FIG. 5.

Packaging of foreign plasmid DNA by VP1 VLP. (A) Packaging of foreign plasmid DNA was performed with ratios of DNA to VP1 VLP of 1:10 (lanes 1 to 3) and 1:20 (lanes 4 to 6) by osmotic shock (lanes 1 and 4), as well as particle dissociation in dissociation buffer (lanes 2 and 5) and double-distilled H₂O (lanes 3 and 6). After reassociation and DNase I digestion, particles were pelleted through a 40% sucrose cushion and packaged DNA was extracted and analyzed. As a control, unpackaged DNA was digested with DNase I (lane 7) or not digested (lane 8). (B) Quantification of packaged DNA by quantitative competitive PCR. VP1 VLP (3 × 10¹¹) were dissociated and incubated with increasing amounts of plasmid molecules for 1 h at 37°C and then subjected to dialysis overnight against reassociation buffer. Packaged VP1 VLP were digested with DNase I and pelleted through a 40% sucrose cushion. DNA was extracted and quantified by quantitative competitive PCR. (C) Analysis of β-galactosidase expression. The reporter plasmid was packaged in VP1 VLP, and COS-7 cells were exposed for 2 days. Cells were stained with 5-bromo-4-chloro-3-indolyl-β-d-galactopyranoside (X-Gal) and then examined microscopically and photographed.