Targeted Large DNA Insertion Figure 3. Propagation of rep68-positive and fiber-modified dHVs. (A) Direct fluorescence microscopy analysis of the PER.tTA.Cre76 cells used for the serial propagation of dHV.F50, dHV.68/5939.F50 and dHV.68/3959.F50. (B) Flow cytometric analysis of HeLa cells employed for the determination of the eGFP transfer activity of clarified producer cell lysates derived from consecutive rounds of vector amplification. P1, passage 1; P2, passage 2; P3, passage 3; P4, passage 4. doi:10.1371/journal.pone.0003084.g003 pression to prevent graft rejection. The use of gene-corrected autologous myoblasts may obviate the need to severely immuno- suppress the patients. We previously showed that dystrophin- defective myoblasts are very well transduced ex vivo by non- integrating hcAd-based vectors carrying the receptor-interacting domains of hAd serotype 50 (hAd50) fibers [11]. This makes it relevant to test the ability of dHV.68/5939.F50 to stably transduce DMD myoblasts through targeted transgene integration. More- over, contrary to HeLa cells, the DMD myoblasts used in this study possess a normal, diploid karyotype [31]. HeLa cells and DMD myoblasts were independently exposed to equal doses of dHV.F50 or dHV.68/5939.F50, either alone or together with a constant amount of hcAd.FLPe.F50. Three days later the initial levels of target cell transduction were determined by eGFP-directed flow cytometry. The frequencies of eGFP- positive cells were within a relatively narrow range, i.e., between 72 and 78% for the HeLa cells and between 76 and 81% for the DMD myoblasts. Next, the stable transduction frequencies (STFs) reached by dHV.F50 and by dHV.68/5939.F50 were determined by extensively subculturing both types of dividing cells and by measuring the percentages of eGFP-positive cells through flow cytometry as a function of time. This experimental setup does not introduce any bias towards the selection of cells carrying chromosomally integrated vector DNA and prevents that cells containing only episomal vector genomes are included in the STF calculations, which is a concern given the relatively long half-life of eGFP [32]. Stable transduction levels were reached at 27 days postinfection in HeLa cells whereas it took 56 days before the percentage of eGFP-positive DMD myoblasts stabilized, most likely reflecting the lower division rate of the latter cell type. The STFs obtained with dHV.68/5939.F50 in the presence of hcAd.FLPe.F50 were 0.6% (n = 2) and 0.4% (n = 1) for HeLa cells and DMD myoblasts, respectively (n events acquired = 10,000). In the absence of FLPe, dHV.68/5939.F50 yielded an STF of 0.05% (n = 2) for HeLa cells and of 0.1% (n = 1) for DMD myoblasts (n events acquired = 10,000). Thus, rep68 gene activation increased by 12- and 4-fold the STF of dHV.68/5939.F50-transduced HeLa cells and DMD myoblasts, respectively. Importantly, infection of both target cell types with the control vector dHV.F50 alone or together with hcAd.FLPe.F50 gave rise to similar STFs as observed for HeLa cells and DMD myoblasts transduced exclusively with dHV.68/5939.F50. To determine the frequencies of targeted DNA integration, stably transduced HeLa cells and DMD myoblasts were isolated from cell cultures initially exposed to both dHV.68/5939.F50 and hcAd.FLPe.F50 and individually expanded. HeLa cell clones derived from cultures infected exclusively with dHV.68/5939.F50 served as controls. Genomic DNA was extracted from the various cell lines and subjected to Eco32I digestion and Southern blot analysis. The transferred DNA was incubated first with an AAVS1- PLoS ONE | www.plosone.org 5 August 2008 | Volume 3 | Issue 8 | e3084