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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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Abstract

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.

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Data Availability

All plasmid DNA used in this study is available upon reasonable request.

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Funding

This study was partly sponsored by MEXT (18H05545 and 20H00430) and JST (JPMJMI21C1).

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Author notes

  1. Ryo Fujihara and Naoto Katayama contributed equally.

Authors and Affiliations

  1. Department of Marine Biosciences, Tokyo University of Marine Science and Technology, 4-5-7 Konan, Minato-Ku, Tokyo, 108-8477, Japan

    Ryo Fujihara, Naoto Katayama, Sakiko Sadaie, Misako Miwa, Gabriela Angelica Sanchez Matias & Goro Yoshizaki

  2. Institute for Reproductive Biotechnology for Aquatic Species (IRBAS), Tokyo University of Marine Science and Technology, 4-5-7 Konan Minato-ku, Tokyo, 108-8477, Japan

    Kensuke Ichida & Goro Yoshizaki

  3. Laboratory of Applied Genetics, Department of Animal Resource Sciences, Graduate School of Agricultural and Life Sciences, The University of Tokyo, Tokyo, Japan

    Wataru Fujii & Kunihiko Naito

  4. Life Science Center for Survival Dynamics, Tsukuba Advanced Research Alliance (TARA), University of Tsukuba, Tsukuba, Ibaraki, 305-8577, Japan

    Makoto Hayashi

Authors
  1. Ryo Fujihara
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  2. Naoto Katayama
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  3. Sakiko Sadaie
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  4. Misako Miwa
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  5. Gabriela Angelica Sanchez Matias
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  7. Wataru Fujii
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  9. Makoto Hayashi
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  10. Goro Yoshizaki
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Correspondence to Makoto Hayashi or Goro Yoshizaki.

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Fujihara, R., Katayama, N., Sadaie, S. et al. Production of Germ Cell-Less Rainbow Trout by dead end Gene Knockout and their Use as Recipients for Germ Cell Transplantation. Mar Biotechnol 24, 417–429 (2022). https://doi.org/10.1007/s10126-022-10128-w

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  • DOI: https://doi.org/10.1007/s10126-022-10128-w

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