FIGURE 3

Quantitation of hFAH vector genomes in HTI mouse livers by Southern blot analysis. HTI mouse livers injected with each hFAH-expressing vector were harvested 6 weeks postinjection (before in vivo selection, A and B) and after in vivo selection (C and D), and the numbers of total vector genomes (A, C, and D) and replicated vector genomes (B) were analyzed by DraI digestion and a combination of DraI and DpnI digestion, respectively. The results from the samples collected after in vivo selection were verified by two Southern blots (C and D) performed independently using DNA samples extracted from different portions of the liver from individual mice. The numbers (0.0–3.0) above the lanes are hFAH vector copy number standards. Each lane represents a sample from individual mice. In B, a 2.2-kb fragment is DpnI-resistant replicated vector genome, while 0.5- and 0.6-kb bands are DpnI-sensitive genomes. Lanes a and b are 1.0 vector genome per diploid genomic equivalent of hFAH-C- in 20 g of total mouse genomic DNA digested with BglII and DpnI, demonstrating that the DNA was digested to completion. A DraI-hFAH probe was used. In D, the blot was also hybridized with a mouse agouti gene probe. The signals at 0.6 kb are from the endogenous mouse agouti gene.