Plasmid 11643: pLVCT-tTR-KRAB

Transgenes can be expressed from any RNA Pol II promoter as part of
bicistronic unit comprising the KRAB-based repressor; tetO sequences
are inserted into the vector LTR. Tet-on and Tet-off versions rely on
repressors that bind in the absence or the presence of doxycycline,
respectively. Addition of Pol III promoter-small hairpin RNA cassette
allows for drug-controllable RNA interference (Tet-on shRNA).

Cloning shRNA into pLVET, pLVCT, and pLVPT vectors: first clone
shRNA into pLVTHM downstream of the tetO-H1 region. Then cut pLVTHM
containing your shRNA with MscI-FspI and clone the insert containing
the 3'LTR to target plasmid opened with MscI-FspI. FspI cuts into
AmpR. This means for inverted clones AmpR will not be restored; after
selection you will be left with clones with the shRNA cassette and
functional AmpR.

pLVTHM and packaging plasmids for this system are also available at Addgene http://www.addgene.org/rnaitools Please note that the full sequence for this plasmid is approximated and not fully verified. Please visit the Trono lab website http://tronolab.epfl.ch to see frequently asked
questions on cloning strategies and packaging.
You may also visit LentiWeb http://www.lentiweb.com for discussions on cloning strategies and protocols.

Addgene has sequenced a portion of this plasmid for verification. Full plasmid sequence is available only if provided by the depositing laboratory.

Article: A versatile tool for conditional gene expression and knockdown. Szulc et al (Nat Methods. 2006 Feb . 3(2):109-16. PubMed)

Please acknowledge the principal investigator and cite this article if you use this plasmid in a publication. Also, please include the text "Addgene plasmid 11643" in your Materials and Methods section.