Fisheries Research Institute of Karatsu, Department of Joint Research, Faculty of Agriculture, Kyushu University, Saga, 847-0132, Japan.
National Research Institute of Fisheries and Environment of Inland Sea, Hakatajima Station, Imabari, 794-2305, Japan.
Sci Rep. 2019 Mar 20;9(1):4948. doi: 10.1038/s41598-019-41468-8.
A comprehensive experimental system for Japanese anchovy, a promising candidate model organism for marine teleosts, was established. Through the design of a rearing/spawning facility that controls the photoperiod and water temperature, one-cell eggs were continuously obtained shortly after spawning throughout the rearing period. The stages of eggs are indispensable for microinjection experiments, and we developed an efficient and robust microinjection system for the Japanese anchovy. Embryos injected with GFP mRNA showed strong whole-body GFP fluorescence and the survival rates of injected- and non-injected embryos were not significantly different, 87.5% (28 in 32 embryos) and 90.0% (45 in 50 embryos), respectively. We verified that the Tol2 transposon system, which mediates gene transfer in vertebrates, worked efficiently in the Japanese anchovy using the transient transgenesis protocol, with GFP or DsRed as the reporter gene. Finally, we confirmed that genome-editing technologies, namely Transcription Activator-Like Effector Nucleases (TALEN) and Clustered Regulatory Interspaced Short Palindromic Repeats (CRISPR)/Cas9, were applicable to the Japanese anchovy. In practice, specific gene-disrupted fishes were generated in the F generation. These results demonstrated the establishment of a basic, yet comprehensive, experimental system, which could be employed to undertake experiments using the Japanese anchovy as a model organism for marine teleost fish.
建立了一套完整的日本鯷实验系统，该系统以日本鯷为研究对象，有望成为海洋硬骨鱼类的模式生物。通过设计一个可以控制光周期和水温的养殖/产卵设施，我们可以在整个养殖期间在产卵后不久持续获得单细胞卵。卵的各个阶段对于微注射实验都是必不可少的，因此我们开发了一种高效、稳健的日本鯷微注射系统。注射 GFP mRNA 的胚胎表现出强烈的全身 GFP 荧光，并且注射和未注射胚胎的存活率没有显著差异，分别为 87.5%（32 个胚胎中的 28 个）和 90.0%（50 个胚胎中的 45 个）。我们使用瞬时转基因方案验证了 Tol2 转座子系统在日本鯷中的有效性，该系统介导了脊椎动物中的基因转移，使用 GFP 或 DsRed 作为报告基因。最后，我们证实了基因组编辑技术，即转录激活子样效应物核酸酶（TALEN）和成簇规则间隔短回文重复（CRISPR）/Cas9，适用于日本鯷。实际上，在 F1 代中产生了特定基因缺失的鱼类。这些结果证明了建立了一个基本但全面的实验系统，可以使用日本鯷作为海洋硬骨鱼类的模式生物进行实验。
