FIG. 4.
Analysis of ΔAd.AAV1 genomes after transduction of SKHEP-1 cells by Southern blotting with a SEAP-specific probe. (A) SKHEP-1 cells were infected with 105 genomes per cell. Genomic DNA from single colonies was expanded to 106 cells and was analyzed by Southern blotting with a SEAP-specific probe. Genomic DNA (10 μg) was digested with BamHI, EcoRI, or XhoI and was separated in 0.8% agarose gels. Genomic DNA from diploid SKHEP-1 (11) contains two copies of the SEAP gene resulting in endogenous signals, labeled by asterisks. XhoI fragments were between 4 and 20 kb. The localizations of BamHI, EcoRI, and XhoI sites in the ΔAd.AAV1 genome (containing the SEAP cDNA) and in the endogenous genomic SEAP gene are indicated. The XhoI digestion product of the endogenous SEAP gene was larger than 23 kb and does not appear on the blot. The diagram illustrates the structure of episomal ΔAd.AAV1 vector DNA and integrated tandems forming junctions with chromosomal DNA. The complementary region for the SEAP-specific probe is indicated.