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Genetic Insert: HIV-1 GAG/POL
Vector Backbone Description: Backbone Size:8895; Vector Backbone:pMD; Vector Types:Mammalian Expression, Lentiviral, Packaging; Bacterial Resistance:Ampicillin
Defining Citation: PMID:9765382
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.
Please note that the ClaI site in this plasmid is blocked by Dam methylation. Do not use this enzyme for diagnostic digests.
Proper citation: RRID:Addgene_12251 Copy
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