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Fisheries Science

, Volume 85, Issue 1, pp 217–226 | Cite as

An effective microinjection method for genome editing of marine aquaculture fish: tiger pufferfish Takifugu rubripes and red sea bream Pagrus major

  • Kenta Kishimoto
  • Youhei Washio
  • Yu Murakami
  • Takashi Katayama
  • Miwa Kuroyanagi
  • Keitaro Kato
  • Yasutoshi Yoshiura
  • Masato KinoshitaEmail author
Original Article Aquaculture

Abstract

Genome editing technology is becoming increasingly accepted as a way to improve traits in marine fish aquaculture. In fish, microinjection is a major method for introducing RNA or protein into eggs for genome editing; however, this method has not yet been established in aquaculture fish. We successfully established microinjection methods achieving high survival rates for tiger pufferfish and red sea bream by optimizing the following three parameters: (1) the soaking solution of fertilized eggs during microinjection, (2) the elapsed time from in vitro fertilization to microinjection, (3) the elapsed time from stripping to microinjection. In tiger pufferfish, Iwamatsu solution or diluted sea water is effective as the soaking solution. In vitro fertilization can be performed at intervals of 15 min from fertilization until 2.5 h after stripping. Similarly, in red sea bream, Leibovitz’s L-15 medium or Iwamatsu solution is effective as the soaking solution and in vitro fertilization can be performed at intervals of 10 min from fertilization until 2.5 h after stripping. We anticipate our findings will contribute to effectively establish genome edited aquaculture breeds.

Keywords

CRISPR/Cas9 Knockout Teleost Fertilized egg Microinjection Genome-editing 

Notes

Acknowledgements

We thank Tadashi Imai for maintaining the tiger pufferfish parents and the stripping method. This work was partially supported by a Grant-in-Aid for Scientific Research (B) 26292104 (Kinoshita M, Kato K, Yoshiura Y), JSPS KAKENHI Grant Number JP 17J10249 (Kishimoto K), and the Science and Technology Research Promotion Program for Agriculture, Forestry, Fisheries, and Food Industry (26047A) (Kinoshita M, Kato K).

Author contributions

KK designed and carried out the experiment. KK, YW, YM, and MK performed microinjection. TK, MK, TI, and YY performed in vitro fertilization and bred the tiger pufferfish. YW and KK performed in vitro fertilization and bred the red sea bream. KK and MK wrote the manuscript. MK conceived the study and guided the overall project.

Compliance with ethical standards

Conflict of interest

The authors declare no competing financial interests.

Supplementary material

12562_2018_1277_MOESM1_ESM.pdf (760 kb)
Supplementary material 1 (PDF 759 kb)

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Copyright information

© Japanese Society of Fisheries Science 2018

Authors and Affiliations

  • Kenta Kishimoto
    • 1
  • Youhei Washio
    • 1
    • 2
  • Yu Murakami
    • 1
  • Takashi Katayama
    • 3
    • 4
  • Miwa Kuroyanagi
    • 3
  • Keitaro Kato
    • 2
  • Yasutoshi Yoshiura
    • 3
  • Masato Kinoshita
    • 1
    Email author
  1. 1.Division of Applied Bioscience, Graduate School of AgricultureKyoto UniversityKyotoJapan
  2. 2.Aquaculture Research InstituteKindai UniversityNishimuroJapan
  3. 3.Yashima Station, Stock Enhancement and Management Department, National Research Institute of Fisheries and Enhancement of Inland SeaJapan Fisheries Research and Education AgencyTakamatsuJapan
  4. 4.Miyazu Laboratory, Japan Sea National Fisheries Research InstituteJapan Fisheries Research and Education AgencyMiyazuJapan

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