Blood flow-mediated gene transfer and siRNA-knockdown in the developing vasculature in a spatio-temporally controlled manner in chicken embryos
Blood flow-mediated gene transfer and siRNA-knockdown in the developing vasculature in a spatio-temporally controlled manner in chicken embryos
复制标题
鸡胚胎中发育中的脉管系统中血流介导的基因转移和 siRNA 敲低以时空控制的方式
DOI：
10.1016/j.ydbio.2019.08.001
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发表时间：
2019
影响因子：
2.7
通讯作者：
Takahashi Yoshiko
中科院分区：
文献类型：
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作者：
Takase Yuta;Takahashi Yoshiko
我们描述了一种方法，通过该方法可以以时空控制的方式独立于心脏对早期发育的脉管系统进行基因操作。 Lipofectamine 2000 或 3000 是一种易于使用的脂质试剂，被发现在脂质浓度的临界范围内与 GFPDNA 共注射时可产生高转染效率。通过利用脉管系统和血流的发育变化模式，我们成功地控制了转染位点：在血液循环开始之前或之后向心脏注射脂质-DNA 混合物，分别导致有限和广泛传播的转染模式。此外，将鸡尾酒注射到右背主动脉会导致胚胎脉管系统右半部分的转基因。此外，这种方法与 siRNA 技术相结合，首次能够敲低 VE-钙粘蛋白（也称为 Cdh5）的内源性表达，该蛋白与新生血管的组装有关：当 Cah5siRNA 注射到右背主动脉时，可以观察到右半脉管系统的明显缺陷，但没有心脏缺陷。之前报道的输注介导的基因转染方法使用的是自己精心制备的脂质试剂，而 Lipofectamine 是一种易于使用的试剂，不需要特殊的专业知识。这里报道的方法将克服传统血管转基因动物（例如小鼠和斑马鱼）的缺点，其中泛内皮增强子驱动的转基因常常导致心脏畸形，进而由于流动缺陷而间接影响外周脉管系统。由于脉管系统中的各种亚型越来越受到重视，本研究中描述的时空可控基因操作为理解脉管系统如何在分子水平上建立提供了强大的工具。
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 withGFPDNA 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 ofVE-cadherin(also calledCdh5), which has been implicated in assembly of nasant blood vessels: whenCah5siRNA 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.