Optimization of siRNA and mRNA transfection in Atlantic salmon erythrocytes as an approach for studying antiviral responses

• Main Author: Joseph, Jeremiah Paul
• Other Authors: Espen Rimstad, Maria Krudtå Dahle
• Format: Master Thesis
• Language: English
• Published: Norwegian University of Life Sciences 2024
• Subjects: Atlantic salmon
• Online Access: https://hdl.handle.net/11250/3147925

Atlantic salmon have nucleated erythrocytes (Red blood cells, RBCs), possessing not only respiratory function, but also some immune functions. Piscine orthoreovirus-1 (PRV-1) is a dsRNA virus that infects A. salmon RBCs and the heart, resulting in Heart and skeletal muscle inflammation (HSMI). PRV induces an innate antiviral response in RBCs, a response also mimicked by poly(I:C). The pattern recognition receptors (PRR) that activate the innate antiviral response against dsRNA in RBC have not been determined, and functional gene studies can increase that knowledge. Transfection of small interfering RNA (siRNA) can result in silencing of a gene, and this mechanism is termed RNA interference (RNAi). RNAi could potentially be used for functional gene-studies in A. salmon RBCs. This thesis aims to characterize the expression of the siRNA system in A. salmon RBCs and optimize transfection of siRNA. The aim is to ultimately silence the dsRNA receptors TLR3, RIG-I, RLR3, and the RIG-I mediator MAVS in A. salmon RBCs and study the antiviral response. It was first determined that genes involved in the siRNA system was expressed in RBCs, andsiRNA transfection in A. salmon RBCs was successfully established using electroporation. To control the function of the siRNA system in RBCs, mRNA-GFP was synthesized by in vitro transcription, and mRNA transfection was also optimized, aiming for a co-transfection with anti-GFP siRNA. The experiment was also set up in Chinook Salmon Embryo – 214 (CHSE-214) cells. Silencing was not observed for either A. salmon RBCs or CHSE-214. Despite the failed control experiment, siRNAs were designed against the target genes as planned. For each target gene, three 21 nt siRNA was designed, ordered, pooled together for each target, and transfected in A. salmon RBCs. Additionally, three longer siRNAs (27 nt) were ordered for MAVS to test the hypothesis that longer siRNAs could be more efficient. No silencing could be shown at the mRNA level or functional level (i.e. effects of poly(I:C) stimulation). ...