Zebrafish: a new paradigm for disease analysis

Abstract

Model organisms provide an important platform for the analysis of human disease processes and the development of therapies. The use of model organisms for the analysis of disease relies on an underlying conservation of pathways and processes between the organism and humans. Through the development of gene targeting via homologous recombination the knock-out mouse has become a paradigm of disease modelling. Many studies have highlighted the difficulty of recapitulating the pathology of human diseases in mice. This problem has prompted the use of multiple model organisms to study disease in an effort to highlight conserved vs. organism-specific processes. The work described here proposes the zebrafish as a critical vertebrate species for the modelling of human disease. The use of zebrafish to study disease is limited by the current lack of gene targeting tools. Current gene targeting methods in zebrafish rely on transient antisense directed gene knockdown, and at present no method exists for the stable directed targeting of genes in zebrafish. This work presents the investigation of short interfering RNA (siRNA) directed gene silencing methods in zebrafish. The effectiveness of siRNA directed gene targeting is demonstrated by targeting the zebrafish dystrophin gene. This gene is involved in the human neuromuscular disorder Duchenne Muscular Dystrophy (DMD), and zebrafish present an ideal system for the further analysis of the role that dystrophin transcripts play in this disease. As part of this study it was also shown that zebrafish share with mammals an interferon response when challenged with double stranded RNA in excess of 30bp in length. The demonstration of effective siRNA directed gene targeting provided the framework for the subsequent design and construction of zebrafish siRNA expression vectors. The zebrafish was also investigated as a potential model system for a human neurodegenerative disorder, adrenoleukodystrophy (ALD), where research has been hindered by a lack of diverse animal models. In this context, the family of ALD-like genes was identified and characterized in zebrafish and transcriptional expression analysis was undertaken. Together this work provides the background for undertaking gene targeting studies in the zebrafish.