Abstract
Repressible knockdown approaches were investigated for transgenic sterilization in channel catfish, Ictalurus punctatus. Two primordial germ cell (PGC) marker genes, nanos and dead end, were targeted for knockdown, and an off-target gene, vasa, was monitored. Two potentially salt sensitive repressible promoters, zebrafish adenylosuccinate synthase 2 (ADSS) and zebrafish racemase (Rm), were each coupled with four knockdown strategies: ds-sh RNA targeting the 5′ end (N1) or 3′ end (N2) of channel catfish nanos, full-length cDNA sequence of channel catfish nanos for overexpression (cDNA) and ds-sh RNA targeting channel catfish dead end (DND). Each construct had an untreated group and treated group with sodium chloride as the repressor compound. Spawning rates of full-sibling P1 fish exposed or not exposed to the constructs as treated and untreated embryos were 93% and 59%, respectively, indicating potential sterilization of fish and repression of the constructs. Although the mRNA expression data of PGC marker genes were inconsistent in P1 fish, most F1 individuals were able to downregulate the target genes in untreated groups and repress the knockdown process in treated groups. The results indicate that repressible transgenic sterilization is feasible for reproductive control of fish, but more data from F2 or F3 are needed for evaluation.
Highlights
Since the commercialization of AquaBounty Technologies’s AquAdvantage® Salmon [1], the control of escapement of transgenic offspring has received as much attention as the advantages of transgenic fish
Dead end was downregulated 3.35X at 48 hpf and 12.91X at 120 hpf compared to 24 hpf (Figure 1)
F1 individuals were produced containing the knockdown constructs. These constructs were designed to be repressed by application of salt to accomplish repressible transgenic sterilization
Summary
Since the commercialization of AquaBounty Technologies’s AquAdvantage® Salmon [1], the control of escapement of transgenic offspring has received as much attention as the advantages of transgenic fish. A major concern regarding the use of transgenic fish is potential ecological impact from escapees or released individuals on natural populations [2,3]. All transgenic organisms should be adequately confined both physically and genetically to minimize environmental risk. Physical confinement cannot guarantee that transgenic fish will never establish in the wild since the possibility of theft, human error and catastrophic events could compromise the physical confinement, leading to escape of transgenic animals from aquaculture systems [4]. The use of both and redundant systems further decreases the risk of establishment of transgenes. If used outside their native geographical range, all species are potentially invasive. Different permutations of transgenic sterilization can potentially be used to prevent spread of invasive species
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