Gene knockdown via electroporation of short hairpin RNAs in embryos of the marine hydroid Hydractinia symbiolongicarpus
Gene knockdown via electroporation of short hairpin RNAs in embryos of the marine hydroid Hydractinia symbiolongicarpus
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DOI：
10.1038/s41598-020-69489-8
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发表时间：
2020-07-30
影响因子：
4.6
通讯作者：
Schnitzler, Christine E.
中科院分区：
文献类型：
--
作者：
Quiroga-Artigas, Gonzalo;Duscher, Alexandrea;Schnitzler, Christine E.
在广泛的研究生物体中分析基因功能对于理解基因的生物功能及其进化至关重要。最近的研究表明，短发夹状RNA(ShRNAs)可以在两个蛇类物种中诱导基因特异性击倒。我们已经开发出一种详细、直接和可扩展的方法，通过电穿孔将shRNA输送到水生动物共生放线虫的受精卵中，产生有效的基因靶向敲除，可以持续整个胚胎发生。我们的电穿孔方案允许将shRNAs同时导入数百个受精的共生孢子虫卵，胚胎死亡最少，对发育中的动物没有长期有害影响。我们展示了RT-qPCR和详细的表型证据，证明我们的方法成功地诱导了一个外源基因(EGFP)和一个内源基因(Nanos2)的有效敲除，以及通过RT-qPCR对另外两个内源基因的敲除确认。我们还提供了通过电穿孔成功地将shRNA导入胚胎内的可视确认。我们的共生共生孢子虫胚胎shRNAs电穿孔的详细方案为水生动物群落提供了重要的实验资源，同时也为开发其他海洋无脊椎动物基因功能的类似方法提供了一个成功的模式。
Analyzing gene function in a broad range of research organisms is crucial for understanding the biological functions of genes and their evolution. Recent studies have shown that short hairpin RNAs (shRNAs) can induce gene-specific knockdowns in two cnidarian species. We have developed a detailed, straightforward, and scalable method to deliver shRNAs into fertilized eggs of the hydrozoan cnidarian Hydractinia symbiolongicarpus via electroporation, yielding effective gene-targeted knockdowns that can last throughout embryogenesis. Our electroporation protocol allows for the transfection of shRNAs into hundreds of fertilized H. symbiolongicarpus eggs simultaneously with minimal embryo death and no long-term harmful consequences on the developing animals. We show RT-qPCR and detailed phenotypic evidence of our method successfully inducing effective knockdowns of an exogenous gene (eGFP) and an endogenous gene (Nanos2), as well as knockdown confirmation by RT-qPCR of two other endogenous genes. We also provide visual confirmation of successful shRNA transfection inside embryos through electroporation. Our detailed protocol for electroporation of shRNAs in H. symbiolongicarpus embryos constitutes an important experimental resource for the hydrozoan community while also serving as a successful model for the development of similar methods for interrogating gene function in other marine invertebrates.