Tet-on inducible system combined with in ovo electroporation dissects multiple roles of genes in somitogenesis of chicken embryos
Tet-on inducible system combined with in ovo electroporation dissects multiple roles of genes in somitogenesis of chicken embryos
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DOI：
10.1016/j.ydbio.2007.01.042
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发表时间：
2007-05-15
影响因子：
2.7
通讯作者：
Takahashi, Yoshiko
中科院分区：
文献类型：
--
作者：
Watanabe, Tadayoshi;Saito, Daisuke;Takahashi, Yoshiko
鸡胚胎的卵内电穿孔技术使研究人员能够揭示许多发育基因的功能。在这种技术中，普遍存在的启动子CAGGS （CMV碱基）经常用于过表达实验。然而，如果一个给定的基因在发育的多个步骤中发挥作用，如果该基因的过度表达在电穿孔时导致致命的后果，那么它在发育的后期步骤中的作用就会被忽视。因此，需要制定一种技术，该技术可以用特定阶段的方式控制电穿孔DNA的表达。在这里，我们首次证明了四环素控制的表达方法，“tet-on”和“tet-off”，有效地调节了电穿孔鸡胚胎的基因表达。我们证明了电穿孔DNA表达的开始或终止可以通过定时给药四环素进入鸡蛋来精确控制。此外，通过这项技术，我们已经揭示了RhoA、cMeso-1和Pax2在早期体细胞发生中的未知作用。特别是，cMeso-1似乎通过调节Pax2和NCAM的表达参与了新形成的体的细胞凝聚。因此，本研究提出的新的鸡分子技术为研究发育基因的阶段特异性作用提供了一个有用的工具。(c) 2007爱思唯尔公司版权所有。
The in ovo electroporation technique in chicken embryos has enabled investigators to uncover the functions of numerous developmental genes. In this technique, the ubiquitous promoter, CAGGS (CMV base), has often been used for overexpression experiments. However, if a given gene plays a role in multiple steps of development and if overexpression of this gene causes fatal consequences at the time of electroporation, its roles in later steps of development would be overlooked. Thus, a technique with which expression of an electroporated DNA can be controlled in a stage-specific manner needs to be formulated. Here we show for the first time that the tetracycline-controlled expression method, "tet-on" and "tet-off', works efficiently to regulate gene expression in electroporated chicken embryos. We demonstrate that the onset or termination of expression of an electroporated DNA can be precisely controlled by timing the administration of tetracycline into an egg. Furthermore, with this technique we have revealed previously unknown roles of RhoA, cMeso-1 and Pax2 in early somitogenesis. In particular, cMeso-1 appears to be involved in cell condensation of a newly forming somite by regulating Pax2 and NCAM expression. Thus, the novel molecular technique in chickens proposed in this study provides a useful tool to investigate stage-specific roles of developmental genes. (c) 2007 Elsevier Inc. All rights reserved.