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Editing of the Luteinizing Hormone Gene to Sterilize Channel Catfish, Ictalurus punctatus, Using a Modified Zinc Finger Nuclease Technology with Electroporation

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Abstract

Channel catfish (Ictalurus punctatus) is the most important freshwater aquaculture species in the USA. Genetically enhanced fish that are sterile could both profit the catfish industry and reduce potential environmental and ecological risks. As the first step to generate sterile channel catfish, three sets of zinc finger nuclease (ZFN) plasmids targeting the luteinizing hormone (LH) gene were designed and electroporated into one-cell embryos, different concentrations were introduced, and the Cel-I assay was conducted to detect mutations. Channel catfish carrying the mutated LH gene were sterile, as confirmed by DNA sequencing and mating experiments. The overall mutation rate was 19.7 % for 66 channel catfish, and the best treatment was ZFN set 1 at the concentration 25 μg/ml. To our knowledge, this is the first instance of gene editing of fish via plasmid introduction instead of mRNA microinjection. The introduction of the ZFN plasmids may have reduced mosaicism, as mutated individuals were gene edited in every tissue evaluated. Apparently, the plasmids were eventually degraded without integration, as they were not detectable in mutated individuals using PCR. Carp pituitary extract failed to induce spawning and restoration of fertility, indicating the need for developing other hormone therapies to achieve reversal of sterility upon demand. This is the first sterilization achieved using ZFN technology in an aquaculture species and the first successful gene editing of channel catfish. Our results will help understand the roles of the LH gene, purposeful sterilization of teleost fishes, and is a step towards control of domestic, hybrid, exotic, invasive, and transgenic fishes.

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Acknowledgments

This material is based upon work that is supported by the Biotechnology Risk Assessment Grants (BRAG) program from the U.S. Department of Agriculture, under award number 2014-33522-22263, and by the Auburn University-IGP program-Office of Vice President for Research. Zhenkui Qin is supported by a scholarship from the China Scholarship Council (CSC).

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  1. Zhenkui Qin

    Present address: Ministry of Education Key Laboratory of Marine Genetics and Breeding, College of Marine Life Sciences, Ocean University of China, Qingdao, 266003, China

  2. Yun Li

    Present address: Laboratory of Fisheries Physiology and Reproduction-Breeding, Fisheries College, Ocean University of China, Qingdao, 266003, China

  3. Baofeng Su & Mei Shang

    Present address: Key Laboratory of Freshwater Aquatic Biotechnology and Genetic Breeding, Ministry of Agriculture, Heilongjiang Fisheries Research Institute, Chinese Academy of Fishery Sciences, Harbin, 150070, China

  4. Dayan A. Perera

    Present address: Research and Development Corporation, Gus R. Douglass Land-Grant Institute, West Virginia State University, Institute, WV, 25112, USA

Authors and Affiliations

  1. School of Fisheries, Aquaculture and Aquatic Sciences, Auburn University, Auburn, AL, 36849, USA

    Zhenkui Qin, Yun Li, Baofeng Su, Qi Cheng, Zhi Ye, Dayan A. Perera, Michael Fobes, Mei Shang & Rex A. Dunham

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  1. Zhenkui Qin
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Qin, Z., Li, Y., Su, B. et al. Editing of the Luteinizing Hormone Gene to Sterilize Channel Catfish, Ictalurus punctatus, Using a Modified Zinc Finger Nuclease Technology with Electroporation. Mar Biotechnol 18, 255–263 (2016). https://doi.org/10.1007/s10126-016-9687-7

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