HEK293 cells were seeded in 24-well plates at 2.0 × 105 cells per well in complete DMEM. After 24 hours, the cells were incubated with the AAV, bioAAV, T4, or T4-AAV vectors at different multiplicities of infection in antibiotic-free Opti-MEM for 6 hours. Thereafter, Opti-MEM was removed and replaced with complete DMEM. The cells were further incubated at 37°C for an additional 48 hours. GFP/mCherry transgene expression was observed by fluorescence microscopy (Carl Zeiss, Germany) at 48 hours after transduction, and the mean fluorescence intensities were quantified using ImageJ software. The nucleus was stained using Hoechst 33342 (Thermo Fisher Scientific, MA). To analyze luciferase gene delivery into cells by T4 or T4-AAV, we measured luciferase activity with the Luciferase Assay System (Promega, WI) according to the manufacturer’s instructions. Briefly, growth medium was removed, and cells were rinsed with PBS buffer. After removing the wash buffer, 150 μl of passive lysis buffer was added to each well, followed by gentle shaking at RT for 20 min. Twenty microliters of the cell lysate was then transferred to a 96-well white opaque plate and mixed with 80 μl of Luciferase Assay Reagent, and the luminescence signal was recorded using the GloMax-Multi Detection System (Promega, WI). The activity of the Soc-β-gal enzyme displayed on the T4 head in cells was determined by staining with X-Gal using the β-Galactosidase Staining Kit (Sigma-Aldrich, MO). Triplicate measurements were applied to each group.

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