Figure 4
Gene expression of osteocytic markers in cell fractions enzymatically isolated from mouse long bone, and in the osteocyte-enriched cell population from wild type and tamoxifen-inducible Ccn2-deficient mice. (A) After bone marrow had been flashed out, femurs were cut into small pieces and digested by 0.2% collagenase for 20 min. The supernatant was saved as Fr1, and the bone pieces were treated with 5 mM EDTA for 20 min. The supernatant was saved as Fr2. This was repeated and the cells of Fr5/6 and Fr7/8 were collected. After Fr5/6 and Fr7/8 had been collected and placed in 2-D cultures for a few days, these cells were embedded into collagen gel and cultured for 5 days. Total RNA was isolated, and real-time RT-PCR analysis was performed by using specific primers. The amounts of osteocyte marker transcripts were normalized to that of Gapdh mRNA. Data show the value from independent samples of n = 6, and graph presents the mean and standard deviation. The asterisk indicates a significant difference from the Fr5/6 sample (p < 0.05). (B) Femurs were collected from tamoxifen-inducible Ccn2-deficient and wild type mice 1 month after injection with tamoxifen. As described in “A,” osteocytes in osteocyte-enriched cell population (Fr7/8) were isolated. After 2-D culture for a few days, these cells were embedded into collagen gel and cultured for 5 days. Total RNA was isolated and real-time RT-PCR analysis was performed by using specific primers. Data show the value from independent samples of n = 6, and the graph presents the mean and standard deviation. The asterisk indicates a significant difference from WT osteocyte-enriched cells (p < 0.05).
