Figure 4.

Transduction efficiency of rAAV-β-gal following freeze-drying onto allografts and implants in vitro and in vivo. 5 × 10⁷ transducing units of rAAV-β-gal was lyophilized onto mouse femoral allografts (a) or stainless steel pins (b). The transduction efficiency was determined in vitro by incubating the coated pins on top of a monolayer of confluent 293 human embryonic kidney cells for 72 h. Photographs of the X-gal-stained cells distal (c) and proximal (d) to the coated pin, as well as an uncoated control pin (e) are shown. The transduction efficiency of the coated pins was also quantified after the indicated storage time at −80 °C. RLU, relative light units. (f). As a control, 5 × 10⁷ transducing units of rAAV-β-gal in 50 μl PBS was directly placed on a monolayer of 293 cells. The β-galactosidase activity in the cultures was determined using the Galacto-Light system. No significant differences were observed. The efficiency of in vivo transduction 14 d after transplantation is shown at ×10 (g) and ×40 (h) magnification, where the blue staining indicates transduction of the fibroblasts (f) between the allograft (a) and the muscle (m).