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Lentiviral Packaging Plasmids

Overview

2nd Generation Packaging Systems

3rd Generation Packaging Systems

2nd vs 3rd Generation Packaging Systems

Overview

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):

1. A lentiviral vector or “transfer vector” containing the shRNA or transgene and the flanking LTRs
2. A packaging vector or set of packaging plasmids
3. An envelope vector

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.

Figure 1. Lentiviral Plasmids: An Overview. At the top of this diagram is an illustrated version of a lentiviral genome, which includes the LTRs and all the important viral components. Below are the lentiviral plasmids necessary to generate lentivirus in the lab- the transfer plasmid, the packaging plasmid(s), and the envelope plasmid- all of which contain some part of the lentiviral genome.

2nd Generation Packaging Systems

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:

2nd generation system deposited by the Weinberg Lab:

Figure 2. 2nd Generation Lentiviral Plasmids. Depicted in this diagram are the components required to generate virus in the second generation system: a transfer plasmid, a single packaging plasmid, and an envelope plasmid.

3rd Generation Packaging Systems

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:

• The Tat protein has been eliminated from the packaging completely
• The Rev protein is expressed on an independent plasmid
• The 5’LTR of the transfer plasmid has been modified to include a conventional promoter and delete a portion of the LTR

Figure 3. 3rd Generation Lentiviral Plasmids. Depicted in this diagram are the components required to generate virus in the third generation system: a transfer plasmid, two packaging plasmids: one encoding Gag and Pol and a second encoding Rev, and an envelope plasmid.

2nd vs 3rd Generation Packaging Systems