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.
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.
School of Fisheries, Aquaculture, and Aquatic Sciences, Auburn University, Auburn, Alabama, USA; Key Laboratory of Freshwater Aquatic Biotechnology and Genetic Breeding, Ministry of Agriculture, Heilongjiang Fisheries Research Institute, Chinese Academy of Fishery Sciences, Harbin, China.
Baofeng
B
Su
School of Fisheries, Aquaculture, and Aquatic Sciences, Auburn University, Auburn, Alabama, USA; Key Laboratory of Freshwater Aquatic Biotechnology and Genetic Breeding, Ministry of Agriculture, Heilongjiang Fisheries Research Institute, Chinese Academy of Fishery Sciences, Harbin, China.
Mei
M
Shang
CSIRO Oceans and Atmosphere Flagship, Hobart, Tasmania, Australia.
Peter M
PM
Grewe
CSIRO Oceans and Atmosphere Flagship, Hobart, Tasmania, Australia; National Centre for Marine Conservation and Resource Sustainability, University of Tasmania, Launceston, Australia.
Jawahar G
JG
Patil
School of Fisheries, Aquaculture, and Aquatic Sciences, Auburn University, Auburn, Alabama, USA.
Eric
E
Peatman
School of Fisheries, Aquaculture, and Aquatic Sciences, Auburn University, Auburn, Alabama, USA; Research and Development Corporation, Gus R. Douglass Land-Grant Institute, West Virginia State University, Institute, West Virginia, USA.
Dayan A
DA
Perera
School of Fisheries, Aquaculture, and Aquatic Sciences, Auburn University, Auburn, Alabama, USA.
Qi
Q
Cheng
School of Fisheries, Aquaculture, and Aquatic Sciences, Auburn University, Auburn, Alabama, USA; Marine Science and Engineering College, Qingdao Agricultural University, Qingdao, China.
Chao
C
Li
School of Fisheries, Aquaculture, and Aquatic Sciences, Auburn University, Auburn, Alabama, USA.
Chia-Chen
CC
Weng
School of Fisheries, Aquaculture, and Aquatic Sciences, Auburn University, Auburn, Alabama, USA; Cotton Fiber Bioscience Research Unit, New Orleans, Louisiana, USA.
Ping
P
Li
School of Fisheries, Aquaculture, and Aquatic Sciences, Auburn University, Auburn, Alabama, USA.
Zhanjiang
Z
Liu
School of Fisheries, Aquaculture, and Aquatic Sciences, Auburn University, Auburn, Alabama, USA. Electronic address: dunhara@auburn.edu.
Rex A
RA
Dunham
D000595:Amino Acid Sequence; D000818:Animals; D030801:Animals, Genetically Modified; D001483:Base Sequence; D027682:Cation Transport Proteins / Q000235:genetics / Q000378:metabolism*; D003300:Copper / Q000378:metabolism*; D004625:Embryo, Nonmammalian; D005260:Female; D018507:Gene Expression Regulation, Developmental / Q000502:physiology; D055785:Gene Knockdown Techniques; D007059:Ictaluridae / Q000235:genetics / Q000378:metabolism*; D008297:Male; D008969:Molecular Sequence Data; D013242:Sterilization / Q000379:methods*
D027682:Cation Transport Proteins; D003300:Copper
Channel catfish embryo; Electroporation; Gene expression; Primordial germ cell; RNA interference; Transgenic sterilization