ABSTRACT
The zebrafish (Danio rerio) has become an attractive model for human disease modeling as there are a large number of orthologous genes that encode similar proteins to those found in humans. The number of tools available to manipulate the zebrafish genome is limited and many currently used techniques are only effective during early development (such as morpholino-based antisense technology) or it is phenotypically driven and does not offer targeted gene knockdown (such as chemical mutagenesis). The use of RNA interference has been met with controversy as off-target effects can make interpreting phenotypic outcomes difficult; however, this has been resolved by creating zebrafish lines that contain stably integrated miRNA constructs that target the desired gene of interest. In this study, we show that a commercially available miRNA vector system with a mouse-derived miRNA backbone is functional in zebrafish and is effective in causing eGFP knockdown in a transient in vivo eGFP sensor assay system. We chose to apply this system to the knockdown of transcripts that are implicated in the human cardiac disorder, Long QT syndrome.
Subject(s)
Gene Knockdown Techniques/methods , MicroRNAs/genetics , Zebrafish Proteins/genetics , Analysis of Variance , Animals , Disease Models, Animal , Embryo, Nonmammalian , Ether-A-Go-Go Potassium Channels/genetics , Ether-A-Go-Go Potassium Channels/metabolism , Green Fluorescent Proteins/genetics , Green Fluorescent Proteins/metabolism , Humans , Long QT Syndrome , Mice , MicroRNAs/chemistry , RNA Interference , Zebrafish , Zebrafish Proteins/metabolismABSTRACT
Some genes can encode multiple overlapping transcripts, and this can result in challenges in identifying transcript-specific developmental expression profiles where tools such as RNA in situ hybrisations are inapplicable. Given this difficulty, we have undertaken a preliminary analysis of the developmental expression profile of selected transcripts of the dystrophin and utrophin genes of the zebrafish (Danio rerio) by targeting unique and common regions of each of these transcripts. The dystrophin and utrophin genes of zebrafish were identified by bioinformatic analysis and the dystrophin gene predictions were confirmed by transcript sequencing. These data enabled primer pairs to be designed in order to determine the expression profiles of unique, but overlapping transcripts, throughout embryonic development using quantitative real time reverse transcription PCR (qRT-PCR). The data indicated the early expression of the short carboxyl-terminal dystrophin transcript, with expression of the full length muscle transcript occurring during myogenesis. Importantly, a composite of these two profiles appeared to comprise the major transcriptional load of the zebrafish dystrophin gene. In contrast, utrophin gene expression was dominated by the full length transcript throughout embryogenesis. The approach described here provided a means by which a gene's transcriptional complexity can be deconvoluted to reveal transcriptional diversity during embryogenesis. This approach, however, required the identification of unique regions for transcript-specific targeting, and an appreciation of alternative splicing events that may compromise the design of primers for qRT-PCR.
Subject(s)
Dystrophin/genetics , Gene Expression Regulation, Developmental , Transcription, Genetic , Utrophin/genetics , Zebrafish/embryology , Zebrafish/genetics , Amino Acid Sequence , Animals , Base Sequence , Exons , Gene Expression Profiling , Gene Order , Humans , Molecular Sequence DataABSTRACT
The modeling of human disease in the zebrafish (Danio rerio) is moving away from chemical mutagensis and transient downregulation using morpholino oligomers to more targeted and stable transgenic methods. In this respect, zinc finger nucleases offer a means of introducing mutations at targeted sites at high efficiency. We describe here the development of zinc finger nucleases and their general use in model systems with a focus on the zebrafish.
Subject(s)
Deoxyribonucleases/metabolism , Gene Targeting , Mutagenesis , Zebrafish/genetics , Zinc Fingers/genetics , Animals , Animals, Genetically Modified , Humans , Zebrafish/embryologyABSTRACT
Long QT syndrome is a disorder that is characterised by a prolonged QT-interval and can lead to fatal cardiac arrhythmias. Many animal models have been created to study congenital long QT syndrome. Of these, zebrafish models have involved targeting two different KCNH2 gene (long QT syndrome 2) orthologues, termed zerg-2 and zerg-3, with differing cardiac phenotypes. In order to clarify this situation, this study uses a bioinformatic approach to search the current zebrafish genome sequence (Zv7 and Zv8 builds) to investigate and locate all likely zebrafish orthologues of the human KCNH2 gene. Quantitative real-time RT-PCR was also used to determine the temporal and spatial gene expression profile of the zebrafish orthologues. The data support the conclusion that zerg-2 and zerg-3 are apparent orthologues of different human genes encoding potassium ion channels, but that their functions have switched compared to the respective human proteins.
