Repressible Transgenic Sterilization in Channel Catfish, Ictalurus punctatus, by Knockdown of Primordial Germ Cell Genes

Abstract

Genetic engineering has great potential for development of fish with higher growth rate, better disease resistance and increased nutritional value. However, genetically modified animals could pose ecological risk to the environment upon escapement, and fail-safe confinement needs to be developed to minimize this risk. Knockdown approaches utilizing overexpression and shRNAi approaches were investigated to attempt repressible transgenic sterilization in channel catfish, Ictalurus punctatus. Two primordial germ cell marker genes, nanos and dead end were targeted for knockdown and an off-target gene, vasa, was monitored. Their expression was evaluated at 3 time points during embryonic development using real-time PCR. Seven potentially repressible promoters, zebrafish Adss2 and racemase (sodium chloride), yeast ctr3 and ctr3-reduced (copper sulfate), channel catfish nanos and vasa coupled with a Tet-off system and salmon transferrin (cadmium chloride) were each coupled with 4 knockdown strategies including: (1) a ds-sh RNA targeting the 5’ end of channel catfish nanos gene (N1), (2) a ds-sh RNA targeting the 3’ end of channel catfish nanos gene (N2), (3) a full length cDNA sequence of channel catfish nanos gene to overexpress nanos (cDNA) and (4) a ds-sh RNA targeting channel catfish dead end gene (dnd). Except for the nanos cDNA sequence, all constructs have a short hairpin structure and double stranded RNA to produce 28 different constructs for evaluation as repressible transgenic sterilization systems. Each construct was divided into two groups: untreated group and treated group with sodium chloride, cadmium chloride, copper sulfate or doxycycline as repressor compounds. Constructs were electroporated into embryos to produce the P1 generation and artificial spawning used to make the F1 and F2 generations. For most systems and gene constructs, rates of P1 fish exposed to the constructs as embryos spawning full-siblings, 88% and 56%, respectively, indicating potential sterilization and repression of the constructs. In F1 fish, mRNA expression levels of PGC marker genes for most of constructs were significantly down regulated in untreated group and the knockdown was repressed in treated group. The downregulation in the F1 transgenic untreated embryos was sometimes similar, but often greater than what was observed in their parents (Su 2012, Su et al. 2015) that were exposed to the constructs via electroporation the previous generation. The repression was also more effective in treated F1 embryos than for the P1. In three F2 families that were produced, knockdown and repression for the constructs TDND and McDNA were similar as the same constructs in the F1, but the treated ADSSN2 embryos had strong upregulation rather than repression of the Adss promoter. Constructs with the knockdown strategies N2 and cDNA were the most effective for knockdown of primordial germ cell genes, and the promoters, ADSS, Mctr and M were the most responsive to the chemicals applied for repression. When considering the combination of knockdown and repression, the constructs ADSSN2, MctrN2 and McDNA showed the most potential as repressible transgenic sterilization systems. Gonad development in transgenic untreated F1 channel catfish was significantly reduced compared to non-transgenic fish for MctrN2, MN1, MN2, MDND and TDND. For 3-year-old adults, gonad size in the transgenic untreated group was 93.4% smaller than the non-transgenic group for females, and 92.3% for males. However, body size of transgenic females (782g) and males (884g) were smaller than non-transgenic counterparts (984g and 1254g) at three years of age, a 25.8% and 41.9% difference for females and males, respectively. This negative pleiotropic effect would negate the usefulness of the repressible transgenic sterilization unless this growth reduction is more than compensated for by insertion of growth related transgenes.