Tol2-mediated gene transfer and in ovo electroporation of the otic placode: a powerful and versatile approach for investigating embryonic development and regeneration of the chicken inner ear.
Tol2-mediated gene transfer and in ovo electroporation of the otic placode: a powerful and versatile approach for investigating embryonic development and regeneration of the chicken inner ear.
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DOI：
10.1007/978-1-61779-980-8_10
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发表时间：
2012-01-01
期刊：
影响因子：
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通讯作者：
Daudet, Nicolas
中科院分区：
文献类型：
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作者：
Freeman, Stephen;Chrysostomou, Elena;Daudet, Nicolas
脊椎动物的内耳由几个特化的上皮细胞组成，这些上皮细胞含有对声音和头部运动敏感的机械感觉“毛”细胞。在哺乳动物中，例如在衰老期间或在噪声创伤之后毛细胞的损失是不可逆的，并且导致永久性感觉缺陷。相比之下，鸟类，鱼类和两栖动物可以有效地再生创伤后失去的毛细胞。鸡内耳是研究内耳发育和再生的细胞和分子机制的经典模型系统，但直到最近，它一直受到相对缺乏灵活的遗传研究工具的影响。随着卵内电穿孔和Tol2转座子载体在禽类细胞中的基因转移的引入，该模型的实验可能性领域现已显着扩大。在这里，我们提供了一个一般的协议，在卵电穿孔的鸡耳基板，并说明这种方法，结合Tol2载体，可用于驱动长期和诱导基因表达的胚胎鸡内耳。这种方法将是特别有用的，以调查候选基因的功能，调节祖细胞的行为和感觉细胞分化的内耳。
The vertebrate inner ear is composed of several specialized epithelia containing mechanosensory "hair" cells, sensitive to sound and head movements. In mammals, the loss of hair cells for example during aging or after noise trauma is irreversible and results in permanent sensory deficits. By contrast, avian, fish, and amphibians can efficiently regenerate lost hair cells following trauma. The chicken inner ear is a classic model system to investigate the cellular and molecular mechanisms of inner ear development and regeneration, yet it suffered until recently from a relative lack of flexible tools for genetic studies. With the introduction of in ovo electroporation and of Tol2 transposon vectors for gene transfer in avian cells, the field of experimental possibilities has now expanded significantly in this model. Here we provide a general protocol for in ovo electroporation of the chicken otic placode and illustrate how this approach, combined with Tol2 vectors, can be used to drive long-term and inducible gene expression in the embryonic chicken inner ear. This method will be particularly useful to investigate the function of candidate genes regulating progenitor cell behavior and sensory cell differentiation in the inner ear.