Purpose3rd generation lentiviral packaging plasmid; Contains Gag and Pol; also requires pRSV-Rev (Addgene#12253) and envelope expressing plasmid (Addgene#12259)
|Item||Catalog #||Description||Quantity||Price (USD)|
|Plasmid||12251||Plasmid sent as bacteria in agar stab||1||$65|
This material is available to academics and nonprofits only.
- Backbone size w/o insert (bp) 8895
Vector typeMammalian Expression, Lentiviral ; Packaging
Growth in Bacteria
Copy numberHigh Copy
Gene/Insert nameHIV-1 GAG/POL
- Cloning method Restriction Enzyme
- 5′ sequencing primer CMV Fwd (Common Sequencing Primers)
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 note that the ClaI site in this plasmid is blocked by Dam methylation. Do not use this enzyme for diagnostic digests.
These plasmids were created by your colleagues. Please acknowledge the Principal Investigator, cite the article in which the plasmids were described, and include Addgene in the Materials and Methods of your future publications.
For your Materials & Methods section:pMDLg/pRRE was a gift from Didier Trono (Addgene plasmid # 12251)
For your References section:A third-generation lentivirus vector with a conditional packaging system. Dull T, Zufferey R, Kelly M, Mandel RJ, Nguyen M, Trono D, Naldini L. J Virol. 1998 Nov . 72(11):8463-71. PubMed 9765382