Comments: 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 full sequence for this plasmid is approximated and not fully verified. 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.

Addgene has sequenced a portion of this plasmid for verification. Click here for the sequencing result.