A versatile protocol for mRNA electroporation of Xenopus laevis embryos.
A versatile protocol for mRNA electroporation of Xenopus laevis embryos.
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DOI：
10.1101/pdb.prot067694
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发表时间：
2012-04-01
影响因子：
--
通讯作者：
Levin, Michael
中科院分区：
文献类型：
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作者：
Chernet, Brook T;Levin, Michael
非洲爪蟾是研究模式形成机制的理想模式系统。表达外源mRNA或将morpholinos引入卵裂期非洲爪蟾胚胎的能力已经允许了揭示发育和再生的分子遗传控制的功能获得和丧失实验。然而，将mRNA注射到卵裂期胚胎中提供了有限的时空控制：难以将靶向限制在小区域（例如，诱导表达灶），并且命运图不利于靶向某些组织，例如尾的那些组织。同样，早期注射可能导致不必要的发育缺陷，因为mRNA可以在所需时间点之前很久翻译。当研究原肠胚到尾芽阶段的发育和再生过程时，这些是特别重要的限制。虽然转基因技术允许在适当的启动子可用时精确控制基因的时空表达，但创建稳定的转基因动物的过程是耗时的。电穿孔提供了一种用于递送mRNA和其他核酸的替代方法，使得能够在任何发育阶段靶向单个细胞或细胞群。该方案描述了详细的电穿孔参数，用于将mRNA转染到原肠胚至尾芽阶段的胚胎中的广泛组织中，具有高效率和早在电穿孔后4小时的表达。
Xenopus laevis is an ideal model system for investigating the mechanisms of pattern formation. The ability to express exogenous mRNA or introduce morpholinos into cleavage-stage Xenopus embryos has allowed gain- and loss-of-function experiments that reveal molecular-genetic control of development and regeneration. However, injection of mRNAs into cleavage-stage embryos provides limited spatio-temporal control: It is difficult to limit targeting to small regions (e.g., inducing foci of expression) and the fate map does not facilitate targeting some tissues, such as those of the tail. Likewise, early injection can result in unwanted developmental defects because mRNA can be translated long before the desired time point. These are especially important limitations when studying developmental and regenerative processes during the gastrula to tailbud stages. Although transgenic techniques allow precise control over spatio-temporal expression of genes when the appropriate promoter is available, the process of creating stable transgenic animals is time-consuming. Electroporation provides an alternative method for delivering mRNA and other nucleic acids, enabling the targeting of single cells or groups of cells at any stage of development. This protocol describes detailed electroporation parameters for the transfection of mRNA into a wide range of tissues in embryos at gastrula to tailbud stages, with high efficiency and expression as early as 4 h post electroporation.