Addgene - pLVPT-rtTR-KRAB Plasmid Data

Gene/insert name:
mPGK, GFP, rtTR-KRAB, Tet-off
Vector backbone:
None
(Search Vector Database)
Vector type:
Mammalian Expression, Lentiviral
Backbone size w/o insert:
11627
5' sequencing primer:
See map List of Sequencing Primers
Bacterial resistance(s)
Ampicillin
Growth strain(s)
Stbl3
Growth temperature (℃):
37
Growth instructions:
Use Stbl3 or HB101 to reduce chance of recombination. Grow at 37C
High or low copy:
High Copy
Sequence: View sequences (3)
Map:
View map
Supplemental document:
Notes from Addgene (1)
Principal Investigator:
Patrick Aebischer
Principal Investigator:
Didier Trono
Terms and Licenses:
MTA
Tet Systems Limited Use Label License

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.

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 11777" in your Materials and Methods section.