Production of reproductively sterile fish by a non-transgenic gene silencing technology

We developed a novel bath-immersion technology to produce large numbers of infertile fish. As seafood consumption shifts from fishery harvests towards artificially propagated species, optimization of aquaculture practices will be necessary to maximize food production and minimize ecological impact. Farming infertile fish is the most effective genetic-containment strategy to support the development of environmentally-responsible aquaculture. We discovered that a molecular transporter, Vivo, can effectively carry the Morpholino oligomer (MO) across the chorion, enter the embryo and reach target cells. Vivo-conjugated MO against zebrafish deadend (dnd-MO-Vivo) effectively caused primordial germ cell mis-migration and differentiation into somatic cells, which resulted in generation of infertile fish. Optimal conditions were achieved when embryos, immediately after fertilization, were immersed with dnd-MO-Vivo at the initial concentration of either 60 or 40 μM followed by a lower serially diluted concentration. Under these conditions, 100% induced sterility was achieved even when the total immersion time was reduced from 24 to 5 hours. In 8 independent experiments, 736 adults developed from these conditions were all found to be infertile fish that possessed minimally-developed gonads that lacked any gametes. The results demonstrate that dnd-MO-Vivo bath immersion is an effective strategy to produce infertile fish without introducing transgenic modifications.