Characterization of Mesenchymal-Fibroblast Cells Using the Col1a2 Promoter/Enhancer.

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.