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Develop a protocol for generating endothelial cell-specific gene knockout mouse models using Cas9 technology.

May 10, 2026Open Access

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

Key Points

Develop a protocol for generating endothelial cell-specific gene knockout mouse models using Cas9 technology.
Generate Cas9/Cdh5-Cre mouse models for targeted gene knockout.
Design sgRNA for plasmid vector construction and prepare liposome complexes for injection.
Perform lung tissue harvest, homogenization, and protein verification through western blotting.
Successfully created endothelial-specific knockout models verified through western blot analysis of target proteins.
Demonstrated effective gene knockout in adult mice using the described methodology.

Abstract

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

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Wang et al. (Fri,) studied this question.

synapsesocial.com/papers/6a001ff2c8f74e3340f9b2f4 https://doi.org/https://doi.org/10.1016/j.xpro.2026.104551

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