Characterization of Mesenchymal-Fibroblast Cells Using the Col1a2 Promoter/Enhancer
Characterization of Mesenchymal-Fibroblast Cells Using the Col1a2 Promoter/Enhancer
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DOI：
10.1007/978-1-4939-7113-8_10
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发表时间：
2017-01-01
期刊：
影响因子：
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通讯作者：
Bou-Gharios, George
中科院分区：
文献类型：
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作者：
Li, Ian M. H.;Horwell, Amy L.;Bou-Gharios, George
细胞外基质（ECM）的过度沉积是各种器官纤维化疾病的共同标志。ECM中的嵌合体是I型和III型胶原蛋白，由局部成纤维细胞分泌，以及其他用于修复目的的间充质细胞。在过去的二十年中，为了控制这些细胞中ECM的调节并限制纤维化过程的瘢痕形成，对成纤维细胞特异性启动子/增强子的研究已经加强。在我们以前的工作中，我们的特点是增强子区域17 kb上游的Col 1a 2基因转录起始位点。这种增强子在转基因小鼠中主要在发育期间的间充质细胞中表达，并且在损伤后的成年中表达。当驱动转基因如β-半乳糖苷酶或荧光素酶时，该构建体充当胶原蛋白转录的信息报告者，并预测I型胶原蛋白沉积。在这一章中，我们提供了详细的协议，以确定类似的增强子，并使用序列，以产生一个构建转染和生产转基因动物。我们还提供了在转基因小鼠中使用发光、组织处理以及使用cre/lox系统获得小鼠的条件性获得和丧失功能的信息。
Excessive deposition of extracellular matrix (ECM) is a common hallmark of fibrotic diseases in various organs. Chiefly among this ECM are collagen types I and III, secreted by local fibroblasts, and other mesenchymal cells recruited for repair purposes. In the last two decades, the search for a fibroblast-specific promoter/enhancer has intensified in order to control the regulation of ECM in these cells and limit the scarring of the fibrotic process. In our previous work, we characterized an enhancer region 17 kb upstream of the Col1a2 gene transcription start site. This enhancer in transgenic mice is expressed mainly in mesenchymal cells during development and in adults upon injury. When driving transgenes such as beta-galactosidase or luciferase, this construct acts as an informative reporter of collagen transcription and is predictive of collagen type I deposition. In this chapter, we provide detailed protocols for identifying similar enhancers and using the sequence to generate a construct for transfection and producing transgenic animals. We also provided information on the use of luminescence in transgenic mice, tissue processing, as well as using cre/lox system to obtain conditional gain and loss of function in mice.