Expression and knockdown of primordial germ cell genes, vasa, nanos and dead end in common carp (Cyprinus carpio) embryos for transgenic sterilization and reduced sexual maturity

Su, Baofeng; Peatman, Eric; Shang, Mei; Thresher, Ron; Grewe, Peter M.; Patil, Jawahar G.; Pinkert, Carl A.; Irwin, Michael H.; Li, Chao; Perera, Dayan A.; Duncan, Patricia; Fobes, Michael; Dunham, Rex A.

doi:10.1016/j.aquaculture.2013.07.008

Cited by 25 publications

(27 citation statements)

References 25 publications

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“…All construct systems included four alternative knock-down gene components, short hairpins targeting 5 0 nanos (N1), 3 0 nanos (N2), DND, or a full-length double-stranded nanos RNA (dsRNA). The three short-hairpin structures used in this research were described by Su et al (2014). Expression of the constructs was driven by different promoters, zebrafish alpha-methylacyl-coenzyme A racemase (located on D. rerio chromosome 21: 14822249-14822812; Vega Danio rerio version 56.201105) (RM, salt-responsive), copper transport protein CTR3 (CTR3) but with the first 869 bp removed from yeast (Saccharomyces cerevisiae) (MCTR, copper less-responsive), Atlantic salmon (Salmo salar) transferrin (GenBank: L26909.1) (TF, cadmium sensitive), and channel catfish nanos (GenBank: KM874266) and vasa (GenBank: KM874268) (nanos and vasa, respectively), which were linked to the aforementioned knock-down gene components, either targeting N1, N2, DND, or full-length doublestranded nanos RNA to overexpress nanos mRNA of channel catfish.…”

Section: Construct Design and Preparationmentioning

confidence: 99%

“…Artificial spawning procedures were as described by Lambert et al (1999), Dunham et al (2000), Kristanto et al (2009), andSu et al (2013b). LHRHa was diluted with a 0.9 % NaCl physiological saline solution (0.9 % sodium chloride injection, Hospira, Inc., Lake Forest, IL, USA) and Su et al (2014)). Blocker: knock-down sequences included double-stranded short-hairpins targeting the 5 0 nanos (N1), 3 0 nanos (N2), or dead end (DND), or a full-length double-stranded nanos RNA (dsRNA) to overexpress nanos mRNA.…”

Section: Brood Fish Preparationmentioning

confidence: 99%

“…These compounds could be used as repressors to restore fertility of the transgenic knockout embryos. Su (2012) and Su et al (2014) evaluated alternative repressible knock-down systems for which the promoters would theoretically be sensitive to sodium chloride, copper sulfate or cadmium chloride in common carp, Cyprinus carpio. The constructs caused down-regulation of the targets, but the repressors were not evaluated.…”

Section: Introductionmentioning

confidence: 99%

“…The knock-down of target and off-target genes could have effects on embryonic development, percentage hatch, hatch rate and survival. Constitutive promoters used for the salt, copper and cadmium repressor systems were evaluated by Su (2012) and Su et al (2014). Long-term expression of the knock-down constructs may or may not affect short-term and long-term viability.…”

Section: Introductionmentioning

confidence: 99%

“…Su (2012) and Su et al (2014) transiently knocked down the primordial germ cell migration-related genes, nanos, vasa and dead end during early embryonic development in channel catfish and common carp. These constructs, which are the subject of the current study, had been designed to specifically knock down one of the primordial germ cell marker genes, nanos, vasa or dead end, however, they had off-target effects, knocking down other primordial germ cell marker genes.…”

Section: Introductionmentioning

confidence: 99%

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Effects of transgenic sterilization constructs and their repressor compounds on hatch, developmental rate and early survival of electroporated channel catfish embryos and fry

