Characterization of primary cultures of chondrocytes from type II collagen/beta-galactosidase transgenic mice.
Studies on the function of extracellular matrix components of cartilages 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 primary cultures of mouse chondrocytes isolated from rib growth plates of newborn mice harboring a transgene in which type II collagen gene regulatory sequences were driving expression of an E. coli beta-galactosidase reporter gene. Primary chondrocytes expressed a fully differentiated phenotype in monolayer culture, producing mRNAs for the collagen types II, IX and X, and for the transgene. Transgenic cells also synthesized high levels of E. coli beta-galactosidase, easily quantifiable and also detectable in individual cells by X-gal staining. When chondrocytes were isolated from transgenic mice in which beta-galactosidase was fused to the product of the neomycin resistance gene, they displayed resistance to G418. After one to two weeks in culture, chondrocytes progressively lost expression of the transgenes, in parallel with that of cartilage-specific genes, and started expressing high levels of type I collagen RNA. The use of transgenic chondrocytes allowed us to easily score phenotypic changes by assaying beta-galactosidase activity and neomycin resistance. Cultures of mouse chondrocytes, such as those reported here, should also help characterize biochemically the phenotypes of other transgenic mice in studies of genetic diseases of cartilages and of mechanisms involved in chondrogenesis.
Lefebvre
V
V
Department of Molecular Genetics, University of Texas, M.D. Anderson Cancer Center, Houston.
Garofalo
S
S
Zhou
G
G
Metsäranta
M
M
Vuorio
E
E
De Crombrugghe
B
B
D000818:Animals; D001426:Bacterial Proteins / Q000096:biosynthesis / Q000235:genetics; D002478:Cells, Cultured; D003094:Collagen / Q000096:biosynthesis* / Q000235:genetics; D004351:Drug Resistance / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D005786:Gene Expression Regulation; D017930:Genes, Reporter; D005813:Genes, Synthetic; D005839:Gentamicins / Q000494:pharmacology; D006132:Growth Plate / Q000166:cytology* / Q000378:metabolism; D019868:Kanamycin Kinase; D051379:Mice; D008822:Mice, Transgenic; D009355:Neomycin / Q000494:pharmacology; D010641:Phenotype; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000096:biosynthesis / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis*; D001616:beta-Galactosidase / Q000096:biosynthesis / Q000235:genetics
D001426:Bacterial Proteins; D005839:Gentamicins; D011993:Recombinant Fusion Proteins; D003094:Collagen; C010680:antibiotic G 418; D017853:Phosphotransferases (Alcohol Group Acceptor); D019868:Kanamycin Kinase; D001616:beta-Galactosidase; D009355:Neomycin