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Title: Vector-mediated RNA interference in zebrafish : a feasibility study
Author: Kelly, Amanda
ISNI:       0000 0004 2698 8176
Awarding Body: University of Manchester
Current Institution: University of Manchester
Date of Award: 2010
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Zebrafish are becoming an increasingly popular model organism in which to model diseases with a genetic component. Their use is hindered however, by the lack of an efficient, reliable, stable and cost-effective method to carry out reverse genetics and model diseases which arise from a loss of function of a gene. RNAi is a method of post-transcriptional gene regulation and has been widely manipulated in other systems to knockdown genes at will. This thesis therefore looks at the feasibility of vector-mediated RNAi in zebrafish by attempting to knockdown green fluorescent protein (GFP) and the Parkinson‟s disease-associated gene PTEN Induced Kinase 1 (PINK1).Initial results in HEK 293 cells and in G0 animals were encouraging, however low expression of the self-reporting vector made the identification of transgenic animals difficult. To improve expression levels the vector was modified to contain a Gal4-VP16/UAS amplification cassette. Inclusion of this cassette led to increased expression and knockdown capabilities of the vector in HEK 293 cells and led to the successful identification of transgenic zebrafish. Despite high level expression however, no knockdown of GFP or PINK1 was detected in transgenic zebrafish larvae expressing the RNAi vectors out to 5 dpf. This lack of knockdown was shown to be despite the expression of the main components of the RNAi pathway and the production of customised miRNAs throughout development and across tissues. Interestingly however, in adult transgenic zebrafish 50% knockdown of PINK1 was detected in brains expressing two independent PINK1 miRNAs compared to the control miRNA and wild type zebrafish brains. This knockdown coincided with increased transcript expression of the RNAi components and increased production of customised mature miRNA in the brain compared to embryos.In an attempt to improve vector-mediated RNAi in zebrafish, the effect of over-expression of components of the RNAi machinery, including Argonaute 2, Dicer, Drosha and Exportin 5 was assessed in zebrafish cells. Of these, only over-expression of Argonaute 2 improved knockdown in HEK 293 cells and resulted in moderate knockdown in two independent zebrafish cell lines, PAC.2 and ZFL cells. This improvement in knockdown was shown to be a result of the RNase activity of Argonaute 2 as mutation of this domain abrogated the effect of Argonaute 2 over-expression. Despite the encouraging results in zebrafish cell lines, injection of Argonaute 2 mRNA into transgenic zebrafish failed to produce knockdown, suggesting perhaps, that in zebrafish embryos other factors apart from Argonaute 2 are also limitingGiven the difficulties of vector-mediated RNAi in zebrafish, this technology is at present not a feasible approach to knocking down genes in zebrafish, at least not to an extent as to model complete loss of gene function.
Supervisor: Hurlstone, Adam Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available