Transient Production of Retroviral- and Lentiviral-Based Vectors for the Transduction of Mammalian Cells

The genera of the retroviridae comprise oncoretroviruses and lentiviruses ( 1 ). Oncoretroviruses can only infect dividing cells, as they require the breakdown of the nuclear membrane to access the cellular chromosomal deoxyribonucleic acid (DNA; ref. 2 ). Conversely, lentiviruses can also infect nondividing cells ( 3 ). Retroviral and lentiviral vectors are among the most powerful techniques for gene delivery into mammalian cells ( 1 ). For the sake of simplicity, we refer to retroviral and lentiviral vectors as retroviridae-based vectors. This chapter describes a method for the transient production of high titer, helper-free retroviridae-based vectors ( 4 – 6 ). The transient system for the production of retroviridae-based vectors is fast, reliable, and safe ( 1 , 4 , 5 ). In fact, the transient nature of this system greatly minimizes the possible formation of replication-competent viruses, which may occur via homologous recombination ( 1 , 4 , 5 ). The engineering of recombinant retroviridae vectors based on Moloney murine leukemia virus and HIV-1 is shown in Figs. 1 and 2 , respectively ( 7 ). Retroviridae vector stocks can be easily generated in 48 h after the transfection of packaging cells. Three plasmids are simultaneously co-transfected into the packaging cells: one plasmid encodes for the viral envelope, a second plasmid encodes for the components of the viral core (gag-pol), and a third plasmid contains the transgene of interest, which could be either a reporter gene, a therapeutic factor, or a combination of both. The function of the third plasmid consists in providing the chimerical viral genome for the packaging into the recombinant retroviridae-based vector.

---

---