cytometry [11,14] on the basis of eGFP expression and are expressed as HeLa-cell transducing units (HTUs) per ml. The fiber-modified hcAd vector hcAd.FLPe.F50 served as source of FLPe [17]. hcAd.FLPe.F50 particles were produced in PER. tTA.Cre76 cells using the hcAd vector shuttle plasmid pAd. FLPe.dAgeI together with Ad.floxedY.F50. pAd.FLPe.dAgeI was derived from construct pAdFLPe [18] by removal of a 3.3-kb AgeI fragment comprising stuffer sequences derived from the human hypoxanthine guanine phosphoribosyltransferase 1 gene. As a consequence, the FLPe-encoding hcAd.FLPe.F50 vector particles contain a 27.4-kb genome. Titration of the hcAd.FLPe.F50 preparation was performed on HeLa cells containing the FLP-responsive construct pGS.pA+.DsRed [28]. Induction of the synthesis of the fluorescent reporter protein DsRed.T4 was determined through flow cytometry and the corresponding functional titers were expressed as gene switch-activating units (GSAUs) per ml. Flow cytometry Quantification of eGFP- or DsRed.T4-positive cells was carried out by using a BD LSR II flow cytometer (Becton Dickinson). In all instances, 10,000 events were acquired. Data was analysed with the aid of BD FACSDiva Software version 5.0.1 (Becton Dickinson). DNA transfections Plasmid DNA transfections were carried out with Lipofectamine (Invitrogen) as specified before [14]. AAV-dependent DNA replication assay The functionality of the FLP-activatable AAV serotype 2 (AAV2) rep68 expression plasmid pGS.pA+.Rep68 and of hcAd.FLPe.F50 were evaluated as follows. PER.tTA.Cre76 cells were seeded in 6-well plates (Greiner) at a density of 2.56106 cells per well. The next day, these cells were transfected with 1.5 mg of pGS.pA+.Rep68 and 1.5 mg of the rAAV vector shuttle construct pAAV.DsRed. pAAV.DsRed contains an expression unit consist- ing of a chimeric human elongation factor 1a (hEF1a) gene promoter [47], the DsRed.T4 ORF and the rabbit b-globin (rbG) gene polyadenylation (pA) signal. The following day, the transfected cells were co-infected with Ad.floxedY.F50 (5 infectious units [IUs]/cell) and hcAd.FLPe.F50 (50 GSAUs/cell). PER.tTA.Cre76 cells transfected with 1.5 mg of pAAV.DsRed and 1.5 mg of the AAV2 rep78/68 expression plasmid pGAPDH.Rep78/68 and infected with Ad.floxedY.F50 at an MOI of 5 IUs per cell served as positive control. pGAPDH.Rep78/68 directs the constitutive synthesis of Rep78 and Rep68 from the promoter of the human glyceraldehyde-3-phosphate dehydrogenase (hGAPDH) gene [14]. Nega- tive controls consisted of pGS.pA+.Rep68- and pAAV.DsRed- transfected PER.tTA.Cre76 cells infected with Ad.floxedY.F50 alone and of pGAPDH.Rep78/68- and pAAV.DsRed-transfected PER.tTA.Cre76 cells that were not exposed to hcAd.FLPe.F50 or Ad.floxedY.F50. Three days posttransfection, the cells were harvested and processed for extrachromosomal DNA extraction. Extrachromosomal DNA extraction and analysis The purification of extrachromosomal DNA was performed as previously described [48]. To selectively degrade the prokaryotic input DNA, the samples were treated for 1 h at 37uC with DpnI. After agarose gel electrophoresis through 1% agarose gels in 16 Tris-acetate-EDTA buffer, the DNA was transferred by capillary action onto nylon membranes (Amersham Hybond-XL; GE Healthcare). These membranes were subsequently incubated with blocking solution, exposed to the DNA probe specified below and Targeted Large DNA Insertion repeatedly washed using a standard Southern hybridization procedure. Finally, the Southern blots were exposed to autoradi- ography films (Amersham Hyperfilm MP; GE Healthcare). rAAV-specific replication products were detected with the aid of the 0.7-kb NcoI6Eco52I fragment from pAAV.DsRed, which includes the entire DsRed.T4 ORF. This DNA probe was labeled with [a-32P]dATP (GE Healthcare) by using the HexaLabel DNA Labeling Kit (Fermentas). Transduction experiments HeLa cells and DMD myoblasts were seeded at densities of 1.06105 and 4.06105 cells per well of 24-well plates (Greiner), respectively. After an overnight incubation period the cells were exposed for 24 hours to dHV.50 and dHV.68/5939.F50 at 4, 20 and 100 HTUs/cell in a total volume of 500 ml. Direct fluorescence microscopy and eGFP-directed flow cytometry were carried out 72 hours postinfection. Mock-infected HeLa cells and DMD myoblasts processed in parallel served to set the background levels of each assay. Light microscopy and immunostaining eGFP direct fluorescence microscopy and AAV Rep immuno- fluorescence microscopy were performed with an Olympus IX51 inverse fluorescence microscope. Images were captured by a ColorView II Peltier-cooled charge-coupled device camera and were archived using analySIS software (Soft Imaging Systems). Cells were processed for immunostaining as described before [11] except that the primary antibody solution consisted of a 1:50 dilution of murine monoclonal antibody 303.9 (PROGEN Biotechnik; catalog number: 61069), which recognizes all four AAV Rep proteins. Western blot analysis Two-cm2 monolayers of HeLa cells were washed once with phosphate-buffered saline and dissolved in 100 ml ice-cold lysis buffer consisting of 20 mM Tris-HCl (pH 7.6), 150 mM NaCl, 0.1% sodium dodecyl sulfate (SDS), 0.5% sodium deoxycholate, 1% Nonidet P40 and 10% glycerol plus a cocktail of protease inhibitors (Complete Mini, Roche Applied Science). The protein lysates were spun for 10 min at 4uC in a minicentrifuge at 25,0006 g. Fifteen ml of the resulting supernatants were mixed with 3 ml of 66 sample buffer (360 mM Tris-HCl [pH 6.7], 60% glycerol and 12% SDS), which was supplemented immediately prior to use with 150 ml of b-mercaptoethanol and 150 ml of a saturated bromophenol blue solution per 2.5 ml. Next, the samples were heated for 5 min at 100uC and subjected to electrophoresis through an SDS-10% polyacrylamide gel. Re- solved proteins were subsequently transferred onto an Immobilon- P membrane (Millipore) by electroblotting. After incubation for 2 h at room temperature (RT) with blocking solution (10 mM Tris-HCl [pH 8.0], 150 mM NaCl and 0.05% Tween-20 [TBST]) containing 10% non-fat dry milk powder (Elk, Campina), the Western blot was incubated overnight at 4uC with monoclonal antibody 303.9 diluted 1:50 in blocking solution. Next, the Immobilon membrane was washed 3 times for 5 min each with TBST at RT and subjected to a 1-h incubation with horseradish peroxidase-conjugated goat-anti-mouse IgG secondary antibodies (Santa Cruz Biotechnoloy; catalog number: sc-2005) diluted 1:10,000 in blocking solution. After three 20-min washes with TBST, the Western blot was incubated for 5 min with chemiluminescence substrate solution (100 mM Tris-HCl [pH 8.6], 250 mg/ml 5-amino-2,3-dihydro-1,4-phthalazinedione [Sigma-Aldrich]) which was supplemented immediately prior to use with 20 ml of a 30% H2O2 solution and 1 ml of a 0.33% 3-(4- PLoS ONE | www.plosone.org 12 August 2008 | Volume 3 | Issue 8 | e3084