generation Tet-on or the operon-like version. The operon version is always active without the tetR protein because system uses a full active cmv promoter that is simply steric blocked by the tetR protein (-> tetO sites downstream of the core promoter), also without DOX and present tetR this system is terrible leaky. In contrast the 3rd generation system uses an inactive minimal CMV promoter with several tetO sites upstream the minimal promoter, this one should be very silent without the rtTA-VP16 protein and is only active if the is also a rtTA-VP16 chimera and DOX present in the cell.
In you case it looks like you are using an old system with an operon-type promoter ...
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