Examination of targeted versus random dHV.68/5′3′.F50 DNA insertion through Southern blot analysis of genomic DNA.

<p>(A) Upper panel, Eco32I restriction map of the AAV DNA preintegration region (i.e. the <i>AAVS1</i> locus) on human chromosome 19 at 19q13.3-qter. Vertical line and solid oval, trs and RBE, respectively. The vertical solid arrows indicate Eco32I recognition sites delimiting a 5.6-kb DNA segment. The solid horizontal bar denotes the <i>AAVS1</i>-specific probe drawn in relation to its target sequence (diagram not on scale). Lower panel, Eco32I restriction map of dHV.68/5′3′.F50 DNA showing the various hybridization probes (horizontal solid bars) drawn in relation to their cognate target sequences (drawing not on scale). For an explanation of symbols and abbreviations, see the legend of <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0003084#pone-0003084-g001" target="_blank">Fig. 1</a>. (B) Ten micrograms of total cellular DNA from 10 representative clones derived from HeLa cells co-infected with dHV.68/5′3′.F50 and hcAd.FLPe.F50 were digested with Eco32I. Following agarose gel electrophoresis, the resolved DNA fragments were subjected to Southern blot analysis with the <i>AAVS1</i>-specific probe (<i>AAVS1</i> probe; upper panel). After removal of the <i>AAVS1</i>-specific probe, the transferred DNA was incubated simultaneously with a probe derived from the AAV p5IEE plus the <i>rβG</i> gene pA signal and a probe specific for the <i>eGFP</i> ORF (5′ and 3′ vector probes; middle panel). Finally, following a second stripping step, the transferred DNA was exposed to a fourth probe corresponding to the 1.9-kb Eco32I fragment in the dHV.68/5′3′.F50 genome (internal vector probe; lower panel). Clones derived from HeLa cells with AAV Rep68-induced disruptions in the <i>AAVS1</i> locus are indicated by solid arrows, whereas those that also have dHV.68/5′3′.F50 DNA inserted into this specific region of human chromosome 19 are discriminated by open arrows. Marker, GeneRuler DNA Ladder Mix molecular weight marker. (C) Ten micrograms of total cellular DNA from 10 clones of DMD myoblasts co-infected with dHV.68/5′3′.F50 and hcAd.FLPe.F50 were digested with Eco32I. Following agarose gel electrophoresis, the resolved DNA species were subjected to Southern blot analysis with the <i>AAVS1</i>-specific probe (<i>AAVS1</i> probe; upper panel) and, after its removal, with the two probes binding close to the termini of the dHV.68/5′3′.F50 genome (5′ and 3′ vector-specific probes; lower panel). Clones derived from DMD myoblasts with AAV Rep68-induced disruptions in the <i>AAVS1</i> locus are indicated by solid arrows, whereas those that also have dHV.68/5′3′.F50 DNA inserted into this specific region of human chromosome 19 are discriminated by open arrows. Marker, GeneRuler DNA Ladder Mix molecular weight marker. (D) Ten micrograms of total cellular DNA from 9 representative clones derived from HeLa cells infected with dHV.68/5′3′.F50 alone were digested with Eco32I. Following agarose gel electrophoresis, the resolved DNA fragments were subjected to Southern blot analysis with the <i>AAVS1</i>-specific probe. Marker, GeneRuler DNA Ladder Mix molecular weight marker.