Protocol to generate endothelial cell-specific knockout mouse models using Cas9/Cdh5-Cre mice coupled with sgRNA

The vascular endothelium is a critical regulator of vascular homeostasis and tissue fluid balance, and mouse models are essential for studying these processes in vivo. Here, we present a protocol to generate adult endothelial cell (EC)-specific gene knockout (KO) mouse models. We describe steps for Cas9-active and Cdh5-Cre-positive (Cas9/Cdh5-Cre) mouse line generation, single guide RNA (sgRNA) design for vector construction, plasmid DNA generation, and liposome preparation. We then detail procedures for liposome/plasmid complex injection, lung harvest, homogenization, protein quantification, and verification with western blotting. For complete details on the use and execution of this protocol, please refer to Wang et al.¹.