EGFP Reporter Assay in Transgenic Zebrafish Embryos for Assessing Formulation-Dependent dsRNA Bioavailability

RNA interference (RNAi) is a sequence-specific gene silencing mechanism increasingly exploited for the development of sprayable, species-selective pest control products. For many applications, the trigger molecule double-stranded RNA (dsRNA) must be stabilized via suitable formulation strategies to ensure delivery to the target site and enable biological activity. Currently, there is no standardized approach to assess dsRNA bioavailability as a function of its formulation in fish. However, this is a critical step in assessing the environmental risks posed by formulated dsRNA to fish and other non-target organisms. Here, we present a zebrafish embryo-based assay using the Tg(fli1:EGFP)^y1 reporter line to assess dsRNA bioavailability by quantifying RNAi-mediated gene silencing. Based on OECD Test Guideline 236, zebrafish embryos are exposed to sublethal concentrations of nano-lipid formulated dsRNA designed to target the EGFP messenger RNA (mRNA). After 96 h of exposure via immersion, embryos are either assessed microscopically or homogenized to quantify EGFP signal, providing a rapid, high-throughput readout without the need for dissection or individual embryo handling. EGFP downregulation at the protein level, analyzed by fluorescence signal quantification, is complemented by quantifying EGFP mRNA levels using RT-qPCR. Representative results show a concentration-dependent reduction of EGFP signal along with decreased EGFP transcript levels, indicating effective uptake and functional delivery of the nano-lipid formulated dsRNA. This workflow provides a reliable method for assessing the cellular bioavailability of dsRNA as a function of its formulation. Additionally, the microplate reader-based fluorescence measurement offers a straightforward and time-efficient readout, reducing animal use. Utilizing zebrafish embryos, the method is furthermore a refined approach, reducing the need for experiments on protected life stages of fish, supporting the 3Rs principle. More broadly, this protocol is adaptable for additional applications, including the modification of the reporter line, target gene, or nanoparticle-based delivery system for use in developmental and (eco‑)toxicological studies.