Figure 3From: SOX9 gene transfer via safe, stable, replication-defective recombinant adeno-associated virus vectors as a novel, powerful tool to enhance the chondrogenic potential of human mesenchymal stem cellsExpression analyses in differentiated aggregate cultures of human mesenchymal stem cells (hMSCs). Aggregate cultures were prepared and transduced with rAAV-lacZ or rAAV-FLAG-hSOX9, as described in Figure 1, or left untreated, and processed on day 21 for (a) immunodetection of type II, type I, and type X collagen (all at magnification ×4), and (b) gene-expression analysis by real-time RT-PCR amplification after total cellular RNA extraction and cDNA synthesis, as described in Materials and methods. The genes analyzed included the transcription factor SOX9, and types II, I, and X collagen (COL2A1, COL1A1, COL10A1), alkaline phosphatase (ALP), matrix metalloproteinase 13 (MMP13), osteopontin (OP), the transcription factor RUNX2, β-catenin, parathyroid hormone-related protein (PTHrP), lipoprotein lipase (LPL), and the peroxisome proliferator-activated receptor gamma 2 (PPARG2), with GAPDH serving as a housekeeping gene and internal control (primers are listed in Materials and methods). Ct values were obtained for each target and GAPDH as a control for normalization, and fold inductions (relative to untreated aggregates) were measured by using the 2-ΔΔCt method. Statistically significant compared with (a) condition without vector treatment or (b) rAAV-lacZ.Back to article page