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).
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:
|12260||psPAX2||2nd generation packaging plasmid for producing viral particles. psPAX2 contains a robust CAG promoter for efficient expression of packaging proteins. Trono lab and Aebischer lab lentiviral vectors require psPAX2. Produces higher titer than pCMV-dR8.2 dvpr.|
|12259||pMD2.G||Envelope plasmid for producing viral particles.|
2nd generation system deposited by the Weinberg lab:
|8455||pCMV-dR8.2 dvpr||2nd generation packaging plasmid for producing viral particles|
|8454||pCMV-VSVG||Envelope plasmid for producing viral particles|
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 / pSicoR, pCL, pCS, and pLove.
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.
|12251||pMDLg/pRRE||3rd generation packaging plasmid for producing viral particles|
|12253||pRSV-Rev||3rd generation packaging plasmid for producing viral particles|
|12259||pMD2.G||Envelope plasmid for producing viral particles|
The 293T cell line for producing lentiviral particles can be obtained from GenHunter.