Targeted Large DNA Insertion Figure 7. Integration of exogenous DNA into AAVS1 after gene transfer with rep68-positive dHV.68/5939.F50 particles. (A) Overview of the PCR-based targeted DNA integration assay. Genomic DNA extracted from HeLa cell clone N+2 and DMD myoblast clone 3C5 served as control templates derived from cells genetically modified through vector DNA insertion outside and inside the AAVS1 locus, respectively. The experimental samples consisted of chromosomal DNA from DMD myoblasts infected with dHV.68/5939.F50 alone (40 HTUs/cell) or co-infected with dHV.68/ 5939.F50 (40 HTUs/cell) and hcAd.FLPe.F50 (40 GSAUs/cell). The upper part of the boxed pictogram represents the section of human chromosome 19 at 19q13.3-qter harboring the AAVS1 locus. Vertical line and adjacent oval, trs and RBE, respectively; horizontal fading bar, AAV DNA preintegration region; green box, representation of a targeted vector DNA insertion event leading to an AAVS1-foreign DNA junction; numbered black and green arrows symbolize AAVS1- and vector DNA-specific primers, respectively. (B) Agarose gel electrophoresis of PCR products resulting from amplifications carried out on chromosomal DNA from HeLa cell clone N+2 (lanes 1), DMD myoblast clone 3C5 (lanes 2), DMD myoblast population transduced with dHV.68/5939.F50 alone (lanes 3) and DMD myoblast population co-transduced with dHV.68/5939.F50 and hcAd.FLPe.F50 (lanes 4). Lanes M, Gene Ruler DNA Ladder Mix molecular weight marker. Upper panel, internal control PCR products resulting from amplifications performed with the AAVS1- specific primers # 211 and # 196. Lower panel, nested PCR products of amplifications carried out with oligodeoxyribonucleotides # 212 and # 188 on DNA synthesized with the aid of primers # 211 and # 651. (C) Nucleotide sequence analysis of junctions generated between AAVS1 and exogenous DNA following co-transduction of DMD myoblasts with dHV.68/5939.F50 and hcAd.FLPe.F50. The DNA sequencing data in the upper and lower panel correspond to inserts derived from PCR amplifications on chromosomal DNA from myoblast clone 3C5 and from the DMD myoblast population co-transduced with dHV.68/5939.F50, respectively. The black and green bars indicate AAVS1 and vector DNA, respectively. Vertical arrows points to the junction between AAVS1 and dHV.68/5939.F50 DNA present in each individual clone. doi:10.1371/journal.pone.0003084.g007 ITR-flanked DNA into the AAVS1 locus in 14/39 (i.e. 35%) clones of stably transduced HepG2 cells [38]. Similar results were obtained with a single-particle version of this hcAd/AAV hybrid vector system accommodating a 3.8-kb rAAV genome and an inducible rep78 expression unit regulated by a doxycycline- controlled transcriptional silencer [40]. Recently, Wang and Lieber generated capsid-modified hcAd vectors containing a GFP-encoding rAAV genome significantly larger than those tested by Recchia and co-workers [39]. In parallel, they succeeded in rescuing and propagating a Rep78-encoding hcAd vector by relying on a strategy based again on a transcriptional regulatory element with low basal activity in producer cells (i.e. the human b- globin locus control region). Co-infection of human megakaryo- blastic leukemia (Mo7e) cells with the rAAV- and rep78-containing hcAd vectors at an MOI of 300 and 800 genomes per cell, respectively, followed by their clonal expansion yielded 25.8% cell PLoS ONE | www.plosone.org 10 August 2008 | Volume 3 | Issue 8 | e3084