Retroviral Vector Production

Abstract

In this unit, the basic protocol generates stable cell lines that produce retroviral vectors that carry selectable markers. Also included are an alternate protocol that applies when the retroviral vector does not carry a selectable marker, and another alternate protocol for rapidly generating retroviral vector preparations by transient transfection. A support protocol describes construction of the retroviral vectors. The methods for generating virus from retroviral vector plasmids rely on the use of packaging cells that synthesize all of the retroviral proteins but do not produce replication-competent virus. Additional protocols detail plasmid transfection, virus titration, assay for replication-competent virus, and histochemical staining to detect transfer of a vector encoding alkaline phosphatase.

Biosafety in Handling Gene Transfer Vectors

“…Their use is attractive because these retroviral vectors are easy to design and easy to produce in large quantities. Vector DNA more or less randomly integrates into the host genome, and thus can be expressed for long periods or indefinitely (UNIT 12.5;Miller, 2014). Early-generation murine retroviral vectors containing dual long terminal repeats (LTRs) with transcriptional enhancer activity have a propensity to integrate at sites proximal to actively transcribed genes and promoters (Bushman et al, 2005).…”