8 Lentivirus and astrocyte cells in culture has been demonstrated, and subsequent transplantation of these cells results in partial behavioural recovery in the 6-OHDA model of Parkinson’s disease (Wolff et al. 1989). Lentiviruses are a subgroup of the retrovirus family. Their genomes are slightly more complicated, contain- ing accessory genes that regulate viral gene expression, control the assembly of infectious particles, modulate viral replication in infected cells and contribute to the persistence of infection (Kay et al. 2001). For example, human immunodefiency virus-1 (HIV-1), a commonly used lentiviral vector, encodes six additional proteins (tat, rev, vif, vpr, nef and vpu; Frankel and Young 1998; Verma and Weitzman 2005). These proteins are involved with budding, maturation and integration steps of the viral life cycle, enhancing the rate of transcription and nuclear export of the viral RNA as well as assisting in the assembly of the virion (Blesch 2004). In addition, lentiviral vectors do not require the breakdown of the nuclear membrane in order to integrate (Lewis and Emerman 1994). The lentivirus- encoded gag matrix protein, integrase enzyme (Gallay et al. 1997) and vpr protein (Heinzinger et al. 1994) interact with the nuclear import machinery of the target Cell Biol Toxicol (2010) 26:1–20 cell and facilitate active transport of the pre-integration complex through the nucleopore. This enables trans- fection of non-dividing cells, and as a result, such ‘complex retroviruses’ have shown promise as gene transfer vectors, especially for post-mitotic cells such as neurons (Blomer et al. 1996; Naldini et al. 1996; Vigna and Naldini 2000). Lentiviral vectors were developed by the removal of all viral genes, except those required (in cis) in order to complete a single round of replication. All other components are supplied in trans from either transient transfection or stable cell lines. HEK293T cells, a derivative of HEK293 cells that contain the SV40 large T-antigen, are often used for lentiviral production. In addition, the viral rev protein is provided in trans, as are gag and pol, achieved by co-transfection of helper cell lines (Pacchia et al. 2001; Fig. 3). Following co-transfection of helper plasmids with the shuttle plasmid, expressing the foreign transgene of choice, the viral vector particles are produced and released into the media by the budding process. The resulting lentiviral vectors are then purified and concentrated by ultracentrifugation. Further advances in lentiviral vector design involving the deletion of further non-essential pathogenic genes reduced the HIV-derived packaging component of second-generation vectors to the gag, pol, tat and rev ψ CMV/LTR RRE cPPT CMV Transgene WPRE LTR Cotransfection into HEK293T cells Supernatant Harvest and Centrifugation Concentration of viral pellet CMV Δψ gag RSV rev pol polyA CMV VSVG polyA Packaging Constructs RRE polyA Fig. 3 Production of lentiviral vectors. Lentiviral vectors are produced by co-transfection of packaging constructs containing viral elements needed for one round of replication, together with the shuttle vector containing the transgene into a packaging cell line, e.g. HEK293T cells. The newly formed viral particles are released into the supernatant by the budding process and can be collected (harvested) and concentrated by centrifugation. Addition of viral components such as the cPPT, RRE and WPRE can enhance the production and subsequent transgene expression