FIG. 1.

Effects of the method of T-cell activation on Ad5/F35 vector transduction and expression efficiency. Primary CD4 T cells were activated overnight with 5 μg/ml phytohaemagglutinin (PHA) or anti-CD3/28 antibody-conjugated beads at a 3:1 bead:cell ratio and then infected at a multiplicity of infection (MOI) of 600 with Ad5/F35 vector. (A) Ad5/F35 vector-expressing green fluorescent protein (GFP) was used to measure transduction efficiency by fluorescence activity at Days 5 to 11 post-stimulation. The percent of cells expressing GFP are shown, as well as the mean fluorescence intensity (MFI), which reflects the level of GFP expression in each cell. (B) Ad5/F35 vector expressing the CCR5-specific ZFN, SB-728, was used to measure nuclease activity after transduction. Boxes above the gel show the method of T-cell stimulation, ZFN treatment, and the day of culture. Nuclease activity was measured by using the surveyor nuclease assay on CCR5. Cleavage fragments are indicated by the arrows to the right of the gel. Percent disruption was determined by image analysis as described in the methods and is indicated at the bottom of the gel.