Lentiviral vectors can mediate the efficient delivery, integration and stable or controlled expression of transgenes or shRNAs in dividing as well as nondividing cells, either in vitro or in several organs in vivo. As such, it opens exciting possibilities for both basic research and the genetic treatment of human diseases.
HOW TO PRODUCE LENTIVECTOR PARTICLES
Lentivector particles are generated by co-transfection of plasmids coding for the virion packaging system (LV packaging), the vector genome carrying your gene of interest (LV vector) and the plasmid coding for an envelope, in a cell used as producer, for instance 293T (human embryonic kidney cell). In the case of HIV-1 based vectors, the structural and enzymatic components ot the virion come from HIV-1, while the envelope is derived from a heterologous virus, most often vesicular stomatitis virus (VSV) due to the high stability and broad tropism of its G protein.
Three generations of HIV-based LV packaging systems have been successively developed for production of lentivectors by transient transfections:
First generation: LV packaging system encompasses all HIV-1 genes except the envelope
Second generation: will fit most of the experiments. LV packaging system is additionally deleted for all viral auxilliary genes, i.e. vpr, vif, vpu and nef (example: pCMV-dR8.91, pCMV-dR8.74, psPAX2)
Second Generation Lentiviral Plasmids
Third generation: LV packaging system comprises only gag, coding for the virion main sturctural proteins and pol, coding for the retrovirus-specific enymes. A cDNA encoding rev, which encodes a post-transcriptional regulator necessary for efficient gag and pol expression, is provided on a separate plasmid. The third generation packaging system offers maximal biosafety but is more cumbersome, involving the transfection of four different plasmids in the producer cells. (example :pMDL g/p RRE + pRSV-Rev + pMD2G + pLV vector).
Third Generation Lentiviral Plasmids