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Protocol to generate endothelial cell-specific knockout mouse models using Cas9/Cdh5-Cre mice coupled with sgRNA
Dong-Mei Wang1, Chinnaswamy Tiruppathi1
1Department of Pharmacology and Regenerative Medicine and the Center for Lung and Vascular Biology, University of Illinois College of Medicine, Chicago, IL 60612, USA.
STAR Protocols
|May 9, 2026
Summary
This study details a protocol for creating endothelial cell-specific gene knockout (KO) mouse models. This method enables in vivo research into vascular homeostasis and fluid balance using Cas9/Cdh5-Cre mice.
Area of Science:
- Vascular Biology
- Genetics
- Animal Models
Background:
- The vascular endothelium regulates vascular homeostasis and tissue fluid balance.
- Mouse models are crucial for in vivo studies of endothelial cell (EC) function.
- Generating EC-specific gene knockout (KO) models is essential for detailed research.
Purpose of the Study:
- To present a detailed protocol for generating adult EC-specific gene KO mouse models.
- To outline the methodology for creating Cas9-active and Cdh5-Cre-positive (Cas9/Cdh5-Cre) mouse lines.
- To provide a comprehensive guide for researchers studying ECs in vivo.
Main Methods:
- Generation of Cas9/Cdh5-Cre mouse lines.
- Single guide RNA (sgRNA) design and vector construction.
- Liposome/plasmid complex preparation and injection.
- Lung tissue harvest, homogenization, and protein analysis via Western blotting.
Main Results:
- Successful generation of a protocol for EC-specific gene KO mouse models.
- Detailed steps for CRISPR-Cas9 based gene editing in ECs.
- Verification of gene knockout through protein quantification and Western blotting.
Conclusions:
- The presented protocol enables the creation of sophisticated EC-specific KO mouse models.
- This methodology facilitates in vivo investigation of endothelial cell functions.
- The protocol serves as a valuable resource for vascular biology research.