Shang

et al. 2014

Transgenic Res

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Channel catfish (Ictalurus punctatus) embryos were electroporated with sterilization constructs targeting primordial germ cell proteins or with buffer. Some embryos then were treated with repressor compounds, cadmium chloride, copper sulfate, sodium chloride or doxycycline, to prevent expression of the transgene constructs. Promoters included channel catfish nanos and vasa, salmon transferrin (TF), modified yeast Saccharomyces cerevisiae copper transport protein (MCTR) and zebrafish racemase (RM). Knock-down systems were the Tet-off (nanos and vasa constructs), MCTR, RM and TF systems. Knock-down genes included shRNAi targeting 5' nanos (N1), 3' nanos (N2) or dead end (DND), or double-stranded nanos RNA (dsRNA) for overexpression of nanos mRNA. These constructs previously were demonstrated to knock down nanos, vasa and dead end, with the repressors having variable success. Exogenous DNA affected percentage hatch (% hatch), as all 14 constructs, except for the TF dsRNA, TF N1 (T), RM DND (C), vasa DND (C), vasa N1 (C) and vasa N2 (C), had lower % hatch than the control electroporated with buffer. The MCTR and RM DND (T) constructs resulted in delayed hatch, and the vasa and nanos constructs had minimal effects on time of hatch (P < 0.05). Cadmium chloride appeared to counteract the slow development caused by the TF constructs in two TF treatments (P < 0.05). The 4 ppt sodium chloride treatment for the RM system decreased % hatch (P < 0.05) and slowed development. In the case of nanos constructs, doxycycline greatly delayed hatch (P < 0.05). Adverse effects of the transgenes and repressors continued for several treatments for the first 6 days after hatch, but only in a few treatments during the next 10 days. Repressors and gene expression impacted the yield of putative transgenic channel catfish fry, and need to be considered and accounted for in the hatchery phase of producing transgenically sterilized catfish fry and their fertile counterparts. This fry output should be considered to ensure that sufficient numbers of transgenic fish are produced for future applications and for defining repressor systems that are the most successful.

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Section: Construct Design and Preparationmentioning

confidence: 99%

Section: Brood Fish Preparationmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Effects of transgenic sterilization constructs and their repressor compounds on hatch, developmental rate and early survival of electroporated channel catfish embryos and fry

Shang

et al. 2014

Transgenic Res

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Genetically Engineered Fish: Potential Impacts on Aquaculture, Biodiversity, and the Environment

Dunham

2020

Topics in Biodiversity and Conservation

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Testicular germ line cell identification, isolation, and transplantation in two North American catfish species

Shang

Perera

et al. 2018

Fish Physiol Biochem

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Our aim was to transplant blue catfish germ line stem cells into blastulae of triploid channel catfish embryos to produce interspecific xenogenic catfish. The morphological structure of the gonads of blue catfish (Ictalurus furcatus) in ~ 90- to 100-day-old juveniles, two-year-old juveniles, and mature adults was studied histologically. Both oogonia (12-15 μm, diameter with distinct nucleus 7-8 μm diameter) and spermatogonia (12-15 μm, with distinct nucleus 6-7.5 μm diameter) were found in all ages of fish. The percentage of germ line stem cells was higher in younger blue catfish of both sexes. After the testicular tissue was trypsinized, a discontinuous density gradient centrifugation was performed using 70, 45, and 35% Percoll to enrich the percentage of spermatogonial stem cells (SSCs). Four distinct cell bands were generated after the centrifugation. It was estimated that 50% of the total cells in the top band were type A spermatogonia (diameter 12-15 μm) and type B spermatogonia (diameter 10-11 μm). Germ cells were confirmed with expression of vasa. Blastula-stage embryos of channel catfish (I. punctatus) were injected with freshly dissociated blue catfish testicular germ cells as donor cells for transplantation. Seventeen days after the transplantation, 33.3% of the triploid channel catfish fry were determined to be xenogenic catfish. This transplantation technique was efficient, and these xenogenic channel catfish need to be grown to maturity to verify their reproductive capacity and to verify that for the first time SSCs injected into blastulae were able to migrate to the genital ridge and colonize. These results open the possibility of artificially producing xenogenic channel catfish males that can produce blue catfish sperm and mate with normal channel catfish females naturally. The progeny would be all C × B hybrid catfish, and the efficiency of hybrid catfish production could be improved tremendously in the catfish industry.

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Expression and knockdown of primordial germ cell genes, vasa, nanos and dead end in common carp (Cyprinus carpio) embryos for transgenic sterilization and reduced sexual maturity

Cited by 25 publications

References 25 publications

Effects of transgenic sterilization constructs and their repressor compounds on hatch, developmental rate and early survival of electroporated channel catfish embryos and fry

Effects of transgenic sterilization constructs and their repressor compounds on hatch, developmental rate and early survival of electroporated channel catfish embryos and fry

Genetically Engineered Fish: Potential Impacts on Aquaculture, Biodiversity, and the Environment

Testicular germ line cell identification, isolation, and transplantation in two North American catfish species

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