CHARACTERIZATION OF PRIMARY CULTURES OF CHONDROCYTES FROM TYPE-II COLLAGEN BETA-GALACTOSIDASE TRANSGENIC MICE

Studies on the function of extracellular matrix components of cartilag es and on chondrocyte-specific regulatory mechanisms will benefit from approaches in which transgenic mice and cell cultures will complement each other. We therefore established and extensively characterized pr imary cultures of mouse chondrocytes isolated from rib growth plates o f newborn mice harboring a transgene in which type II collagen gene re gulatory sequences were driving expression of an E. coli beta-galactos idase reporter gene. Primary chondrocytes expressed a fully differenti ated phenotype in monolayer culture, producing mRNAs for the collagen types II, IX and X, and for the transgene. Transgenic cells also synth esized high levels of E. coli beta-galactosidase, easily quantifiable and also detectable in individual cells by X-gal staining. When chondr ocytes were isolated from transgenic mice in which beta-galactosidase was fused to the product of the neomycin resistance gene, they display ed resistance to G418. After one to two weeks in culture, chondrocytes progressively lost expression of the transgenes, in parallel with tha t of cartilage-specific genes, and started expressing high levels of t ype I collagen RNA. The use of transgenic chondrocytes allowed us to e asily score phenotypic changes by assaying beta-galactosidase activity and neomycin resistance. Cultures of mouse chondrocytes, such as thos e reported here, should also help characterize biochemically the pheno types of other transgenic mice in studies of genetic diseases of carti lages and of mechanisms involved in chondrogenesis.