Genomic integration of the rHS432Aγ* genome. Individual clones, derived from K562 cells transduced at an MOI of 3 × 108, were cultured in semisolid medium for 14 days and then expanded in liquid culture for 3 to 4 weeks. DNA was extracted and analyzed by the Southern blot methodology after restriction with EcoRI and Bgl II (A) or Stu I (B). DNA extracted from a cell line containing a single integrated copy of an rAAV genome that includes the Aγ* gene (K562 HS2Aγ*) served as a positive control (left lane), whereas K562 cells provided DNA that served as a negative control (second lane from left). The bands of 2.6 and 3.1 kb (A) or the 4.0- and 4.9-kb bands (B) present in each lane contain the endogenous γ globin genes. The diagram below indicates the organization of the rAAV vector genome and the location of specific restriction endonuclease sites. EcoRI and Bgl II (A) release an internal rAAV band of 4.4 kb, whereas Stu I releases a junction fragment of variable length that includes a portion of the rAAV genome and human chromosomal DNA.
