Lentiviral Vector Production and Cell Transduction Review
2011
Lentiviral vectors derived from the human immunodeficiency virus (HIV-1) have become major tools for gene delivery in mammalian cells. The advantageous feature of lentivirus vectors is the ability to mediate potent transduction and stable expression into dividing and non-dividing cells both in vitro and in vivo.
Lentiviral vectors are typically produced in HEK 293T cells. Essential lentiviral (HIV-1) genes must be expressed in these cells to allow the generation of lentiviral particles.
These genes are usually expressed by several plasmids:
- a lentiviral cloning plasmid, such as pLV-Green, containing the psi (ψ) packaging sequence and the transgene gene inserted between the lentiviral LTRs for target cell integration.
- a packaging plasmid, such as pLV-HELP, encoding the pol, gag, rev and tat viral genes and containing the rev-response element (RRE).
- a pseudotyping plasmid, such as pLV-iVSV-G, encoding the G protein of the Vesicular Stomatitis Virus (VSV-G) envelope gene. Unlike the HIV envelope, the VSV-G envelope has a broad cell host range extending the cell types that can be transduced by VSV-G-expressing lentiviruses.
Two days after transfection of HEK 293T cells, the cell supernatant contains recombinant lentiviral vectors, which can be used to transduce the target cells.
Once in the target cells, the viral RNA is reverse-transcribed, imported into the nucleus and stably integrated into the host genome. One or two days after the integration of the viral RNA, the expression of the recombinant protein can be detected.
