Document Type

Thesis

Degree Name

Master of Science (MSc)

Department

Biology

Program Name/Specialization

Integrative Biology

Faculty/School

Faculty of Science

First Advisor

Stephanie DeWitte-Orr

Advisor Role

Supervisor

Abstract

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

2020

Convocation Season

Fall

Download

Share

COinS