VSV delta G rescue

The protocol for VSV-G PP rescue was adapted from Whelan et al., 1995. VSV rescue plasmids pVSV-eGFP-dG (#31842), pMD2.G (#12259), pCAG-VSV-P (#64088), pCAG-VSV-L (#64085), pCAG-VSV-N (#64087), and pCAGGS-T7Opt (#65974) were ordered from Addgene. Briefly, a 70% confluent 10 cm dish of HEK-293T cells was transfected with 10 µg pVSV-eGFP-dG, 2 µg pCAG-VSV-N (nucleocapsid), 2 µg pCAG-VSV-L (polymerase), 2 µg pMD2.G (glycoprotein, VSV-G), 2 µg pCAG-VSV-P (phosphoprotein), and 2 µg pCAGGS-T7Opt (T7 RNA polymerase) using PEI at a ratio of 1:3 (DNA:PEI) in Opti-MEM I (1×) + GlutaMAX. Forty-eight hours post-transfection, the supernatant was transferred onto new plates transfected 24 hr prior with VSV-G. After a further 48 hr, these plates were re-transfected with VSV-G. After 24 hr the resulting PPs were collected, cleared by centrifugation at 2000 × g for 5 min, and stored at −80°C. Subsequent VSV-G PP batches were produced by infecting VSV-G transfected HEK-293T cells with VSV-G PPs at an MOI of 0.1. Titers were determined by preparing 10-fold serial dilutions in Opti-MEM I (1×) + GlutaMAX. Aliquots of each dilution were added to monolayers of 2 × 10⁴ Vero cells in the same medium in a 96-well plate. Three replicates were performed per PP stock. Plates were incubated at 37°C overnight and then scanned using an Amersham Typhoon scanner. Individual infected cells were quantified using ImageQuant TL software. All PP work was performed in a Class II Biosafety Cabinet under BSL-2 conditions at Erasmus Medical Center.