Expressing exogenous genes in newts by transgenesis
Expressing exogenous genes in newts by transgenesis
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DOI：
10.1038/nprot.2011.334
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发表时间：
2011-05-01
期刊：
影响因子：
14.8
通讯作者：
Chiba, Chikafumi
中科院分区：
文献类型：
--
作者：
Casco-Robles, Martin Miguel;Yamada, Shouta;Chiba, Chikafumi
蝾螈巨大的再生能力为分子水平的研究提供了动力。然而，有效的基因调控方法在历史上一直相当有限。在这里，我们描述了一种在蝾螈中转基因表达外源基因的方案。这种方法很简单：将I-SceI巨核酶与携带酶识别位点两侧的转基因盒的质粒DNA的反应混合物直接注射到受精卵中。该方案实现了高效率的转基因，可与其他动物系统中使用的方案相媲美，并提供了实际数量的转基因蝾螈（类似于20%的注射胚胎），这些蝾螈可以在变态后存活，并可应用于再生研究。获得转基因成年蝾螈的整个过程需要45个月。
The great regenerative abilities of newts provide the impetus for studies at the molecular level. However, efficient methods for gene regulation have historically been quite limited. Here we describe a protocol for transgenically expressing exogenous genes in the newt Cynops pyrrhogaster. This method is simple: a reaction mixture of I-SceI meganuclease and a plasmid DNA carrying a transgene cassette flanked by the enzyme recognition sites is directly injected into fertilized eggs. The protocol achieves a high efficiency of transgenesis, comparable to protocols used in other animal systems, and it provides a practical number of transgenic newts (similar to 20% of injected embryos) that survive beyond metamorphosis and that can be applied to regenerative studies. The entire protocol for obtaining transgenic adult newts takes 45 months.