pMDLg/pRRE
(Plasmid #12251)

• Purpose: 3rd generation lentiviral packaging plasmid; Contains Gag and Pol; also requires pRSV-Rev (Addgene#12253) and envelope expressing plasmid (Addgene#12259)
• Depositing Lab: Didier Trono
• Publication: Dull et al J Virol. 1998 Nov . 72(11):8463-71.

Backbone

• Vector backbone: pMD
• Backbone size w/o insert (bp): 8895
• Vector type: Mammalian Expression, Lentiviral ; Packaging

Growth in Bacteria

• Bacterial Resistance(s): Ampicillin, 100 μg/mL
• Growth Temperature: 37°C
• Growth Strain(s): DH5alpha
• Copy number: High Copy

Gene/Insert

• Gene/Insert name: HIV-1 GAG/POL

Cloning Information

• Cloning method: Restriction Enzyme
• 5′ sequencing primer: CMV Fwd

Resource Information

• Addgene Notes:
  • Addgene diagnostic digest
• Articles Citing this Plasmid:
  • 764 References

Terms and Licenses

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

Depositor Comments

Please note that this plasmid runs as a dimer (>17kb). While this may reduce DNA yield, it is not expected to impact function.

This 3rd generation LV packaging plasmid includes gag, coding for the virion main structural proteins; pol, responsible for the retrovirus-specific enzymes; and RRE, a binding site for the Rev protein which facilitates export of the RNA from the nucleus. This plasmid does not include Rev. The third generation packaging system offers maximal biosafety but is more cumbersome, as it involves the transfection of four different plasmids in the producer cells.
Example of a 3rd generation packaging combo: pMDLg/pRRE + pRSV-Rev + pMD2.G.

Please note that most Trono lab lentivectors contain a wt 5'LTR and can be packaged only using the 2nd generation packaging system (as the wt 5'LTR requires TAT for activation).
If you wish to use the 3rd generation packaging system, you need to have a lentivector with a chimeric 5'LTR (e.g. CCL-, RRL-, etc) in which the HIV promoter is replaced with CMV or RSV, thus making it TAT-independent. The lentivectors carrying the chimeric 5'LTR can be packaged into either a 2nd or 3rd generation packaging system.

Please visit the Trono lab http://tronolab.epfl.ch for cloning strategies, protocols, publications, and more. See LentiWeb http://www.lentiweb.com for discussions on cloning strategies and protocols.

Please note that the ClaI site in this plasmid is blocked by Dam methylation. Do not use this enzyme for diagnostic digests.