Stable integration and conditional expression of electroporated transgenes in chicken embryos
Stable integration and conditional expression of electroporated transgenes in chicken embryos
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DOI：
10.1016/j.ydbio.2007.01.043
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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：
Sato, Yuki;Kasai, Toshiharu;Takahashi, Yoshiko
鸡胚的卵内电穿孔作为研究基因在胚胎发生过程中的作用的有力工具而被广泛使用。然而，传统的电穿孔技术不能保留转基因的表达超过几天，因为转基因没有整合到基因组中。为了克服这一缺点，我们开发了转座子介导的基因转移，这是一种新的鸡操作技术。先前有报道称，最初在medaka鱼中发现的转座子To12，当与To12转座酶共同作用时，促进转基因整合到基因组中。在这项研究中，我们将含有To12构建体中克隆的CAGGS-EGFP盒的质粒与转座酶编码质粒共电穿孔到鸡胚胎的早期中胚层或视神经囊泡中。这导致EGFP在somite衍生组织和发育中的视网膜中分别持续表达至少到胚胎第8天（E8）和E12。利用鸡培养细胞的基因组Southern印迹法证实了该基因的整合。我们进一步将这种转座子介导的基因转移与我们最近开发的四环素依赖的条件表达系统结合起来。通过这种组合方法，可以在相对较晚的阶段，如E6，在各种器官发生正在进行的阶段，四环素给药可以实验性地诱导稳定整合的转基因的表达。因此，本研究提出的技术为研究晚期器官发生机制提供了一种新的途径，而鸡是最适合的模型动物。(c) 2007爱思唯尔公司版权所有。
The in ovo electroporation in chicken embryos has widely been used as a powerful tool to study roles of genes during embryogenesis. However, the conventional electroporation technique fails to retain the expression of transgenes for more than several days because transgenes are not integrated into the genome. To overcome this shortcoming, we have developed a transposon-mediated gene transfer, a novel technique in chicken manipulations. It was previously reported that the transposon To12, originally found in medaka fish, facilitates an integration of a transgene into the genome when co-acting with To12 transposase. In this study, we co-electroporated a plasmid containing a CAGGS-EGFP cassette cloned in the To12 construct along with a transposase-encoding plasmid into early presomitic mesoderm or optic vesicles of chicken embryos. This resulted in persistent expression of EGFP at least until embryonic day 8 (E8) and E12 in somite-derived tissues and developing retina, respectively. The integration of the transgene was confirmed by genomic Southern blotting using chicken cultured cells. We further combined this transposon-mediated gene transfer with the tetracycline-dependent conditional expression system that we also developed recently. With this combined method, expression of a stably integrated transgene could be experimentally induced upon tetracycline administration at relatively late stages such as E6, where a variety of organogenesis are underway. Thus, the techniques proposed in this study provide a novel approach to study the mechanisms of late organogenesis, for which chickens are most suitable model animals. (c) 2007 Elsevier Inc. All rights reserved.