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

Abstract

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.

Spatial Transcriptomics to define transcriptional patterns of zonation and structural components in the liver

“…Cluster 3 shows enrichment for genes associated with hemoglobin, whereas cluster 4 shows enriched expression of genes involved in immune-related processes 23,24 . Cluster 5 displays enrichment for mesenchymal genes [25][26][27] (Figure 1c, Supplementary table 1).…”

Insights into Fibroblast Plasticity

“…For lineage tracing, mice hemizygous for a tamoxifendependent Cre recombinase under the control of a Col1a2 18 or Sox2 (Institut Clinique de la Souris, Alsace, France) promoter [Col1a2-Cre(ER)-T; Sox2-Cre(ER)-T] were bred with mice homozygous for a double-fluorescent reporter transgene (mTmG) integrated into the Gt(ROSA) 26Sor locus (Jackson Laboratories, Bar Harbor, ME), thereby generating Col1a2-Cre(ER)T; Rosa26mTmG or Sox2-Cre(ER)T; Rosa26mTmG mice. Please note Col1a2-Cre(ER)-T mice contain a fibroblast-specific far upstream enhancer, initially identified in the laboratory of Benoit de Crombrugghe, subcloned upstream of the Col1a2 minimal promoter; previous publications have extensively shown that this construct permits transgene expression only in fibroblasts and not in other collagen-expressing cells, such as bone and cartilage.…”

“…SKP spheroids were cultured in SKP base media: three parts DMEM glutaMAX and one part Hams F12 (Invitrogen); supplemented with 1% antibiotic-antimycotic, 2% B27 (Invitrogen; A35828-01), 1% N2 (Invitrogen; 17502048), epidermal growth factor (20 ng/mL; Invitrogen; PHG0311), and basic fibroblast growth factor (20 ng/mL; Invitrogen; PHG0026). 18,19 Alternatively, to generate larger quantities of SKPs, similar to a previously described report, 20 confluent 10-cm plates of fibroblasts were removed with 0.25% trypsin (Invitrogen) and resuspended in SKP base media, as described above. Cells were then seeded into 3 wells of a 6well plate per 10-cm plate of fibroblasts.…”

“…15 In human melanoma patients, CCN2 expression does not correlate with BRAF mutational status, but instead correlates with expression of stroma and angiogenic gene subsets, and negatively correlates with survival. 16,17 In mice, conditional knockout strategies, using a tamoxifen-dependent Cre recombinase expressed under the control of fibroblast-specific Col1a2 promoter/ enhancer, 18 have shown that loss of CCN2 expression by fibroblasts results in resistance to melanoma metastasis and tumor-induced neovascularization, concomitant with a reduced production of the proangiogenic mediators periostin and vascular endothelial growth factor. 16,17 Accordingly, CCN2 represents a novel therapeutic target for BRAF inhibitoreresistant melanoma.…”

“…In this report, conditional knockout and lineage tracing strategies using a tamoxifen-dependent Cre recombinase expressed under the control of a fibroblast-specific promoter/enhancer derived from the Col1a2 gene 18 were used to investigate how dermal fibroblasts contribute, in a CCN2-dependent manner, to the appearance of progenitorlike cells and CAFs in tumor stroma. Skin-derived precursor (SKP) spheroids were further used as an in vitro model of fibroblast plasticity to demonstrate the mechanistic role of CCN2 in myofibroblast differentiation of progenitor cells.…”