FIG. 3.

Clonal analysis of GFP expression and vector DNA in MO7e cells. (A) Analysis of integration copy numbers in GFP-positive clones transduced with Ad.LCR-β-GFP. GFP-expressing cells from 20 clones were sorted and expanded, and 10 μg of genomic DNA from each clone was analyzed by Southern blotting using a GFP-specific probe. EcoRI cuts twice within the vector sequence and releases a characteristic 4.1-kb fragment containing the GFP gene. The right side of the gel shows a standard curve for vector genomes using defined concentrations of plasmid DNA (pAd5GFP/F35) corresponding to 0.5, 1, 2.5, 5, and 10 vector copies per cell. Loading differences were equalized by rehybridizing the filters with a probe specific for the human α₁-antitrypsin gene. The blots were analyzed using phosphorimaging, and the calculated number of vector copies per cell is shown for each clone. (B) Mean GFP fluorescence was measured by flow cytometry. The values were divided by the vector copies present in the given clone. The number of Ad.LCR-β-GFP and Ad.HCA-CMV-GFP clones that expressed GFP levels in the indicated range was plotted. (C) Analysis of integrated vectors in GFP-positive clones transduced with Ad.LCR-β-GFP. Ten micrograms of genomic DNA was digested with NheI, which cuts only once within the vector sequence. A GFP probe was used for hybridization. On the right is shown the localization of NheI sites in the vector and chromosomal globin LCR region. Theoretically, vector integration can result in a band that is larger than 6.5 kb (in the range of 7 to 9 kb).