慢病毒包装教程(Lentivirus-Packaging-and-Production)

Lentivirus Packaging and Production

The laboratories of Didier Trono (EPFL) and Robert Weinberg (Whitehead Institute) have deposited plasmids for the production of lentiviral particles. These plasmids can be used with many lentiviral vectors, including The RNAi Consortium shRNA vectors being distributed by Sigma (i.e. MISSION shRNAs) and Open Biosystems (i.e. TRC shRNAs).

Overview

For producing lentiviral particles, you typically need three components: 1) a lentiviral vector, such as pLKO.1 or pLVTHM, containing the shRNA or transgene, 2) a packaging vector, such as psPAX2 or pCMV-dR8.2 dvpr, and 3) an envelope vector, such as pMD2.G or pCMV-VSVG.

For most applications, you can produce viral particles by transient transfection of

293T cells with a 2nd generation packaging system (e.g. packaging plasmid psPAX2 and envelope plasmid pMD2.G).

2nd Generation Packaging System

In general, lentiviral vectors with a wildtype 5' LTR need the 2nd generation packaging system because these vectors require TAT for activation. All lentiviral vectors from the Trono or Aebischer lab require packaging with a 2nd generation system.

Below are two 2nd generation systems. Lentiviral plasmids based on pLKO.1 can be packaged with either system, although the first system has been reported to produce higher titer. See Addgene's pLKO.1 Protocol for producing lentiviral particles.

2nd generation system deposited by the Trono lab:

2nd generation system deposited by the Weinberg lab:

3rd Generation Packaging System

The 3rd generation packaging system offers maximal biosafety but is more cumbersome to use, as it involves the transfection of four different plasmids in the producer cells (two packaging plasmids, an envelope plasmid, and the lentiviral vector).

If you wish to use this system, you need to have a lentiviral vector with a chimeric 5' LTR in which the HIV promoter is replaced with CMV or RSV, thus making it

TAT-independent. Examples of these vectors include pLKO.1, pLL3.7, pLB, pLenti6, pSico, pCL, and pCS. Most Aebischer and Trono Lab lentiviral vectors CANNOT be used with this system. A lentiviral vector carrying a chimeric 5' LTR can be packaged with either the 2nd or 3rd generation packaging system.

More information

∙Click here to browse other RNAi vectors, or search for plasmids using the search bar at the top of the page.

∙Trono Lab website or Lentiweb: information and a discussion forum on cloning, packaging, and other protocols.

∙Moffat J et. al. 2006. A lentiviral RNAi library for human and mouse genes applied to an arrayed viral high-content screen. Cell 124:1283-1298.

(PubMed)

∙Ventura et. al. 2004. Cre-lox-regulated conditional RNA interference from transgenes. PNAS 2004 Jul 13;101(28):10380-5. (PubMed)

∙Naldini L et. al. 1996. In vivo gene delivery and stable transduction of nondividing cells by a lentiviral vector. Science 272:263-267. (PubMed)