Col2a1-CreER^T2 transgene construct. The Cre recombinase cDNA fused with cDNA encoding the ER ligand-binding domain containing three mutation sites was cloned into the PKN-185 vector. The expression of the fusion protein is under control of the 1.0-kb type II collagen promoter. The primer positions for genotyping transgenic mice have been indicated by arrows.

Tamoxifen-induced lacZ expression in Col2a1-CreER^T2 embryos. The male Col2a1-Cre^+/-ER^T2 mice were mated with female Rosa26R reporter mice. The pregnant female mice with E15.5 embryos were injected intraperitoneally with tamoxifen (4 mg/mouse) or corn oil (control). Three days later, the E18.5 embryos were prepared and stained with X-Gal. Only the double transgenic mice exposed to tamoxifen showed X-Gal positive staining. The ribs, spine, and long bones showed positive X-Gal staining (a). A histology section from the knee joint showed positive X-Gal staining of chondrocytes in articular cartilage and patella tissues (b).

Tamoxifen-induced β-Gal activity in chondrocytes. (a) The sternal chondrocytes were isolated from 3-day-old neonatal Col2a1-Cre^+/-ER^T2;R26R^+/- mice. Cells were seeded in 12 well-plates and treated with 4-OH tamoxifen for 24 and 48 h followed by X-Gal staining. Chondrocytes isolated from double transgenic mice were treated with 1 μM 4-OH tamoxifen for 24 and 48 h. Weak X-Gal staining was observed at 24 h time point and strong X-Gal staining was observed when cells were treated with 4-OH tamoxifen for 48 h (n = 3). (b) Primary chondrocytes isolated from Col2a1-Cre^+/-ER^T2;R26R^+/- mice were treated with different concentrations of 4-OH tamoxifen and 1 μM estradiol (E2), the β-Gal activity was measured. 4-OH tamoxifen stimulated β-Gal activity in chondrocytes and estradiol had no significant effect on β-Gal activity. *P < 0.01, compared to corn oil control group; **P < 0.05, compared to 0.01 μM 4-OH tamoxifen group; one way analysis of variance followed by Dunnett’s test, n = 4.

Histological analyses of the Col2a1-Cre^+/-ER^T2;R26R^+/- mice. Two- and eight-week-old double transgenic mice were injected with tamoxifen (1 mg/mouse/day for 5 consecutive days). Mice were killed 5 days after injections were completed. Bone samples and other tissues were fixed, decalcified, and processed for frozen section preparation followed by X-Gal staining. Sections were then counterstained by Nuclear Fast Red. Growth plate and articular chondrocytes from 2- and 8-week-old transgenic mice (a,b) and ribs and spine from 2-week-old transgenic mice (c) received tamoxifen showed X-Gal positive staining. X-Gal-positive chondrocytes were only identified in histological sections of ribs (d, upper panel) and spine (d, lower panel) of transgenic mice received tamoxifen (right panels). Recombination efficiency was analyzed from multiple histological sections of three transgenic mice (n = 3). Other organs, such as heart, kidney, and liver did not show X-Gal positive staining (e). RNA was extracted from multiple tissues including liver, heart, kidney, lung, spleen, skin, spine, long bone, and trachea of the 2-week-old Col2a1-Cre^+/-ER^T2;R26R^+/- mice. The Cre expression was detected in spine, long bone, and trachea but not in other tissues by RT-PCR using the Cre-specific primers (f). The β-Gal activity from tissues rib, spine, long bone, liver, kidney, heart, and skin were also measured and normalized to protein contents. The induction of β-Gal activity by tamoxifen was observed in rib, spine, and long bone but not in other tissues (g).