Generation of transgenic Hydra by embryo microinjection.
Generation of transgenic Hydra by embryo microinjection.
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DOI：
10.3791/51888
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发表时间：
2014-09-11
期刊：
影响因子：
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通讯作者：
Steele, Robert E
中科院分区：
文献类型：
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作者：
Juliano, Celina E;Lin, Haifan;Steele, Robert E
作为刺胞动物门的一员，所有两侧对称动物的姐妹群，水螅可以阐明多细胞动物之间共享的基本生物过程。水螅被用作再生，图案形成和干细胞研究的模型。然而，由于缺乏一种可靠的基因扰动方法来研究分子功能，研究工作受到了阻碍。转基因方法的发展使水螅生物学的研究重新焕发了活力（1）。转基因Hydra允许活细胞的跟踪，分选以产生用于生化分析的纯细胞群体，通过敲低和过表达操纵基因功能，以及分析启动子功能。注入早期胚胎的质粒DNA在发育早期随机整合到基因组中。这导致孵化的幼龟在三个谱系（外胚层上皮、内胚层上皮或间质）中的一个或多个谱系的组织中表达转基因。获得具有转基因组织的幼雏的成功率在10%至20%之间。转基因幼雏的无性繁殖用于在特定谱系中建立均匀的转基因品系。产生转基因水螅是令人惊讶的简单和强大的，在这里，我们描述了一个协议，可以很容易地实现低成本。
As a member of the phylum Cnidaria, the sister group to all bilaterians, Hydra can shed light on fundamental biological processes shared among multicellular animals. Hydra is used as a model for the study of regeneration, pattern formation, and stem cells. However, research efforts have been hampered by lack of a reliable method for gene perturbations to study molecular function. The development of transgenic methods has revitalized the study of Hydra biology(1). Transgenic Hydra allow for the tracking of live cells, sorting to yield pure cell populations for biochemical analysis, manipulation of gene function by knockdown and over-expression, and analysis of promoter function. Plasmid DNA injected into early stage embryos randomly integrates into the genome early in development. This results in hatchlings that express transgenes in patches of tissue in one or more of the three lineages (ectodermal epithelial, endodermal epithelial, or interstitial). The success rate of obtaining a hatchling with transgenic tissue is between 10% and 20%. Asexual propagation of the transgenic hatchling is used to establish a uniformly transgenic line in a particular lineage. Generating transgenic Hydra is surprisingly simple and robust, and here we describe a protocol that can be easily implemented at low cost.