Department of Psychology, University of Illinois, Champaign, IL, 61820, USA.
Department of Molecular & Cellular Biology, University of Illinois, Champaign, IL, USA.
Mar Biotechnol (NY). 2024 Oct;26(5):1067-1078. doi: 10.1007/s10126-024-10357-1. Epub 2024 Aug 19.
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.
普通小丑鱼（Amphiprion ocellaris）是一种具有代表性的珊瑚礁鱼类，广泛存在于海洋水族馆爱好者中，并且对于研究各种生物学过程（例如，共生关系、紫外线视觉和雌雄同体转变）非常有用。最近，开发了 CRISPR/Cas9 方法来敲除特定基因以进行机制研究。在这里，我们通过创建第一个使用 Tol2 转座子系统的转基因系来扩展 A.ocellaris 的遗传工具包。将 Tol2 转座酶 mRNA 和编码延伸因子-1α (Ef1α)：绿色荧光蛋白 (GFP) 盒的质粒以不同浓度、针尖尺寸和受精后时间点共注射到受精卵中。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。然后将基因型为 GFP+的 F0（n=10）饲养至 6 个月龄，并与野生型（WT）雌性杂交以确认生殖系传递。同样以相同的方式饲养和杂交 F1 后代。高效的 Tol2 转座子系统导致存活卵的转基因率达到 37%，相当于所有存活并 GFP+的注射卵（n=160）的 2.7%产量。其中，有 10 个被饲养至成年，8 个产卵，并且 5/8（62.5%）产生 GFP+后代。此外，两个与 WT 雌性杂交的 F1 分别产生了 54.2%和 44.6%的 GFP+后代，证实了稳定系的创建。这是我们所知的第一种在任何珊瑚礁鱼类中产生的转基因系。在标志性海葵鱼中表达感兴趣的转基因的能力为探索其迷人生物学打开了一个新时代的大门。
