FIGURE 3

Functional verification of the rAAV-Gdf5 vector. Human embryonic kidney 293 (HEK293) cells were grown in 6-well plates and transfected with: (1) pUC19, (2) pSPORT6-Gdf5, or (3) pAAV-Gdf5, and 48 hours later total RNA was harvested from the cells. The messenger RNA was reverse transcribed and used as the template for polymerase chain reaction (PCR) with Gdf5-specific primers. (4) The pSPORT-Gdf5 plasmid was used as template in the positive control. (a, top) The ethidium bromide–stained agarose gel shows the predicted 485-base pair PCR product. HEK293 cells were grown in 6-well plates and infected with the indicated amount of rAAV-lacZ or rAAV-Gdf5 (5.0 10⁷ particles/ml). After 48 hours in culture, the supernatants were collected and 30 l was used for Western blotting with GDF-5-specific antibodies. Ten nanograms of recombinant murine GDF-5 was used as a positive control. (a, bottom) Autoradiography of the Western blot reveals the predicted 13.7-kd GDF-5 protein. Microwound monolayer assay: (b) 80% confluent 3T3 cells were growth-arrested for 24 hours, and then microwounded by passing a pipette tip across the culture well and treated with 0.5% bovine calf serum (BCS) and 5.0 10⁷ particles/ml of either rAAV-lacZ or rAAV-Gdf5. (c) The average width of the defect was digitally measured over time and the wound width normalized to the time zero width [w (t)/w (0)] versus time was plotted. (d) Healing time constants () for the different treatments were computed and plotted as mean values SEM. Note that higher values indicate slower wound healing rates. (e) In a separate experiment, 3T3 cells grown to 80% confluence were microwounded and treated with 0.5% BCS and incremental doses of rmGDF-5. The data presented are mean values SEM for the healing time constant () for the different doses of the GDF-5 protein treatments. Asterisks indicate significant differences (P < 0.01; n = 6 per treatment) compared to untreated controls. GDF-5, growth and differentiation factor 5.