Liang Guangyuan, Lu Baoyue, Dai Shengfei, Li Minghui, Yao Jiawen, Liu Hao, Liu Xiayue, Liu Xingyong, Wang Deshou
Integrative Science Center of Germplasm Creation in Western China (Chongqing) Science City, Key Laboratory of Freshwater Fish Reproduction and Development (Ministry of Education), Key Laboratory of Aquatic Science of Chongqing, School of Life Sciences, Southwest University, Chongqing 400715, China.
Integrative Science Center of Germplasm Creation in Western China (Chongqing) Science City, Key Laboratory of Freshwater Fish Reproduction and Development (Ministry of Education), Key Laboratory of Aquatic Science of Chongqing, School of Life Sciences, Southwest University, Chongqing 400715, China.
N Biotechnol. 2025 Jul 31;89:163-176. doi: 10.1016/j.nbt.2025.07.009.
Transparent mutant fish have been obtained from small-sized fish (medaka, zebrafish, and killifish), and are often used as experimental models in biological and medical research. However, transparent fish have never been created in medium- or large-sized fish through gene editing. In this study, mutants without xanthophores, erythrophores and pigmented melanophores were first obtained by crossing to form tyrb;csf1ra double mutant tilapia. Subsequently, single mutants lacking reflective platelets without visible iridophores were obtained by mutation of pnp4a and tfec using CRISPR/Cas9 gene editing, the recovery of iridophores were observed in pnp4a mutants but not in tfec mutants in the later stage. In addition, we also found that tfec mutation led to a decrease in number of melanophores and diameter of melanophores/erythrophores. Finally, transparent triple mutant homozygotes without visible pigment cells were obtained by triple mutation of tyrb;csf1ra;pnp4a (named amber) and tyrb;csf1ra;tfec (named ruby), through crossing of double and single mutants. The two mutant lines were no longer transparent at 60 dpf (days post fertilization) and 120 dpf, respectively, due to the recovery of iridophores in the former, and thickening of the body wall in the latter. In summary, this study created two transparent lines without visible pigment cells through aggregation of multiple gene mutation and crossing. The transparent fish is suitable for in vivo imaging, and is currently the only medium-sized transparent fish obtained through gene editing.
透明突变体鱼已从小型鱼类（青鳉、斑马鱼和鳉鱼）中获得，并经常被用作生物学和医学研究的实验模型。然而，通过基因编辑从未在中型或大型鱼类中培育出透明鱼。在本研究中，首先通过杂交获得了缺乏黄色素细胞、红色素细胞和有色素黑素细胞的突变体罗非鱼，形成tyrb;csf1ra双突变体。随后，利用CRISPR/Cas9基因编辑技术对pnp4a和tfec进行突变，获得了缺乏无可见反光小板的反光血小板的单突变体，后期在pnp4a突变体中观察到反光血小板的恢复，而在tfec突变体中未观察到。此外，我们还发现tfec突变导致黑素细胞数量减少以及黑素细胞/红细胞直径减小。最后，通过双突变体和单突变体杂交，对tyrb;csf1ra;pnp4a（命名为琥珀）和tyrb;csf1ra;tfec（命名为红宝石）进行三重突变，获得了无可见色素细胞的透明三重突变纯合子。这两个突变体系分别在受精后60天（dpf）和120 dpf时不再透明，前者是由于反光血小板的恢复，后者是由于体壁增厚。总之，本研究通过多重基因突变和杂交聚集创建了两条无可见色素细胞的透明品系。这种透明鱼适用于体内成像，是目前通过基因编辑获得的唯一中型透明鱼。
