| dc.description.abstract | 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). Transfection of all siRNAs induced a high antiviral response measured by increased Mx and
ISG15 gene expression, in particular day 1. The longer 27 nt siRNAs led to an even higher antiviral response. All dsRNA receptor target genes, but not the MAVS gene, were induced by siRNA transfection at Day 3 and Day 6, making it hard to evaluate any silencing effects. In
summary, no silencing could be reported in A. salmon RBCs. | |
