Generation of a transgenic zebrafish with a fluorescent TNF-alpha reporter gene
| dc.contributor.advisor | Smith, Carine | en_ZA |
| dc.contributor.advisor | Van Staden, Anton du Preez | en_ZA |
| dc.contributor.author | Ambrone, Wade | en_ZA |
| dc.contributor.other | Stellenbosch University. Faculty of Science. Dept. of Physiological Sciences. | en_ZA |
| dc.date.accessioned | 2022-11-14T10:19:33Z | |
| dc.date.accessioned | 2023-01-16T12:48:40Z | |
| dc.date.available | 2022-11-14T10:19:33Z | |
| dc.date.available | 2023-01-16T12:48:40Z | |
| dc.date.issued | 2022-12 | |
| dc.description | Thesis (MSc)--Stellenbosch University, 2022. | en_ZA |
| dc.description.abstract | 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. | en_ZA |
| dc.description.abstract | AFRIKAANSE OPSOMMING: Gegewe die hoë mate van aangebore immuunstelselbewaring tussen sebravisse en mense, het navorsers 'n groot belangstelling in die gebruik van sebravisse om aangebore immuniteit- verwante meganismes te ontrafel. Veral transgeniese sebravisse wat verslaggewergene uitdruk, word gereeld aangewend vir die monitering van die ruimtelike-tydelike dinamika van aangebore immuniteit-verwante proteïene in vivo, en is dus 'n waardevolle hulpmiddel vir die sebravisnavorsingsgemeenskap. Alhoewel 'n aantal alternatiewe proteïen-metingsmetodes beskikbaar is, is hulle onderhewig aan afleidings van weefselmonsters, wat dikwels deur indringende tegnieke verkry is, en wat dus nie in staat is om die ruimtelike-tydelike dinamika van proteïenuitdrukking in vivo akkuraat te reflekteer nie. Gegewe ons navorsingsgroep se belangstelling in aangebore immuunreaksies, sowel as die voordelige toepassings van transgeniese sebravis, het hierdie studie ten doel gehad om 'n stabiele transgeniese sebravislyn te ontwikkel, wat 'n verslaggewergeen uitdruk onder die transkripsiebeheer van die pro- inflammatoriese sitokien TNF-α. Hiervoor is die transgeenkonstruk, pRSF-zTNFα-mCherry – wat die fluoreserende proteïen mCherry onder die transkripsiebeheer van die sebravis TNF-α1 promotor uitdruk – ontwerp en vervaardig. Sebravis embrio's is getransfekteer met die transgeen konstruk deur mikro- inspuiting op die enkelsel stadium. Na mikro-inspuiting is 'n akute inflammatoriese reaksie in getransfekteerde sebravislarwes geïnduseer. Fluoresensie mikroskopie is ingespan om te bepaal in hoeveel sebravisse die transgeen suksesvol in die genoom geïntegreer is. Net so is 'n genotiperingsmetode, wat gebruik maak van PCR, ontwikkel om alternatiewelik toegang tot die persentasie van suksesvolle transgeen-integrasie te bepaal. Die data wat in hierdie studie aangebied word, illustreer die suksesvolle sintese van die pRSF- zTNFα-mCherry transgeen konstruk. Transfeksie van sebravislarwes met die konstruk hetgeen sebravis opgelewer met waarneembare mCherry-uitdrukking (sigbaar op ‘n stereomikroskoop met fluoreserende ligbron), beide voor en na die induksie van 'n akute inflammatoriese reaksie, nie. Genotipering het egter aan die lig gebring dat die transgeen suksesvol in die genoom van die meerderheid getransfekteerde sebravisse geïntegreer is, ten spyte van die onvermoë om visueel die uitdrukking van die transgeen in genoemde sebravisse te demonstreer. Die transgeen-integrasietempo wat in hierdie studie waargeneem is, was aansienlik hoër as dié wat voorheen in literatuur gesien is, waarin soortgelyke transfeksietegnieke gebruik is. Potensiële redes vir die gebrek aan waarneembare transgeenuitdrukking is deurgaans in hierdie studie bespreek, naamlik: onvoldoende toerusting sensitiwiteit; onvoldoende transgeenstimulasie; integrasie van 'n disfunksionele transgeen. Terwyl die studie wat voorhande nie in staat was om ’n ruimtelike-tydelike TNF-α te demonstreer nie, is baie van die tegnieke wat nodig is vir die ontwikkeling van transgeniese sebravisse vir die eerste keer binne die navorsingsgroep bekendgestel en ondersoek. Net so is die maniere om toegang te verkry tot en te verbeter op toekomstige transgeniese sebravisontwikkeling, ondersoek. Ten slotte, hierdie studie lê die grondslag vir voornemende transgeniese sebravisontwikkeling – 'n model wat in die navorsingsgroep gebruik sal word om vorentoe te beweeg. | af_ZA |
| dc.description.version | Masters | en_ZA |
| dc.format.extent | 107 pages : illustrations (some color) | en_ZA |
| dc.identifier.uri | http://hdl.handle.net/10019.1/126073 | |
| dc.language.iso | en_ZA | en_ZA |
| dc.publisher | Stellenbosch : Stellenbosch University | en_ZA |
| dc.rights.holder | Stellenbosch University | en_ZA |
| dc.subject | Transgenic fish -- Genetics | en_ZA |
| dc.subject | Zebrafish -- Genetic engineering | en_ZA |
| dc.subject | Tumour necrosis factor alpha (TNF) | en_ZA |
| dc.subject | MCherry | en_ZA |
| dc.subject | UCTD | en_ZA |
| dc.title | Generation of a transgenic zebrafish with a fluorescent TNF-alpha reporter gene | en_ZA |
| dc.type | Thesis | en_ZA |
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