2.4.1 Adenoviral transduction and competition assays for AdFZ33βGal
One day prior to transduction fetal human myoblasts (FHM) were seeded in 96-well (1x104 cells per well) flat-bottom tissue culture plates (Corning Glass Works, Corning, USA).
For competition experiments the Ad vector was preincubated with mAb as described and transduction done in the presence of 20µg/ml recombinant Knob protein, 20µg/ml KnobZ33 protein, or 10µg/ml staphylococcal protein A fragment, respectively. Cells were washed twice with Opti-MEM and further incubated with 10% FBS-containing growth medium for 48 hours
at 37°C until the transgene expression was quantified. Transduction efficiencies were evaluated by luminometric quantification of β-galactosidase activity or X-Gal staining.
2.4.2 Virus transduction assay
One day prior transduction fetal human myoblasts (FHM) were seeded in 48-well (1x104 cells per well) flat-bottom tissue culture plates (Corning Glass Works, Corning, USA) and transduced over night (16h) in Opti-MEM medium (Invitrogen Life Technologies, Karlsruhe, Germany) supplemented with 2% FBS with 1250, 2500, and 12500 viral genomes per cell (vg/cell). Cells were washed twice with Opti-MEM and further incubated with 10% FBS- containing growth medium for 48 hours at 37°C until the transgene expression was quantified.
2.4.3 Virus transduction competition assay
One day prior transduction fetal human myoblasts (FHM) were seeded in 48-well (1x104 cells per well) flat-bottom tissue culture plates (Corning Glass Works, Corning, USA).
For competition experiments Ad5EGFP and Ad19aEGFP vectors were preincubated with 10µg/ml Ad19a fiber protein, purified knob proteins (Ad19a knob: 10µg/ml and 100µg/ml; Ad5 knob: 10µg/ml), 2mM GRGDS peptide (Sigma), 10 µg/ml heparin (Sigma), 50µg/ml wheat germ agglutinin (Sigma), α(2-3) neuraminidase (New England Biolabs, Berverly, USA) alone, or combinations thereof, respectively, in Opti-MEM supplemented with 2%FBS. Incubation with competing substances was done for 1h on ice prior to vector addition. Cells were transduced with Ad5EGFP and Ad19aEGFP (MOI=20) for 1h on ice. Then the cells were washed twice with Opti-MEM and further incubated with 10% FBS-containing growth medium for 48 hours at 37°C until the transgene expression was quantified.
Virus internalization-competition assay: Cells were incubated with inhibiting substances for 1h on ice as described above, and transduced with virus (MOI=20) for 1h at 37°C. Then the cells were washed twice with Opti-MEM and further incubated with 10% FBS-containing growth medium for 48 hours at 37°C until the transgene expression was quantified.
2.4.4 Quantification of transgene expression
Transgene expression in cells transduced with EGFP-expressing vectors (Ad5EGFP, Ad19aEGFP) was evaluated using a fluorescence multi-well plate reader (Perseptive Biosystems, Framingham, USA). Fluorescence was stimulated by excitation of EGFP at
488nm and emitted fluorescence measured at 508nm. Transgene expression of cells transduced with vectors expressing β-galactosidase (Ad5βGal, AdFZ33βGal) was evaluated by luminometric quantitation of β-galactosidase activity. For luminometric quantitation of β- galactosidase activity equal amounts of protein were incubated with Galacto-Star reagent (PE Biosystems, Weiterstadt, Germany) according to the manufacturer's instructions and light emission measured in a Berthold plate luminometer (Berthold, Bad Wildbad, Germany). Total amount of cell protein was determined with micro BCA protein assay (Pierce, Rockford, USA).
2.4.5 Flow cytometry (FACS)
For CD11c surface staining of monocyte-derived dendritic cells 5 x 105 cells were labeled with PE-conjugated primary anti-CD11c antibody (50µg/ml) (DakoCytomation, Glostrup, Denmark) for 30min at RT in 500µl FACS buffer (PBS containing 1%FBS). Cells were washed 3 times with PBS and fixed with PBS containing 1% paraformaldehyde (PFA) for 30 min at RT. Next, cells were washed once with PBS and resuspended in 500µl FACS buffer supplemented with 0,01% NaN3 for analysis on a FACScan (Becton Dickinson Bioscience,
Heidelberg, Germany). A total of 10000 cells per experiment were analyzed.
2.4.6 Confocal laser immunofluorescence and immunofluorescent staining of human muscle cells
The protocol for detection of surface NCAM expression on fetal human myoblast-derived myotubes by immunofluorescence was adapted from Walsh et al.228. Human myoblasts and myotubes were grown on laminin-1 coated glass cover slips and incubated with 5.1H11 hybridoma supernatant diluted 1:100 in PBS for 45min at RT. Cells were washed thoroughly with PBS and incubated with Cy3-conjugated sheep-anti mouse secondary antibody (DakoCytomation, Glostrup, Denmark) diluted 1:200 in PBS supplemented with 10% FBS for 45 min at RT. Cells were washed twice with PBS and fixed with 2% PFA/PBS for 5 min at RT. For confocal laser microscopy of virus-infected cells CHOαvβ3 cells were cultured on
cover slips (Menzel Gläser, Braunschweig, Germany). Internalization of Ad5 vector into CHOαvβ3 cells was analysed at 2 time points (15min and 45 min). Ad5βGal vector
(MOI=1000) was added to CHOαvβ3 cells for 1h on ice. Internalization was induced by
incubating the cells at 37°C for the indicated times followed by immediate fixation with 2%
PFA/PBS for 20 min on ice. For staining, coverslips were blocked for 1h at RT with 10%FBS/PBS and co-stained with anti-hexon antiserum and anti-β3 integrin mAb Ab-15
(both 50µg/ml) diluted in PBS containing 0.2% Triton X-100 (Sigma). TRITC-conjugated rat anti-goat and fluorescein isothiocyanate (FITC)-conjugated rabbit anti-mouse secondary antibodies (50µg/ml) were used as secondary antibodies (DakoCytomation, Glostrup, Denmark). Immunofluorescence analysis was performed on a Leica confocal laser microscope with TCS software program.
2.4.7 Visualization of transgene expression (X-Gal) and fluorescence microscopy
β-galactosidase expression in transduced cells was visualized by means of X-Gal staining according to a standard protocol229. Pictures of fluorescent cells were recorded with AxioCam HRC (Zeiss, Jena, Germany) on a Leica DMRBE epifluorescence microscope and analyzed with AxioVision 3.1 software (Zeiss, Jena, Germany).