title: Editing of the Luteinizing Hormone Gene to Sterilize Channel Catfish, Ictalurus punctatus, Using a Modified Zinc Finger Nuclease Technology with Electroporation.
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.
authors: School of Fisheries, Aquaculture and Aquatic Sciences, Auburn University, Auburn, AL, 36849, USA.|Ministry of Education Key Laboratory of Marine Genetics and Breeding, College of Marine Life Sciences, Ocean University of China, Qingdao, 266003, China.
authors: Zhenkui
authors: Z
authors: Qin
authors: School of Fisheries, Aquaculture and Aquatic Sciences, Auburn University, Auburn, AL, 36849, USA.|Laboratory of Fisheries Physiology and Reproduction-Breeding, Fisheries College, Ocean University of China, Qingdao, 266003, China.
authors: Yun
authors: Y
authors: Li
authors: School of Fisheries, Aquaculture and Aquatic Sciences, Auburn University, Auburn, AL, 36849, USA.|Key Laboratory of Freshwater Aquatic Biotechnology and Genetic Breeding, Ministry of Agriculture, Heilongjiang Fisheries Research Institute, Chinese Academy of Fishery Sciences, Harbin, 150070, China.
authors: Baofeng
authors: B
authors: Su
authors: School of Fisheries, Aquaculture and Aquatic Sciences, Auburn University, Auburn, AL, 36849, USA.
authors: Qi
authors: Q
authors: Cheng
authors: School of Fisheries, Aquaculture and Aquatic Sciences, Auburn University, Auburn, AL, 36849, USA.
authors: Zhi
authors: Z
authors: Ye
authors: School of Fisheries, Aquaculture and Aquatic Sciences, Auburn University, Auburn, AL, 36849, USA.|Research and Development Corporation, Gus R. Douglass Land-Grant Institute, West Virginia State University, Institute, WV, 25112, USA.
authors: Dayan A
authors: DA
authors: Perera
authors: School of Fisheries, Aquaculture and Aquatic Sciences, Auburn University, Auburn, AL, 36849, USA.
authors: Michael
authors: M
authors: Fobes
authors: School of Fisheries, Aquaculture and Aquatic Sciences, Auburn University, Auburn, AL, 36849, USA.|Key Laboratory of Freshwater Aquatic Biotechnology and Genetic Breeding, Ministry of Agriculture, Heilongjiang Fisheries Research Institute, Chinese Academy of Fishery Sciences, Harbin, 150070, China.
authors: Mei
authors: M
authors: Shang
authors: School of Fisheries, Aquaculture and Aquatic Sciences, Auburn University, Auburn, AL, 36849, USA. dunhara@auburn.edu.
authors: Rex A
authors: RA
authors: Dunham
mesh_terms: D000818:Animals; D030801:Animals, Genetically Modified; D001483:Base Sequence; D002347:Carps / Q000378:metabolism; D045424:Complex Mixtures / Q000494:pharmacology; D003851:Deoxyribonucleases / Q000235:genetics* / Q000378:metabolism; D018274:Electroporation; D004625:Embryo, Nonmammalian; D005260:Female; D029941:Fish Proteins / Q000037:antagonists & inhibitors / Q000235:genetics* / Q000378:metabolism; D020868:Gene Silencing; D007059:Ictaluridae / Q000235:genetics* / Q000378:metabolism; D007986:Luteinizing Hormone / Q000037:antagonists & inhibitors / Q000235:genetics* / Q000378:metabolism; D008297:Male; D010902:Pituitary Gland / Q000737:chemistry; D010957:Plasmids / Q000737:chemistry / Q000378:metabolism; D012098:Reproduction / Q000187:drug effects; D013245:Sterilization, Reproductive / Q000379:methods*; D016335:Zinc Fingers
chemical_list: D045424:Complex Mixtures; D029941:Fish Proteins; D007986:Luteinizing Hormone; D003851:Deoxyribonucleases
keywords: Ictalurus punctatus; Luteinizing hormone; Sterilization; Targeted gene inactivation; Zinc finger nuclease