Titering replication-defective rSV40 vectors

Abstract

Recombinant simian virus 40 (rSV40)-derived vectors are particularly useful for gene delivery to bone marrow progenitor cells and their differentiated derivatives, certain types of epithelial cells (e.g., hepatocytes), and central nervous system neurons and microglia. They integrate rapidly into cellular DNA to provide long-term gene expression in vitro and in vivo in both resting and dividing cells. Techniques used to produce, purify, and quantitate these vectors are simple, give reproducible results, and may be used to generate vectors that are deleted only for large T antigen (Tag), or for all SV40-coding sequences capable of carrying up to 5 kb of foreign DNA. Viruses are purified by centrifugation using discontinuous sucrose or cesium chloride (CsCl) gradients. Resulting vectors are replication-incompetent and contain no detectable wild-type SV40 revertants. Viruses are titered by quantitative polymerase chain reaction (qPCR), described here. qPCR measures the number of rSV40 genomes in purified viral stocks using primers specific for the rSV40, coupled with SYBR Green detection of PCR products. Sample purity is assessed using qPCR via melt (dissociation) curve analysis. The only specialized equipment necessary is a quantitative real-time PCR machine.