Mathematics-based Creation of Science Program (MACS), Graduate School of Science, Kyoto University, Kitashirakawa, Sakyo-ku, Kyoto, 606-8502, Japan; Department of Zoology, Graduate School of Science, Kyoto University, Kitashirakawa, Sakyo-ku, Kyoto, 606-8502, Japan.
Department of Zoology, Graduate School of Science, Kyoto University, Kitashirakawa, Sakyo-ku, Kyoto, 606-8502, Japan; AMED Core Research for Evolutional Science and Technology (AMED-CREST), Japan Agency for Medical Research and Development (AMED), Chiyoda-ku, Tokyo, 100-0004, Japan.
Dev Biol. 2019 Dec 1;456(1):8-16. doi: 10.1016/j.ydbio.2019.08.001. Epub 2019 Aug 7.
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.
我们描述了一种方法，可以在时空可控的方式下，独立于心脏对早期发育中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，当与 GFP DNA 共同注射在脂质浓度的临界范围内时，已被发现具有很高的转染效率。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的部位：在血液循环开始之前或之后，将脂质-DNA 混合物注射到心脏中，分别导致有限且广泛传播的转染模式。此外，将混合物注射到右背主动脉中会导致胚胎脉管系统的右半部分发生转基因。此外，这种方法与 siRNA 技术相结合，首次允许敲低内源性 VE-钙粘蛋白（也称为 Cdh5）的表达，Cdh5 已被证明参与了新生血管的组装：当 Cah5 siRNA 被注射到右背主动脉中时，会观察到右半部分的血管明显缺陷，而心脏没有缺陷。虽然之前已经报道了使用脂质试剂的基于输注的基因转染方法，但 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的缺点，如小鼠和斑马鱼，其中全内皮增强子驱动的转基因通常会导致心脏畸形，进而由于血流缺陷而间接影响外周血管。由于越来越多的血管亚型得到了认可，本研究中描述的时空可控基因操作提供了一种强大的工具，可以了解血管在分子水平上是如何建立的。
