Retroviral Vector Production by Transient Transfection
This protocol was adapted from “Retroviral Vectors,” Chapter 2, in Gene Transfer: Delivery and Expression of DNA and RNA (eds. Friedmann and Rossi). Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, USA, 2007.INTRODUCTION
This protocol describes vector production by transient transfection. The production of retroviral vectors requires a full-length copy of the vector RNA to be incorporated into virions. This is accomplished by coexpressing vector RNA and the viral proteins required for virion formation from expression plasmids. To avoid generation of replication-competent virus, the viral genes are carried by separate plasmids. Generally, the gag and pol genes are on one plasmid, and the viral envelope gene is on a second plasmid. The viral protein-coding regions can be expressed using various promoters to decrease homology and thereby decrease recombination. Because these plasmids do not contain the packaging (ψ) sequence, the viral genes are unlikely to be incorporated into virions.
