Fig. 3. Expression of the Flag-ICAT transgene in Col2a1-ICAT transgenic mice. (A,B) Primary sternal chondrocytes were isolated from Col2a1-ICAT transgenic mice and their WT littermates. Expression of Flag-ICAT protein was detected in western blot analysis and immunostaining using the anti-Flag M2 antibody in Col2a1-ICAT transgenic chondrocytes. (C) Expression of the Flag-ICAT transgene in growth plate chondrocytes was examined by immunostaining using tissue sections from knee joints of 2-week-old WT and Col2a1-ICAT transgenic mice. The ICAT transgene was specifically expressed in proliferating chondrocytes in the growth plate of Col2a1-ICAT transgenic mice and in articular chondrocytes lining the joint surface. (D) ICAT inhibits canonical Wnt signaling in chondrocytes of Col2a1-ICAT transgenic mice. Primary chondrocytes isolated from 3-day-old WT and Col2a1-ICAT transgenic mice were transfected with TOP-flash reporter and treated with or without Wnt3a (100 ng/ml) for 24 hours. Wnt3a stimulated the reporter activity in WT but not in Col2a1-ICAT transgenic chondrocytes. (E) To determine the specificity of the Flag-ICAT transgene expression, total RNA was extracted from multiple tissues and the expression of Flag-ICAT was examined by PCR. The expression of Flag-ICAT was detected in ribs (strong expression) and brain (weak expression) but not in other tissues. (F) ICAT does not alter cell adhesion. Primary chondrocytes isolated from Col2a1-ICAT transgenic mice and WT littermates were placed in 24-well plates and grown to confluence. Cells were then trypsinized with 0.1% trypsin containing either 1 mM CaCl₂ (TC) or 1 mM EDTA (TE) and incubated at 37°C for 30 minutes. Cells were pipetted gently five times with 10 ml of PBS and the cell clusters were counted. The degree of adhesion was expressed by determining the ratio of cell clusters in TC and TE containing solutions (TC:TE) for each cell type. A similar experiment was also performed using chondrocytes isolated from β-catenin^fx/fx mice and infected with adenovirus expressing Cre recombinase (Ad-Cre) or GFP (Ad-GFP). Results showed that cell adhesion was not changed in Col2a1-ICAT transgenic mice.