FIG. 2.

Transduction of human hematopoietic cell lines. (A) Transduction of MO7e, K562, and HEL cells with Ad5.AAV-b and chimeric Ad5/35.AAV-b. Ad5.AAV-b and Ad5/35.AAV-b contain the same GFP expression cassette (Fig. 1A) but differ in their fiber molecules (Ad5 versus Ad35). Two hundred thousand cells were infected with different MOIs (genomes per cell) of viruses in 200 μl of media for 6 h at 37°C. Virus-containing media were then removed, and the cells were resuspended in fresh media followed by incubation for 18 h at 37°C. Then, the percentage of GFP-expressing cells was determined by flow cytometry. N ≥ 3 (N, number of experiments). (B) Dose-dependent GFP expression after infection with ΔAd5/35.AAV or Ad5/35.AAV-b. Two hundred thousand MO7e, K562, or HEL cells were infected with ΔAd5/35.AAV or Ad5/35.AAV-b at different MOIs (genomes per cell) in 200 μl of growth medium as described above. The percentage of GFP-positive cells was determined 24 h postinfection by flow cytometry. N = 4. (C) Stability of vector DNA in transduced MO7e cells. Two hundred thousand MO7e cells were infected with an MOI of 2,000 genomes per cell of ΔAd5/35.AAV or Ad5/35.AAV-b for 6 h. One set of cells was harvested immediately after virus addition. This sample contained both the free and the adsorbed viruses (Load). The other sets of infected cells were incubated for 6, 24, 48, or 96 h. One-tenth of the purified total genomic DNA from infected cells was analyzed by Southern blotting (upper panel) using a GFP-specific probe. To ensure equivalent gel loading of cellular DNA, the lower panel demonstrates the ethidium bromide-stained agarose gel. Note that for the later time points, the amount of cellular DNA proportionally increases due to cell divisions, which might account for the slight decrease in the amount of Ad5/35.AAV-b vector DNA at 96 h. The sizes for ΔAd5/35.AAV and Ad5/35.AAV-b genomes are 12.4 and 37 kb, respectively.