Spatially and temporally controlled electroporation of early chick embryos
Spatially and temporally controlled electroporation of early chick embryos
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DOI：
10.1038/nprot.2008.10
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发表时间：
2008-01-01
期刊：
影响因子：
14.8
通讯作者：
Stern, Claudio D.
中科院分区：
文献类型：
--
作者：
Voiculescu, Octavian;Papanayotou, Costis;Stern, Claudio D.
十年前卵内电穿孔的引入帮助鸡胚成为研究发育过程中基因调控和功能的强大系统。尽管对于 2 d 孵化的胚胎来说这是一个简单的程序，但早期阶段（从产蛋到早期神经形成，0-1 天）提出了特殊的挑战。在这里，我们描述了一种稳健且可重复的方案，用于将表达载体和吗啉代寡核苷酸电穿孔到胚胎的外胚层中，从产蛋后不久（第 XI 阶段）到第 6-7 阶段（早期神经形成），并具有精确的空间和时间控制。新技术可在3小时内电穿孔并培养约12个胚胎；电穿孔后可立即评估吗啉代的影响，电穿孔后 2-3 小时可观察到质粒 DNA 的强烈过度表达。这些技术可用于延时成像、功能获得和丧失实验以及研究活胚胎中的基因调控元件。
The introduction of in ovo electroporation a decade ago has helped the chick embryo to become a powerful system to study gene regulation and function during development. Although this is a simple procedure for embryos of 2-d incubation, earlier stages (from laying to early neurulation, 0-1 d) present special challenges. Here we describe a robust and reproducible protocol for electroporation of expression vectors and morpholino oligonucleotides into the epiblast of embryos from soon after laying (stage XI) to stages 6-7 (early neurulation), with precise spatial and temporal control. Within 3 h, about 12 embryos can be electroporated and set up for culture by the New technique; the effects of morpholinos can be assessed immediately after electroporation, and robust overexpression from plasmid DNA is seen 2-3 h after electroporation. These techniques can be used for time-lapse imaging, gain- and loss-of-function experiments and studying gene regulatory elements in living embryos.