Dharmafect 3

Bovine sox9 siRNA (ON-TARGETplus Custom SMARTpool) was synthesized by Dharmacon (Table I). To determine whether this sox9 siRNA efficiently silences sox9 expression, adult bovine articular chondrocytes were transfected with sox9 siRNA or non-targeting siRNA (Dharmacon) according to manufacturer’s instructions. After 3 days in culture, the medium was replaced with 2ml complete medium and cells were transfected using 4 µl/well DharmaFECT3 (Dharmacon) and 10 µl of 20 µM sox9 siRNA. ON-TARGETplus non-targeting siRNA was used for mock-transfection. After 16 h, siRNA transfection was stopped by replacing the medium with 4ml of fresh complete medium supplemented with the designated growth factor(s), 50 ng/ml FGF-2 or the combination of 200 ng/ml IGF-I with 100 ng/ml BMP-2 and 100 ng/ml BMP-7. Fresh complete medium without growth factors was used as control. On days 2 and 4 after transfection, conditioned medium was harvested and replaced with basal medium containing 0.1% BSA and the designated growth factor(s) as described above. Basal medium containing 0.1% BSA without growth factors was used as control. On day 6 after transfection, conditioned medium and cell layer were harvested separately.