Dnd shRNA constructs cause altered PGC migration but fail to sterilize

Abstract

The release or escape of genetically modified fish from confined aquaculture facilities presents a potential risk to wild fish stocks and surrounding ecosystems. Thus, reliable biocontainment methods for aquaculture-relevant species are needed. Since physical barriers are not 100% effective, it is critical to develop reliable genetic-based methods for biocontainment. RNA interference (RNAi) could regulate gene expression and target genes that are essential for fish viability and/or reproduction. One such target gene, deadend (dnd), is required for primordial germ cell (PGC) development, migration and survival. While the use of RNAi technology has been successfully reported in C. elegans, M. musculus, and D. melanogaster, its use in zebrafish (Danio rerio) has been met with variable success. We targeted dnd using two different short-hairpin RNA (shRNA) approaches in zebrafish embryos, namely, 1) CMV-driven or a Gal4/UAS constructs with one double short-hairpin RNA stem-loop design consisting of two different target sites, and 2) CMV-driven to test two separate target sites independently flanked by the 5′ and 3′ sequences of the pri-miR-30e miRNA. The first shRNA strategy did not completely nor consistently abolish PGCs, based on vasa-expression, but low vasa expression was observed in some fish at 6 dpf. In contrast, the second shRNA strategy resulted in reduced and/or ectopic positioning of PGCs based on vasa expression at 2 and 6 dpf. However, neither strategy affected fertility in adult fish.