Production of Germ Cell-Less Rainbow Trout by dead end Gene Knockout and their Use as Recipients for Germ Cell Transplantation
Production of Germ Cell-Less Rainbow Trout by dead end Gene Knockout and their Use as Recipients for Germ Cell Transplantation
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DOI：
10.1007/s10126-022-10128-w
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发表时间：
2022-04-05
影响因子：
3
通讯作者：
Yoshizaki, Goro
中科院分区：
文献类型：
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作者：
Fujihara, Ryo;Katayama, Naoto;Yoshizaki, Goro
在生殖细胞移植实验中，使用不产生自身配子的不育受体是一个重要的先决条件。三倍体化和dnd基因敲低（KD）方法已被广泛用于生产不育鱼。然而，在某些鱼类物种中，三倍体化并不产生完全不育，并且基因KD是劳动和时间密集型的，因为它需要显微注射到单个受精卵中。为了克服这些问题，在这项研究中，我们使用成簇调控间隔短回文重复序列/CRISPR相关蛋白9（CRISPR/Cas9）系统在虹鳟鱼（Oncorhynchus mykiss）中产生了死端（dnd）基因的纯合突变体，分析了它们的繁殖能力，并评估了它们作为生殖细胞移植受体的适用性。通过杂交由经历基因组编辑的创始者产生的F1杂合突变体，获得由约1/4纯合敲除突变体（dnd KO）组成的F2代。dnd KO幼仔保留了与野生型（WT）个体相同数量的原始生殖细胞（PGCs），之后数量逐渐减少。在1岁时，生殖细胞在所有分析的个体中完全不存在。为了评估作为生殖细胞移植受体的dnd KO个体，将从供体个体制备的生殖细胞移植到dnd KO孵化的幼雏的腹腔中。这些细胞迁移到受体性腺，在那里它们开始配子发生。成熟的受体个体仅产生与WT虹鳟鱼相同数量的供体来源的精子和卵子。这些结果表明，dnd KO虹鳟鱼是合适的受体候选人，具有高的能力，护士供体来源的生殖细胞。
In germ cell transplantation experiments, the use of sterile recipients that do not produce their own gametes is an important prerequisite. Triploidization and dnd gene knockdown (KD) methods have been widely used to produce sterile fish. However, triploidization does not produce complete sterility in some fish species, and gene KD is labor and time intensive since it requires microinjection into individual fertilized eggs. To overcome these problems, in this study, we generated homozygous mutants of the dead end (dnd) gene in rainbow trout (Oncorhynchus mykiss) using the clustered regulatory interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9) system, analyzed their reproductive capacity, and evaluated their suitability as recipients for germ cell transplantation. By crossing F1 heterozygous mutants produced from founders subjected to genome editing, an F2 generation consisting of approximately 1/4 homozygous knockout mutants (dnd KO) was obtained. The dnd KO hatchlings retained the same number of primordial germ cells (PGCs) as the wild-type (WT) individuals, after which the number gradually decreased. At 1 year of age, germ cells were completely absent in all analyzed individuals. To evaluate the dnd KO individuals as recipients for germ cell transplantation, germ cells prepared from donor individuals were transplanted into the abdominal cavity of dnd KO hatchlings. These cells migrated to the recipient gonads, where they initiated gametogenesis. The mature recipient individuals produced only donor-derived sperm and eggs in equivalent numbers to WT rainbow trout. These results indicate that dnd KO rainbow trout are suitable recipient candidates possessing a high capacity to nurse donor-derived germ cells.