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Blood flow-mediated gene transfer and siRNA-knockdown in the developing vasculature in a spatio-temporally controlled manner in chicken embryos

Takase, Yuta, Takahashi, Yoshiko
Developmental biology 2019
DNA, Danio rerio, abnormal development, aorta, blood, blood flow, chick embryos, genes, heart, lipid content, lipids, mice, small interfering RNA, transfection, transgenesis
We describe a method by which early developing vasculature can be gene-manipulated independently of the heart in a spatio-temporally controlled manner. Lipofectamine 2000 or 3000, an easy-to-use lipid reagent, has been found to yield a high efficiency of transfection when co-injected with GFP DNA within a critical range of lipid concentration. By exploiting developmentally changing patterns of vasculature and blood flow, we have succeed in controlling the site of transfection: injection with a lipid-DNA cocktail into the heart before or after the blood circulation starts results in a limited and widely spread patterns of transfection, respectively. Furthermore, a cocktail injection into the right dorsal aorta leads to transgenesis of the right half of embryonic vasculature. In addition, this method combined with the siRNA technique has allowed, for the first time, to knockdown the endogenous expression of VE-cadherin (also called Cdh5), which has been implicated in assembly of nasant blood vessels: when Cah5 siRNA is injected into the right dorsal aorta, pronounced defects in the right half of vasculature are observed without heart defects. Whereas infusion-mediated gene transfection method has previously been reported using lipid reagents that were elaborately prepared on their own, Lipofectamine is an easy-use reagent with no requirement of special expertise. The methods reported here would overcome shortcomings of conventional vascular-transgenic animals, such as mice and zebrafish, in which pan-endothelial enhancer-driven transgenesis often leads to the heart malformation, which, in turn, indirectly affects peripheral vasculature due to flow defects. Since a variety of subtypes in vasculature have increasingly been appreciated, the spatio-temporally controllable gene manipulation described in this study offers a powerful tool to understand how the vasculature is established at the molecular level.