Authors
Alexander Lohman, Adam Straub, and Brant Isakson
Summary
The vascular endothelium plays an essential role in regulating blood vessel tone, blood flow and blood pressure. Current vascular model systems for examination of endothelial cell biology and blood vessel physiology and pathology rely on cell culture and the generation of genetically modified animals. While these systems are advantageous for studying the endothelium, many cell culture models omit the contribution of other cells types present in the native blood vessel wall and the generation of genetically modified animals can be costly and time consuming. The following protocol outlines a novel ex vivo endothelial cell transfection for the knockdown of endogenously expressed endothelial cell proteins in intact isolated arteries. Briefly, arteries are isolated from the mouse and the lumen is perfused with siRNA and Nucleofector™ transfection reagent. The artery is then ligated at both ends and briefly electroporated to introduce the siRNA specifically into the endothelial cells, followed by perfusion of the lumen to flush excess siRNA. Transfected arteries are then cultured 18-24 hours for knockdown of targeted proteins. This system provides the utility of selectively knocking down a protein(s) of interest specifically from the vascular endothelium, providing the advantages of a genetically modified animal without the long time frame required for generation of tissue specific knockout animals. This protocol requires approximately 1 hour from isolation of the artery from the mouse to culturing of the transfected vessel.Further details
The protocol was published on Protocol Exchange on 6 November 2012. To see the entire protocol, click on the source link.Advertisement
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