Lentiviral Protocols & Resources
Lentiviral Webinar
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Lentiviruses 101: Plasmids and Viral Production Addgene has put together a webinar focused on understanding the components of lentiviruses and how they are produced in the lab. The webinar covers: 1- Plasmids required to generate lentivirus (both 2nd and 3rd generation systems) 2- Safety Concerns 3- Lentiviral-based applications This webinar is a great place to start or build your knowledge on this widely used technique. |
Protocols
The process of producing infectious transgenic lentivirus is outlined in the simple schematic below. Three plasmids (four for a third generation lentiviral system [see packaging systems]) are transfected into A293T cells: one transfer vector, one or two packaging vector(s), and one envelope vector. After media change and a brief incubation period, supernatant containing the virus is removed and stored or centrifuged to concentrate virus. Crude or concentrated virus can then be used to transduce the cells of interest. For determination of viral titer and full details see the protocol available at the Trono lab webpage.
Also see Addgene’s pLKO.1 protocol
Definitions table
| Feature | Description |
|---|---|
| Gag | Precursor structural protein of the lentiviral particle containing the following proteins Matrix (MA): attaches the lipid envelope to the core of the virus Capsid (CA): surrounds the nucleocapsid and RNA genome in the viral core Nucleocapsid (NC): directly binds to the RNA genome in the viral core |
| Pro | Precursor protein containing protease (Pr) which cleaves the gag-pro-pol polyprotein into its individual constituents |
| Pol | Precursor protein containing the following proteins reverse transcriptase (RT): reverse transcribes viral genomic RNA into proviral DNA, also has RNase activity Integrase (IN): Catalyzes the integration of the provirus into the genome of the host cell |
| Env | Envelope protein composed of the surface (SU) and transmembrane (TM); glycoproteins found within the phospholipids bilayer surrounding the viral core |
| Vif, Vpr, Vpu, Nef | HIV-1 virulence genes, deleted in 2nd and 3rd generation lentiviral vectors |
| VSVG | Vesicular somatitis virus G glycoprotein; envelope protein used in the production of most lentiviral vectors. Used for the broad tropism it confers |
| Rev | HIV-1 protein that binds to the rev response element (RRE) within unspliced and partially spliced transcripts from the HIV-1 genome in order to facilitate their nuclear export |
| RRE | Rev response element; sequence to which the Rev protein binds |
| Tat | Trans-activator, a HIV-1 protein that binds to the target sequence for viral transactivation (TAR) in the R region of the HIV-1 provirus and activates transcription from the HIV-1 provirus |
| TAR | Target sequence for viral transactivation; sequence to which Tat binds |
| Psi (Ψ) | Sequence in retroviral genomic RNA to which nucleocapsid binds in order to package it |
| cPPT | Central polypurine tract; region in the center of lentiviral genomes from which reverse transcription occurs. A 3 strand overlap occur here in HIV-1 reverse transcription and the structure it forms is recognized by the nuclear import machinery (acts as a cis-active determinate of nuclear import or HIV-1) |
| LTR | Long terminal repeats; U3-R-U5 regions found on either side of a retroviral provirus |
| U3 | Unique 3’; region at the 3’ end of viral genomic RNA (but found at both the 3’ and 5’ ends of the provirus). Contains sequences necessary for activation of viral genomic RNA transcription |
| R | Repeat region found within both the 5’and 3’ LTRs of retroviral vectors. Tat binds to this region. |
| U5 | Unique 5’; region at the 5’ end of the viral genomic RNA (but found at both the 3’ and 5’ ends of the provirus) |
| PBS | Primer binding site |
| CMV | Cytomegalovirus promoter; promoter used to drive the transient expression of transgenes in a variety of cell types |
| EF1α | Promoter for elongation factor 1α, used to express a reporter gene such asGFP |
| TetO | Binding site for the tet repressor protein or a variant thereof which can be used to turn on or off shRNA expression |
| H1 | Histone H1 promoter used to drive shRNA expression |
| LoxP | Site which is recombined by the Cre recombinase in order to conditionally remove the lentiviral provirus from the host genome after successful infection and incorporation |
| PolyA | Poly adenylation signal; sequence signaling for the addition of a poly A tail to a nascent mRNA |
| hPGK | Human phosphoglycerate kinase promoter; promoter for the ubiquitous expression of a transgene |
| WPRE | Woodchuck hepatitis virus posttranscriptional regulatory element; sequence that stimulates the expression of transgenes via increased nuclear export |
| SIN | Self-inactivating retroviral vector |
Additional Resources
Guides to our most popular lentiviral plasmids
pLVTHM: 2nd generation transfer vector
Publications
Coffin J.M., Hughes S.H., and Varmus H.E., Retroviruses, (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, 1997). Avaliable online at: http://www.ncbi.nlm.nih.gov/bookshelf/br.fcgi?book=rv&part=A288 and http://www.ncbi.nlm.nih.gov/bookshelf/br.fcgi?book=rv&part=A7763#A7782
Cronin J, Zhang XY, Reiser J. 2005. Altering the tropism of lentiviral vectors through pseudotyping. Curr Gene Ther. 5(4): 387-398. (Pubmed)
Dull T, Zufferey R, Kelly M, Mandel RJ, Nguyen M, Trono D, and Naldini L. 1998. A Third Generation Lentivirus Vector with a Conditional Packaging System. J Virol. 72(11):8463-8471. (Pubmed)
Naldini L, Blömer U, Gallay P, Ory D, Mulligan R, Gage FH, Verma IM, and Trono D. 1996. In vivo gene delivery and stable transduction of nondividing cells by a lentiviral vector. Science. 272(5259): 263-267. (Pubmed)
Zennou V, Petit C, Guetard D, Nerhbass U, Montagnier L, Charneau P. 2000. HIV-1 Genome Nuclear Import Is Mediated by a Central DNA Flap. Cell. 101(2): 173-185. (Article)
Zufferey R, Dull T, Mandel RJ, Bukovsky A, Quiroz D, Naldini L, and Trono D. 1998. Self-Inactivating Lentivirus for Safe and Efficient In Vivo Gene Delivery. J Virol. 72(12): 9873-9880. (Article)
Zufferey R, Donello JE, Trono D, and Hope TJ. 1999. Woodchuck Hepatitis Virus Posttranscriptional Regulatory Element Enhances Expression of Transgenes Delivered by Retroviral Vectors. (Pubmed)
Web Resources
Tronolab Lentiviral Production Protocol: http://tcf.epfl.ch/Jahia/site/tcf/op/edit/lang/en/pid/85318 Tronolab Maps Page: http://tronolab.epfl.ch/page58115.html