Genomic integration of the rHS32Aγ*3′RE genome. Individual colonies of K562 cells, transduced at an MOI of 4 × 109, were cultured in semisolid medium for 14 days and then expanded in liquid culture for 2 to 4 weeks. (A) DNA from 7 individual colonies analyzed without restriction (−) or cut with BsaBI (+). The bands at 4.8 and 5.3 kb contain the endogenous globin genes, whereas the band at 3.3 kb is an internal fragment derived from the vector genome (see diagram below). The negative control lanes contain DNA derived from nontransduced K562 cells. (B and C) DNA from the same 7 colonies and the negative control restricted with Stu I or EcoRI, respectively. Stu I releases variable length junction fragments, a 4.7-kb band derived from head to tail concatamers of the rAAV genome, and 4.0- and 4.9-kb fragments containing the endogenous globin genes. EcoRI releases a 3.4-kb fragment derived from tandem repeats of the rAAV genome, variable length junction fragments, and two fragments of 2.6 and 3.1 kb containing the endogenous γ globin genes.
