hydroxyphenyl)-2-propenoic acid (Sigma-Aldrich) solution in dimethylsulfoxide per 100 ml. The chemiluminescent signal was visualized by exposing the Immobilon membrane to an Amersham Hyperfilm MP film. Actin served as loading control and was detected by using a 1:5,000 dilution of a mouse monoclonal antibody (clone: C4; MP Biomedicals; catalog number: 691001). Chromosomal DNA extraction and analysis The purification of chromosomal DNA was carried essentially as previously described [49] except for the use of a different lysis buffer consisting of 100 mM Tris-HCl (pH 8.5), 5 mM EDTA (pH 8.0), 0.2% SDS and 200 mM NaCl. After restriction enzyme digestion with Eco32I (Fermentas) and electrophoresis through 0.8% agarose gels in 16 Tris-acetate-EDTA buffer, the DNA was transferred by capillary action onto Amersham Hybond-XL membranes. These membranes were subsequently incubated with blocking solution, exposed to the DNA probes specified below and repeatedly washed using a standard Southern hybridization procedure. Next, the Southern blots were exposed to autoradiog- raphy films. Before incubating the nylon membranes with new probes, the previous probes were removed by soaking them in a boiling 0.1% SDS solution. After cooling to RT the 0.1% SDS solution was discarded and the procedure was repeated twice. The AAVS1-specific probe (353 bp) was obtained by PCR amplification using the template, primer pair and cycling parameters described elsewhere [14]. The 377-bp probe covering the AAV p5 promoter and rbG gene pA signal was also generated by PCR using construct pAd/AAV.EGFP [14] as template and the oligodeoxyribonucleotides 59-GTCCATTCCTTATTCCA- TAGAAAAGC-39 plus 59-CCATCGATTGGAGTCGTGAC- GTGAATTACGTCATAGGGTTAGGGAGGTCCTGTATTA- GAGGTCACG-39,whereas the eGFP-specific probe (596 bp) was PCR amplified from plasmid pEGFP-N1 (Clontech) with the aid of the primers 59-GAGCTGGACGGCGACGTAAACG-39 and 59-CGCTTCTCGTTGGGGTCTTTGCT-39. The final concen- trations of template, primers (Invitrogen), dNTPs (Fermentas) and GoTaq polymerase (Promega) in the 50-ml PCR mixtures were 20 pg/ml, 0.2 mM, 0.2 mM and 50 mU/ml, respectively. All PCR- derived probes as well as the 1.9-kb human dystrophin cDNA- specific Eco32I fragment used as an internal vector probe were purified by agarose gel electrophoresis followed by gel extraction using the QIAEX II system (Qiagen) according to the instructions provided by the manufacturer. Prior to their addition to the hybridization solution, the radiolabeled DNA probes were separated from unincorporated dNTPs through size-exclusion chromatography by using Sephadex-50 (GE Healthcare) columns. PCR-based targeted DNA integration assay Four hundred thousand DMD myoblasts were seeded in wells of 24-well plates. After overnight incubation, the cells were infected with dHV.68/5939.F50 alone (40 HTUs/cell) or were co-infected with dHV.68/5939.F50 (40 HTUs/cell) and hcAd.FLPe.F50 (40 GSAUs/cell). The cells were exposed to the viral vectors for 24 hours after which the inocula were substituted by fresh culture References Targeted Large DNA Insertion medium. At 72 hours postinfection, genomic DNA was extracted from these cells as described above. Chromosomal DNA was also extracted from HeLa cell clone N+2 and DMD myoblast clone 3C5, which were derived from cells stably transduced with dHV.68/5939.F50 DNA through random and AAVS1-targeted DNA insertion, respectively. Next, samples containing 160 ng of genomic DNA were subjected to PCR with the AAVS1-specific primer # 211 (59-CAGGTCCACCCTCTGCTG-39), together with primer # 651 (59-TTCCTAACCCCAACACTTGC-39) targeting the vector DNA (i.e. hEF1a promoter sequence). PCR amplifications with the AAVS1-specific oligodeoxyribonucleotides # 211 and # 196 (59-GGCTCAACATCGGAAGAGG-39) were carried out in parallel to serve as a control for template integrity and are expected to give rise to a 354-bp PCR product. PCR mixtures of 50 ml containing 200 mM dNTPs, 0.2 mM of each primer, 0.012 U/ml of Phusion High-Fidelity DNA polymerase (Finnzymes) and 16GC buffer (Finnzymes) were placed in a DNA Engine Tetrad 2 thermal cycler (Bio-Rad) and a touchdown PCR program was initiated by a 3-min incubation at 98uC, followed by 19 cycles of 98uC for 20 sec, an annealing period of 30 sec with the temperature decreasing by 0.5uC every cycle from 62 to 52uC, and a 2-min extension step at 72uC. When the PCR program reached the lower annealing temperature of 52uC, 28 additional cycles were performed by using the same cycling conditions except for the use of a constant annealing temperature of 52uC. This was followed by a 5-min incubation at 72uC. Next, 1 ml of each PCR sample was subjected to a nested PCR with the AAVS1- and vector DNA-specific primers #212 (59-GCTTTGCCACCCTATGCT- GAC-39) and # 188 (59-GGATCTGAGGAACCCCTAGT- GATGG-39), respectively. The cycling conditions applied to the nested PCR were as described above except for the use of a maximal annealing temperature of 69uC and a final touchdown temperature of 59uC. Samples from all PCR amplifications were subjected to agarose gel electrophoresis. Subsequently, PCR products were purified from the gel with the aid of the JetSorb gel extraction kit (GENOMED) and the recovered DNA was inserted into cloning vector pCR-Blunt II-TOPO (Invitrogen) according to the instructions supplied by the manufacturers. Finally, the nucleotide sequence of several of the cloned PCR fragments was determined as previously described [45] by using the M13 forward (59-GACGTTGTAAAACGACGGCCAGT-39) or reverse (59-CAGGAAACAGCTATGACCATGA-39) primer. Acknowledgments We thank Florian Kreppel and Stefan Kochanek (Division of Gene Therapy, University of Ulm, Ulm, Germany) for providing us with plasmid pAdFLPe. Author Contributions Conceived and designed the experiments: MAFVG AAFdV. Performed the experiments: MAFVG MH GPvN AAFdV. Analyzed the data: MAFVG MH AAFdV. Contributed reagents/materials/analysis tools: MAFVG MH GPvN RW MGP AAFdV. Wrote the paper: MAFVG. Edited the paper: AAFdV. 1. Kochanek S, Schiedner G, Volpers C (2001) High-capacity ‘gutless’ adenoviral vectors. Curr Opin Mol Ther 3: 454–463. 2. Palmer DJ, Ng P (2005) Helper-dependent adenoviral vectors for gene therapy. Hum. Gene Ther 16: 1–16. 3. Gonc¸alves MAFV, de Vries AAF (2006) Adenovirus: from foe to friend. Rev Med Virol 16: 167–186. 4. Mitani K, Kubo S (2002) Adenovirus as an integrating vector. Curr Gene Ther 2: 135–144. 5. Jager L, Ehrhardt A (2007) Emerging adenoviral vectors for stable correction of genetic disorders. Curr Gene Ther 7: 272–283. 6. Gonc¸alves MAFV (2005) Adeno-associated virus: from defective virus to effective vector. Virol J 2: e43. PLoS ONE | www.plosone.org 13 August 2008 | Volume 3 | Issue 8 | e3084