Document Type


Degree Name

Master of Science (MSc)



Program Name/Specialization

Integrative Biology


Faculty of Science

First Advisor

Stephanie DeWitte-Orr

Advisor Role



RNA interference (RNAi) was first characterized in plants and invertebrates as an antiviral innate immune response. It is a natural antiviral defence mechanism to degrade viral RNA by virus-induced gene silencing. Studies showed synthetic long double-stranded RNA (dsRNA; >30bp) degraded Caenorhabditis elegans messenger RNA (mRNA) transcripts, resulting in sequence-dependent gene silencing. Currently, there is only limited research on RNAi in fish, particularly the use of long dsRNA to trigger sequence-specific gene silencing. This is because studies have shown long dsRNAs trigger interferon response that could mask gene-specific RNAi effects. This thesis explored long dsRNA-mediated RNAi pathways in rainbow trout cells from two aspects. Firstly, the optimal dsRNA concentration that would funnel into RNAi without triggering type 1 interferon responses was determined. Secondly, the ability of sequence-specific dsRNA to mediate gene silencing was identified. Long dsRNA effects were measured in (1) over-expressed, (2) induced and (3) endogenously expressed gene expression systems. Overexpression studies utilized dsRNA-GFP and an expression vector encoding GFP in RTG-2 cells, and subsequent fluorescence intensity was measured. Induced expression studies utilized a luciferase reporter, RTG-P1 cell line and dsRNA-Luc, and subsequent luminescence intensity was measured. Finally, endogenously expressed rainbow trout host genes (IFN1 and Myc) were targeted with either dsRNA-IFN1 or dsRNA-Myc, and knockdown was measured by luciferase ii reporter assay and quantitative real-time PCR respectively. It was found that synthetic long dsRNA-Luc is able to silence luciferase gene expressions. Also, this study demonstrated the potential of dsRNA-IFN1 as an IFN1 silencing tool. This thesis further suggests dsRNA pretreatment and dsRNA concentration are two crucial factors that influence the gene silencing efficiency. RNAi-based strategies could potentially be used to study rainbow trout gene function, develop new drugs and vaccines to control infectious diseases in the future.

Convocation Year


Convocation Season