An effective microinjection method for genome editing of marine aquaculture fish: tiger pufferfish Takifugu rubripes and red sea bream Pagrus major
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.
KeywordsCRISPR/Cas9 Knockout Teleost Fertilized egg Microinjection Genome-editing
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).
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.
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