DNA analysis of vector sequences.
(A) Diagram of AAV-(ApoE)4/hAAT-cFIX vector. Shown are AAV2 inverted terminal repeats (ITRs), a 1.1-kb enhancer/promoter sequence containing 4 repeats of the human ApoE enhancer linked to the human α1-antitrypsin promoter, a chimeric β-globin/CMV intron, the cFIX cDNA, and hGH poly A signal. A 0.9-kb EcoRI fragment used as a probe for Southern hybridization and a unique NcoI restriction site are also indicated. (B) Southern blot hybridization. Lanes 1-3: plasmid pAAV-(ApoE)4/hAAT-cFIX encoding the vector,EcoRI digested, 1 ng, 100 pg, and 10 pg, respectively. Lanes 4-6: genomic DNA isolated from liver of hemophilia B dog Beech, 40 μg/lane. Lane 4, EcoRI digest; lane 5, NcoI digest; lane 6, uncut DNA. Lane 7: genomic DNA isolated from spleen, 40 μg, cut with EcoRI. Indicated on the right margin are high-molecular-weight DNA (HMW), the 4.5-kb fragment representing the unit length of the double-stranded vector, and the 0.9-kbEcoRI fragment used for estimation of gene copy number.
