A versatile chondrogenic rat calvaria cell line R-tTA-24 that permits tetracycline-regulated gene expression

The clonal rat calvaria cell line RCJ3.1C5.18 (RCJ) undergoes chondrogenic differentiation after long-term culture post confluence. To allow flexible genetic manipulation, a tetracycline-regulated gene expression system was e stablished in this cell line. Treatment with tetracycline in operational do ses does not affect the differentiation of RCJ cells with respect to the ma rkers tested. After stable transfection with pUHD15.1 containing the tetrac ycline transactivator (tTA) in the presence of pTK-hyg for hygromycin selec tion, 28 clones were isolated and characterized for alcian blue staining of cartilage-specific proteoglycans and for collagen type II expression. Clon e R-tTA-24 was selected on the basis of phenotype and displayed tetracyclin e-dependent down-regulation of luciferase activity (tet-OFF system) by two orders of magnitude (57-149-fold) after stable transfection with the report er gene pBI-EGFP/luc. The novel, chondrogenic cell line R-tTA-24 may be sta bly transfected with various genes of interest for tetracycline-regulated g ene expression using neomycin selection and may be a valuable tool to study the process of chondro-genic differentiation in vitro.