CHARACTERIZATION OF PRIMARY CULTURES OF CHONDROCYTES FROM TYPE-II COLLAGEN BETA-GALACTOSIDASE TRANSGENIC MICE
CHARACTERIZATION OF PRIMARY CULTURES OF CHONDROCYTES FROM TYPE-II COLLAGEN BETA-GALACTOSIDASE TRANSGENIC MICE
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DOI：
10.1016/0945-053x(94)90199-6
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发表时间：
1994-08-01
期刊：
影响因子：
6.9
通讯作者：
DECROMBRUGGHE, B
中科院分区：
文献类型：
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作者：
LEFEBVRE, V;GAROFALO, S;DECROMBRUGGHE, B
软骨细胞外基质成分的功能和软骨细胞特异性调节机制的研究将受益于转基因小鼠和细胞培养相互补充的方法。因此，我们建立并广泛表征了从携带转基因的新生小鼠肋骨生长板分离的小鼠软骨细胞的原代培养物，其中II型胶原基因调控序列驱动E.大肠杆菌β-半乳糖苷酶报告基因。原代软骨细胞在单层培养中表达完全分化的表型，产生II型、IX型和X型胶原蛋白以及转基因的mRNA。转基因细胞也能合成高水平的E.大肠杆菌β-半乳糖苷酶，易于定量，也可通过X-gal染色在单个细胞中检测到。当软骨细胞从β-半乳糖苷酶与新霉素抗性基因产物融合的转基因小鼠中分离时，它们显示出对G418的抗性。在培养一到两周后，软骨细胞逐渐失去转基因的表达，与软骨特异性基因的表达平行，并开始表达高水平的I型胶原RNA。转基因软骨细胞的使用使我们能够通过测定β-半乳糖苷酶活性和新霉素抗性来容易地对表型变化进行评分。培养的小鼠软骨细胞，如那些在这里报道，也应该有助于生化特性的表型的其他转基因小鼠的遗传性疾病的软骨和参与软骨形成的机制的研究。
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.