培育出第一条标志性珊瑚礁鱼类 Amphiprion ocellaris

来源：好游戏攻略时间：2025-04-02 10:02

IF 2.6 3区生物学 Q3 BIOTECHNOLOGY & APPLIED MICROBIOLOGY

Gabriel J. Graham, Emma M. Ibanez, Laurie J. Mitchell, Karen E. Weis, Lori T. Raetzman, Fabio Cortesi, Justin S. Rhodes

摘要

普通小丑鱼（Amphiprion ocellaris）是一种标志性的珊瑚礁鱼类，在海洋水族馆中无处不在，可用于研究多种生物过程（如互生、紫外线视觉和原生性变）。最近，CRISPR/Cas9 方法被开发出来，用于敲除特定基因以进行机理研究。在这里，我们利用 Tol2 转座子系统创建了第一个转基因品系，从而扩展了鳞栉水母的基因工具包。受精卵在不同浓度、针尖尺寸和受精后时间点上共同注射了 Tol2 转座酶 mRNA 和编码延伸因子-1α（Ef1α）：绿色荧光蛋白（GFP）盒的质粒。我们比较了各种注射参数和消毒方法，以最大限度地提高注射卵的存活率。然后将基因分型为 GFP + 的 F0s（n = 10）养育到 6 个月大，并与野生型（WT）雌性杂交，以确认种系传播。F1 后代也以同样的方式进行饲养和杂交。高效的 Tol2 转座子系统使存活卵子的转基因率达到 37%，在所有注射卵子中，GFP + 存活率为 2.7%（n = 160）。其中，10 个卵子长大成人，8 个卵子产卵，5/8（62.5%）的后代为 GFP +。此外，两个 F1 与 WT 雌性杂交，分别产生了 54.2% 和 44.6% 的 GFP + 后代，证实了稳定品系的建立。据我们所知，这是珊瑚礁鱼类的第一代转基因品系。在标志性的无须鳕中表达感兴趣的转基因的能力，为探索无须鳕迷人的生物学打开了一扇新的大门。

本文章由计算机程序翻译，如有差异，请以英文原文为准。

Generation of the First Transgenic Line of the Iconic Coral Reef Fish Amphiprion ocellaris

The common clownfish, Amphiprion ocellaris, is an iconic coral reef fish, ubiquitous in the marine aquarium hobby and useful for studying a variety of biological processes (e.g., mutual symbiosis, ultraviolet vision, and protandrous sex change). Recently, CRISPR/Cas9 methods were developed for knocking out specific genes for mechanistic studies. Here, we expand the genetic toolkit for A. ocellaris by creating the first transgenic line using the Tol2 transposon system. Fertilized eggs were co-injected with Tol2 transposase mRNA and a plasmid encoding an elongation factor-1α (Ef1α): green fluorescent protein (GFP) cassette at various concentrations, needle tip dimensions, and timepoints post-fertilization. We compared various injection parameters and sterilization methods to maximize the survival of injected eggs. F0s (n = 10) that were genotyped GFP + were then raised to 6 months of age and crossed with wild-type (WT) females to confirm germline transmission. F1 offspring were also raised and crossed in the same manner. The highly efficient Tol2 transposon system resulted in a 37% rate of transgenesis for surviving eggs amounting to a 2.7% yield of all injected eggs surviving and being GFP + (n = 160). Of these, 10 were raised to adulthood, 8 spawned, and 5/8 (62.5%) produced GFP + offspring. Further, two F1s crossed with WT females produced 54.2% and 44.6% GFP + offspring respectively, confirming the creation of a stable line. This is, to our knowledge, the first generation of a transgenic line in any coral reef fish. The ability to express transgenes of interest in the iconic anemonefish opens the door to a new era of exploration into their fascinating biology.

来源期刊

Marine Biotechnology

期刊介绍： Marine Biotechnology welcomes high-quality research papers presenting novel data on the biotechnology of aquatic organisms. The journal publishes high quality papers in the areas of molecular biology, genomics, proteomics, cell biology, and biochemistry, and particularly encourages submissions of papers related to genome biology such as linkage mapping, large-scale gene discoveries, QTL analysis, physical mapping, and comparative and functional genome analysis. Papers on technological development and marine natural products should demonstrate innovation and novel applications.