Suppression and restoration of primordial germ cell marker gene expression in channel catfish,Ictalurus punctatus,using knockdown constructs regulated by copper transport protein gene promoters: Potential for reversible transgenic sterilization |
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| Affiliation: | 1. Institute of Hydrobiology, Chinese Academy of Sciences, University of Chinese Academy of Sciences, Wuhan 430072, China;2. Key Laboratory of Freshwater Biodiversity Conservation, Ministry of Agriculture of China, Yangtze River Fisheries Research Institute, Chinese Academy of Fishery Sciences, Wuhan 430223, China;3. Freshwater Fisheries Research Center, Chinese Academy of Fisheries Science, Wuxi 214081, China;1. School of Fisheries, Aquaculture and Aquatic Sciences, Auburn University, AL 36849, USA;2. Department of Agricultural Botany, Faculty of Agriculture Saba-Basha, Alexandria University, Alexandria City, P.O. Box 2153, Egypt;3. Department of Bioinformatics, Institute of Genetic Engineering and Biotechnology, Minufiya University, Sadat City, P.O. Box 22857-79, Egypt;4. Department of Biology, University of Ottawa, Ottawa, Ontario K1N 6N5, Canada;5. Ministry of Agriculture Key Laboratory of Freshwater Aquatic Biotechnology and Genetic Breeding, Heilongjiang Fisheries Research Institute, Chinese Academy of Fishery Sciences, Harbin 150070, China;6. Agricultural and Food Sciences College, King Faisal University, Al ahsa City, P.O. Box 31982, Saudi Arabia |
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| Abstract: | Complementary DNA overexpression and short hairpin RNA interference approaches were evaluated for decreasing expression of primordial germ cell (PGC) marker genes and thereby sterilizing channel catfish, Ictalurus punctatus, by delivering knockdown constructs driven by a constitutive promoter from yeast and a copper transport protein gene into fish embryos by electroporation. Two PGC marker genes, nanos and dead end, were the target knockdown genes, and their expressions, along with that of an off-target gene, vasa, were evaluated temporally using real-time polymerase chain reaction. Copper sulfate was evaluated as a repressor compound. Some of the constructs knocked down PGC marker gene expression, and some of the constructs were partially repressed by application of 0.1-ppm copper sulfate. When the rate of sexual maturity was compared for three-year-old broodfish that had been exposed to the sterilizing constructs during embryologic development and controls that had not been exposed, several treatments had reduced sexual maturity for the exposed fish. Of two promoter systems evaluated, the one which had been designed to be less sensitive to copper generally was more effective at achieving sterilization and more responsive to repression. Knockdown constructs based on 3′ nanos short hairpin RNA interference appeared to result in the best repression and restoration of normal sexual maturity. We conclude that these copper-based systems exhibited good potential for repressible transgenic sterilization. Optimization of this system could allow environmentally safe application of transgenic technology and might be applicable to other applications for aquatic organisms. |
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| Keywords: | Transgenic sterilization RNA interference Electroporation Channel catfish embryo Primordial germ cell Gene expression |
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