pLVPT-rtTR-KRAB
(Plasmid #11777)

• Depositing Labs: Patrick Aebischer, Didier Trono
• Publication: Szulc et al Nat Methods. 2006 Feb . 3(2):109-16.

Backbone

• Vector backbone: None
• Backbone size w/o insert (bp): 11627
• Vector type: Mammalian Expression, Lentiviral

Growth in Bacteria

• Bacterial Resistance(s): Ampicillin, 100 μg/mL
• Growth Temperature: 37°C
• Growth Strain(s): Stbl3
• Growth instructions: Use Stbl3 or HB101 to reduce chance of recombination. Grow at 37C
• Copy number: High Copy

Gene/Insert

• Gene/Insert name: mPGK, GFP, rtTR-KRAB, Tet-off

Cloning Information

• Cloning method: Restriction Enzyme
• 5′ sequencing primer: See map

Resource Information

• Addgene Notes:
  • Plasmid 11777 sequenced with EGFP-N primer
• Article Citing this Plasmid:
  • 1 Reference

Terms and Licenses

• Academic/Nonprofit Terms:
  • UBMTA
• Industry Terms:
  • Not Available to Industry

Depositor 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.