In ovo electroporation of chicken limb bud ectoderm
In ovo electroporation of chicken limb bud ectoderm
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鸡肢芽外胚层卵内电穿孔
DOI：
10.1002/dvdy.352
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发表时间：
2021
影响因子：
2.5
通讯作者：
Atsuta Yuji
中科院分区：
文献类型：
--
作者：
Tomizawa Reiko R.;Tabin Clifford J.;Atsuta Yuji
了解外胚层组织是如何形成的，以及它们是如何保持其完整性的，对于理解表皮的发育和发病机制至关重要。然而，缺乏简单快速的基因操作技术限制了遗传学研究阐明这些事件背后的机制。结果我们描述了一种简单的鸡肢芽外胚层电穿孔方法，可以在外胚层发育和伤口愈合过程中进行基因操作。利用小的副膜孔对DNA质粒的局部限制和肢体外胚层产生于特定位置的事实，我们通过卵电穿孔瞄准肢体外胚层形成区域。该方法可实现肢体外胚层细胞的局灶性和高效的转基因。此外，使用先前描述的msx2启动子，基因操作可以专门针对顶端外胚层脊（AER），一个调节肢体发育的信号中心。利用电穿孔技术将荧光标记物传递到胚胎肢体外胚层，我们展示了其在伤口愈合过程中进行延时成像的应用。该分析揭示了以前未被认识到的肌动蛋白细胞骨架的动态重塑和伤口边缘的板足形成。我们发现板足的形成需要Rac1 GTPase的活性，这表明它对伤口愈合是必要的。结论本方法简便易行。因此，它将允许对肢体外胚层发育和伤口愈合过程中的基因功能进行高通量测试。
BackgroundDeciphering how ectodermal tissues form, and how they maintain their integrity, is crucial for understanding epidermal development and pathogenesis. However, lack of simple and rapid gene manipulation techniques limits genetic studies to elucidate mechanisms underlying these events.ResultsHere we describe an easy method for electroporation of chick limb bud ectoderm enabling gene manipulation during ectoderm development and wound healing. Taking advantage of a small parafilm well that constrains DNA plasmids locally and the fact that the limb ectoderm arises from a defined site, we target the limb ectoderm forming region byin ovoelectroporation. This approach results in focal and efficient transgenesis of the limb ectodermal cells. Further, using a previously describedMsx2promoter, gene manipulation can be specifically targeted to the apical ectodermal ridge (AER), a signaling center regulating limb development. Using the electroporation technique to deliver a fluorescent marker into the embryonic limb ectoderm, we show its utility in performing time‐lapse imaging during wound healing. This analysis revealed previously unrecognized dynamic remodeling of the actin cytoskeleton and lamellipodia formation at the edges of the wound. We find that the lamellipodia formation requires activity of Rac1 GTPase, suggesting its necessity for wound closure.ConclusionOur method is simple and easy. Thus, it would permit high throughput tests for gene function during limb ectodermal development and wound healing.
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影响因子：
64.8
作者：
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通讯作者：
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影响因子：
9.2
作者：
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通讯作者：
Haase, Ingo
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作者：
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通讯作者：
Robin A. Kimmel;D. Turnbull;V. Blanquet;W. Wurst;C. Loomis;A. Joyner