Department of Physiological Sciences

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Browsing Department of Physiological Sciences by Author "Ambrone, Wade"

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    Generation of a transgenic zebrafish with a fluorescent TNF-alpha reporter gene
    (Stellenbosch : Stellenbosch University, 2022-12) Ambrone, Wade; Smith, Carine; Van Staden, Anton du Preez; Stellenbosch University. Faculty of Science. Dept. of Physiological Sciences.
    ENGLISH ABSTRACT: Given the high degree of innate immune system conservation between zebrafish and humans, researchers have taken a keen interest in utilising zebrafish to unpack innate immunity related mechanisms. Notably, transgenic zebrafish expressing reporter genes are routinely employed for monitoring the spatio-temporal dynamics of innate immunity related proteins in vivo, and thus, are an invaluable tool to the zebrafish research community. While a number of alternative protein detection methods are available, they are subject to inference from tissue samples, often acquired through invasive techniques, which are incapable of accurately monitoring the spatio- temporal dynamics of protein expression in vivo. Given our research group’s interest in innate immune processes, as well as the advantageous applications of transgenic zebrafish, this study aimed to develop a stable transgenic zebrafish line, expressing a reporter gene under the transcriptional control of the pro-inflammatory cytokine TNF-α. For this, the transgene construct, pRSF-zTNFα-mCherry – expressing the fluorescent protein mCherry under the transcriptional control of the zebrafish TNF-α1 promoter – was designed and synthesized. Zebrafish embryos were transfected with the transgene construct by microinjection at the single-cell stage. Following microinjection, an acute inflammatory response was induced in transfected zebrafish larvae, coupled with fluorescence microscopy, to access the number of zebrafish in which the transgene was successfully integrated into the genome. Likewise, a genotyping method, using PCR, was developed to assess the rate of successful transgene integration. The data presented in this study illustrates the successful synthesis of the pRSF-zTNFα- mCherry transgene construct. Transfection of zebrafish larvae with the construct yielded zero zebrafish with detectable mCherry expression, using a stereomicroscope fitted with a fluorescent light source, both before and after the induction of an acute inflammatory response. Genotyping, however, revealed that the transgene was successfully integrated into the genome of the majority of transfected zebrafish, despite the inability to visually illustrate expression of the transgene. The transgene integration rate observed in this study was considerably higher than those previously seen in literature, in which similar transfection techniques were used. Potential reasons for the lack of detectable transgene expression were discussed throughout this study, namely: insufficient equipment sensitivity; insufficient transgene stimulation; integration of a dysfunctional transgene. While the study at hand was unable to demonstrate a TNF-α response spatio-temporally, many of the methodologies needed for the development of transgenic zebrafish were introduced and explored within the research group for the first time. Likewise, the means of both accessing and improving on future transgenic zebrafish development were investigated. In conclusion, this study lays the foundation for prospective transgenic zebrafish development – a model that will be utilized within the research group moving forward.