FIG. 3.

Stability of VSV-G, gag-pol RNA, and authentic pseudotyped virus particles. (A) Stability in incubation at 37°C. VSV-G-containing conditioned medium from 293 cells transfected with pCMV-G and gag-pol RNA (GP/LLRNL)-containing conditioned medium from 293GP/LLRNL cells (0.5 ml of each) were independently incubated at 37°C for the indicated time. After that, they were mixed with equal volumes of the other fresh components, centrifuged at 14,000 rpm for 1 h, and used to infect BHK cells. Authentic pseudotyped virus was made by the conventional method and incubated at 37°C. The activity of each component was determined by luciferase assay and expressed as the percentage of fresh-sample activity. An identical experiment in which samples were incubated at 4°C instead of 37°C resulted in a similar profile (data not shown). (B) Stability during freeze-thaw. Each sample (0.5 ml) was subjected to freezing at −80°C for 15 min and thawing at 37°C for 5 min, and each sample was kept at 4°C until the last samples were prepared. Then VSV-G or gag-pol RNA particles were mixed with equal volumes of fresh other components, centrifuged at 14,000 rpm for 1 h, and subjected to infection. Each value represents the percentage of fresh-sample activity. Results are the mean ± standard error of three independent experiments.