Reproductive Confinement of Common Carp, Cyprinus carpio, and Channel Catfish, Ictalurus punctatus, via Transgenic Sterilization

Abstract

Transgenic animals had shown great potential for increasing growth rate, ability to resist disease, and for use as tool to research in forward and reverse genetics. Genetically modified animals could pose ecological risk to the environment upon escapement. Modified sterile feral constructs based on sterile feral concept and technology has been applied to reproductively control the genetically modified animals in the field of aquaculture. Constructs utilzing short hair-pin structures to produce cDNA, theoretically could control primordial germ cell (PGC) marker genes, nanos, vasa and dead end (dnd) in channel catfish, Ictalurus punctatus. Constructs were electroporated into embryos of common carp and channel catfish. Ideally, the embryos would undergo normal development and the gonad would form and mature in the presence of repressible compounds. However, without repressible chemicals, the gonad would not develop normally. Real-time PCR was used to test the relative temporal expression level of natural PGC marker gene expression between constructs electroporated and no constructs non-electroporated embryos (common carp) and no constructs electroporate embryos (channel catfish). Sexual maturity of electroporated and control channel catfish and common carp were evaluated. In the case of knockdown common carp, two proteins, nanos and dead end were targeted and a third related, but off-target PGC protein, vasa, was assayed. The effectiveness of constructs varied depending upon the promoter. The expression levels of these proteins naturally decreased during development. In some, but not all cases, the expression of targeted mRNA was knocked down to minimal levels or degradation accelerated. Up-regulation was also observed for dead end, possibly due to interferon responses. In other cases, the sterilization constructs had little effect on the targeted gene, but strongly down-regulated one or two of the alternative PGC genes examined in this study. At 19 months of age, common carp males exposed to these constructs had reduced rates of sexual maturity, but evaluation of reduction of female rates of sexual maturity were inconclusive. In the case of channel catfish electroporated with the knockdown constructs some constructs knocked down PGC genes at certain time points. As was the case with common carp, some constructs appeared to induce sterility in catfish at 3-year of age. Further experimentation is needed to evaluate sterility in the F1 generation. Potential pleiotropic effects of these PGC knockdown constructs should be evaluated, especially since off-target effect was observed.