Comments: 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 plasmid for this system are also available at Addgene (http://www.addgene.org/rnaitools). Please visit 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 discussion on cloning strategies and protocols.

Addgene has sequenced a portion of this plasmid for verification. Click here for the sequencing result.