Biological Confinement of Zebrafish Using RNAi

Abstract

The increasing demand for fish in the food industry has resulted in the extensive overfishing of wild fisheries. In efforts to alleviate the demand from the food industry, genetically modified (GM) fish were developed possessing traits such as larger mass, faster growth, and increased resistance to disease. However, the greater fitness advantage of GM fish presents potential risks for wild type populations in the event of release or escape from a confined fish facility. In addition to physical barriers, it is critical to develop a genetic mechanism in order to ensure that the spread of GM transgene to the natural populations does not occur. RNA interference (RNAi) is an endogenous mechanism used to regulate gene expression by destroying targeted mRNA molecules. Manipulation of this biological process has been successfully utilized to knockdown specific genes through the introduction of synthetic transgenes in organisms such as C. elegans, M. musculus, and D. melanogaster. Although the use of RNAi as a biological tool is still relatively new in zebrafish, recent work has explored elements of this mechanism allowing for greater knockdown efficiencies. The deadend (dnd) gene is required for primordial germ cell (PGC) development and survival. Previous studies have shown that zebrafish dnd knockouts develop into sterile adults without disrupting somatic development. In efforts to induce sterility in zebrafish, short hairpin RNA (shRNA) constructs targeting dnd were designed to exploit the endogenous RNAi pathway. Upon qualitative analysis in transient and transgenic zebrafish subjected to the synthetic RNAi construct, a reduction in the germ cell population at early stages of development was observed. However, quantification of dnd mRNA in fish from the same time points did not show significant changes in expression levels compared to their wildtype counterparts. Adult fish subjected to the transgene construct produced viable gametes. The use of RNAi as a tool for bioconfinement relies on sterility among all individuals subjected to the shRNA bearing transgene. Based on the results obtained, the verdict is still unclear as to whether shRNA is a viable mechanism for large scale bioconfinement.