Tissue Regeneration, Department of Hard Tissue Engineering, Tokyo Medical and Dental University, Tokyo 113-8549, Japan.
J Biochem. 2012 Dec;152(6):565-75. doi: 10.1093/jb/mvs110. Epub 2012 Sep 27.
Type II collagen is a major component of cartilage where it is present at a high concentration, which is essential for the functional maintenance of the tissue. Therefore, any fundamental understanding of the physiology of cartilage tissue must include an understanding of the mechanism that allows the high level of expression of type II collagen gene, Col2a1, by chondrocytes. To this end, we developed a new reporter assay system based on the co-transfection of candidate enhancer elements and reporter construct into Swarm rat chondrosarcoma chondrocytes that allowed their stable expression. Using this system, we screened more than 70 kb of the Col2a1 gene and found an enhancer domain that is responsible for the regulation of its expression level. The domain is localized in intron 7, and consists of an 800-bp region that contains within it a previously unidentified domain, ∼140 bp in size.
II 型胶原蛋白是软骨的主要成分,其在软骨中的浓度很高,这对于组织的功能维持至关重要。因此,对软骨组织生理学的任何基本理解都必须包括对允许软骨细胞高水平表达 II 型胶原蛋白基因 Col2a1 的机制的理解。为此,我们开发了一种基于候选增强子元件和报告构建体共转染的新报告基因检测系统,该系统可使 Swarm 大鼠软骨肉瘤软骨细胞稳定表达。使用该系统,我们筛选了超过 70kb 的 Col2a1 基因,发现了一个负责调节其表达水平的增强子域。该域位于内含子 7 中,由一个包含 800bp 区域的增强子组成,其中包含一个以前未被识别的约 140bp 的域。