慢病毒包装教程(lentiviruspackagingandproduction).docVIP

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 Addgenes pLKO.1 Protocol for producing lentiviral particles. 2nd generation system deposited by the Trono lab: ID/FONT td Plasmid Description 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: ID/FONT td Plasmid Description 8455 pCMV-dR8.2 dvpr 2nd generation packaging plasmid for producing viral particles 8454 pCMV-VSVG Envelope