TGF-β-mediated Endothelial to Mesenchymal Transition (EndMT) and the Functional Assessment of EndMT Effectors using CRISPR/Cas9 Gene Editing

In response to specific external cues and the activation of certain transcription factors, endothelial cells can differentiate into a mesenchymal-like phenotype, process that is termed mesenchymal transition (EndMT). Emerging results have suggested EndMT causally linked multiple human diseases, such as fibrosis cancer. addition, endothelial-derived may be applied in tissue regeneration procedures, they further differentiated various cell types (e.g., osteoblasts chondrocytes). Thus, selective manipulation clinical potential. Like epithelial-mesenchymal (EMT), strongly induced by secreted cytokine transforming growth factor-beta (TGF-β), which stimulates expression so-called factors (EndMT-TFs), including Snail Slug. These EndMT-TFs then up- downregulate levels proteins, respectively. Here, we describe methods investigate TGF-β-induced vitro, protocol study role particular TFs EndMT. Using these techniques, provide evidence TGF-β2 murine pancreatic microvascular (MS-1 cells), genetic depletion using clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9)-mediated gene editing, abrogates this phenomenon. This approach serve model interrogate potential modulators biology, used perform or pharmacological screens order identify novel regulators EndMT, with application disease.