Genetic manipulation of betta fish.
Genetic manipulation of betta fish.
复制标题
斗鱼的基因操作。
DOI：
10.1101/2023.02.16.528733
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发表时间：
2023
期刊：
影响因子：
--
通讯作者：
Bendesky,Andres
中科院分区：
文献类型：
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作者：
Palmiotti,Alec;Lichak,MadisonR;Shih,Pei-Yin;Bendesky,Andres
芨芨草斗鱼（Betta splendens），也被称为暹罗斗鱼或“斗鱼”，是一种淡水鱼，以其惊人的形态多样性和极端的攻击性行为而闻名。尽管最近我们对斗鱼遗传学和神经生物学的理解取得了进展，但缺乏操纵其基因组的工具阻碍了功能和机械水平的进展。在这项研究中，我们概述了三种基因操作技术的使用，我们已针对斗鱼进行了优化：CRISPR/Cas9 介导的基因敲除、CRISPR/Cas9 介导的基因敲入和 Tol2 介导的转基因。我们敲除了三个基因：alkal2l、bco1l和mitfa，并分析了它们对活力和色素沉着的影响。此外，我们还在 themitfalocus 中敲入了一种荧光蛋白，这是斗鱼中这项强大技术的原理验证实验。最后，我们使用 Tol2 介导的转基因技术培育出普遍表达 GFP 的鱼，然后开发了一种具有心脏特异性表达红色荧光蛋白的双顺反子质粒，作为成功转基因的可见标记。我们的工作凸显了斗鱼基因操纵的潜力，为在该动物模型中有效利用遗传工具提供了宝贵的资源。
Betta splendens,also known as Siamese fighting fish or “betta,” is a freshwater fish species renowned for its astonishing morphological diversity and extreme aggressive behavior. Despite recent advances in our understanding of the genetics and neurobiology of betta, the lack of tools to manipulate their genome has hindered progress at functional and mechanistic levels. In this study, we outline the use of three genetic manipulation technologies, which we have optimized for use in betta: CRISPR/Cas9-mediated knockout, CRISPR/Cas9-mediated knockin, and Tol2-mediated transgenesis. We knocked out three genes:alkal2l, bco1l,andmitfa,and analyzed their effects on viability and pigmentation. Furthermore, we knocked in a fluorescent protein into themitfalocus, a proof-of-principle experiment of this powerful technology in betta. Finally, we used Tol2-mediated transgenesis to create fish with ubiquitous expression of GFP, and then developed a bicistronic plasmid with heart-specific expression of a red fluorescent protein to serve as a visible marker of successful transgenesis. Our work highlights the potential for the genetic manipulation of betta, providing valuable resources for the effective use of genetic tools in this animal model.