Electroporation of cDNA/Morpholinos to targeted areas of embryonic CNS in Xenopus.
Electroporation of cDNA/Morpholinos to targeted areas of embryonic CNS in Xenopus.
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DOI：
10.1186/1471-213x-7-107
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发表时间：
2007-09-27
影响因子：
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通讯作者：
Holt CE
中科院分区：
文献类型：
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作者：
Falk J;Drinjakovic J;Leung KM;Dwivedy A;Regan AG;Piper M;Holt CE
卵裂球注射mRNA或反义寡核苷酸已被证明是分析爪蟾早期基因功能的有效方法。然而，通过这种方法对参与神经元分化和轴突寻路的基因的功能分析常常受到这些基因在发育过程中早期功能的阻碍。因此，需要对过表达或敲除方法进行精细的时空控制，以具体解决给定基因在这些过程中的作用。我们在这里描述了一种电穿孔程序，可以高效、低毒性地在轴突束首次形成的关键发育时间窗口（受精后22-50小时）将DNA和反义morpholino寡核苷酸（MOs）靶向到爪蟾中枢神经系统的空间限制区域。该方法依赖于“电穿孔室”的设计，该设计能够重复定位固定间距的电极，并结合精确的DNA/MO注射。可以对电穿孔腔进行简单的调整，以适应不同年龄胚胎的形状，并改变目标区域的大小和位置。该方法可用于在同一胚胎中电穿孔中枢神经系统的不同区域，从而可以单独操作正在生长的轴突及其在大脑中的中间和最终目标。我们的研究表明，电穿孔可以作为一种多功能的工具来研究爪蟾胚胎发生过程中参与轴突延伸的分子途径。电穿孔使功能研究的增益或损失可以很容易地监测电穿孔细胞。双靶向转染提供了一个独特的机会来监测体内轴突-靶标相互作用。最后，电穿孔胚胎为体外分析提供了mo负载或DNA转染细胞的宝贵来源。这项技术具有广泛的应用，因为它可以通过对电穿孔室进行简单的调整，很容易地适应其他发育中的器官系统和其他生物体。
Blastomere injection of mRNA or antisense oligonucleotides has proven effective in analyzing early gene function in Xenopus. However, functional analysis of genes involved in neuronal differentiation and axon pathfinding by this method is often hampered by earlier function of these genes during development. Therefore, fine spatio-temporal control of over-expression or knock-down approaches is required to specifically address the role of a given gene in these processes. We describe here an electroporation procedure that can be used with high efficiency and low toxicity for targeting DNA and antisense morpholino oligonucleotides (MOs) into spatially restricted regions of the Xenopus CNS at a critical time-window of development (22–50 hour post-fertilization) when axonal tracts are first forming. The approach relies on the design of "electroporation chambers" that enable reproducible positioning of fixed-spaced electrodes coupled with accurate DNA/MO injection. Simple adjustments can be made to the electroporation chamber to suit the shape of different aged embryos and to alter the size and location of the targeted region. This procedure can be used to electroporate separate regions of the CNS in the same embryo allowing separate manipulation of growing axons and their intermediate and final targets in the brain. Our study demonstrates that electroporation can be used as a versatile tool to investigate molecular pathways involved in axon extension during Xenopus embryogenesis. Electroporation enables gain or loss of function studies to be performed with easy monitoring of electroporated cells. Double-targeted transfection provides a unique opportunity to monitor axon-target interaction in vivo. Finally, electroporated embryos represent a valuable source of MO-loaded or DNA transfected cells for in vitro analysis. The technique has broad applications as it can be tailored easily to other developing organ systems and to other organisms by making simple adjustments to the electroporation chamber.