Lentiviral Packaging Plasmids
Lentiviral plasmids are based off the genomes of lentiviruses (see Figure 1). In most cases, the plasmids have been derived from the HIV-1 genome. HIV lentiviral vectors however have been modified so they are safe to use in research labs. For a list of some of our popular lentiviral transfer vectors click here.
For producing lentiviral particles, you typically need three components (as illustrated in the diagram below):
The transfer plasmid is often the one which requires the most consideration when deciding what to use for your experiment. Often the envelope and packaging plasmids are interchangeable with many types of transfer plasmids.
As you can see in Figure 2, the second generation system has one packaging plasmid which includes all the important packaging components: Gag, Pol, Rev, and Tat. To produce virus, you require a single packaging plasmid, an envelope plasmid, and your transfer vector.
Q: How can you tell if your transfer vector is 2nd generation?
A: In general, lentiviral vectors with a wildtype 5’ LTR need the 2nd generation packaging system because these vectors require TAT for activation.
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:
|psPAX2||Packaging Plasmid||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.|
|pMD2.G||Envelope Plasmid||Envelope plasmid for producing viral particles|
2nd generation system deposited by the Weinberg Lab:
|pCMV-dR8.2||Packaging Plasmid||2nd generation packaging plasmid for producing viral particles, includes Gag, Pol, Rev, and Tat|
|pCMV-VSVG||Envelope Plasmid||Envelope plasmid for producing viral particles|
The main difference in the 3rd generation system is that there are 4 plasmids in total 2 packaging plasmids, an envelope plasmid, and a transfer plasmid (Figure 3) which need to be used to generate virus. 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.
The main differences in the 3rd generation system are as follows:
|pMDLg/pRRE||Packaging Plasmid||3rd generation packaging plasmid for producing viral particles, includes Gag and Pol (NOT Tat)|
|pRSV-Rev||Packaging Plasmid||3rd generation packaging plasmid for producing viral particles; includes ONLY Rev|
|pMD2.G||Envelope Plasmid||2nd or 3rd generation envelope plasmid; encodes for VSVG|
|Feature||2nd Generation||3rd Generation|
|Transfer Plasmid||Can be packaged ONLY by a second generation packaging system that includes TAT||Can be packaged by both 2nd and 3rd generation packaging systems|
|Packaging Plasmid||All on one plasmid: Gag, Pol, Rev, Tat||Two plasmids; one encoding Gag and Pol; and another encoding Rev|
|Envelope Plasmid||Interchangeable: usually encodes for VSV-G||Interchangeable: usually encodes for VSV-G|
|SIN||Large deletion in the 3’LTR makes the virus replication incompetent||Large deletion in the 3’LTR makes the virus replication incompetent|
|Safety||Safe. Uses separate plasmids for encoding various HIV genes||Safer. Eliminates the requirement for Tat. Uses 4 plasmids instead of 3|
|Promoter||Uses the wt 5’LTR||5’LTR is partially deleted and fused to a heterologous enhancer/promoter such as CMV or RSV|