Studies on fluorescent approach for detecting genetic exchange in Giardia duodenalis
Koh, Wan Hon (2008) Studies on fluorescent approach for detecting genetic exchange in Giardia duodenalis. Honours thesis, Murdoch University.
The demonstration of a clonal population structure in epidemiological studies of Giardia duodenalis has been considered to support asexual reproduction in their life cycle. However, the exhibition of remarkable phenotypic and genetic heterogeneity within zoonotic assemblages of G. duodenalis (assemblages A and B) suggests the possible existence of sexual reproduction in the Giardia life cycle. To identify such a possibility, the aim of this study was to develop a modified transient and stable fluorescent expression systems to examine the possibility of sexual exchange between G. duodenalis isolates.
A commercial mammalian fluorescent plasmid was modified by cloning the glutamate dehydrogenase promoter upstream of the red fluorescent protein (pRFP). The plasmid was then transfected into the trophozoites of assemblages A and B by electroporation. Transient fluorescent trophozoites were observed in the assemblage B isolate but not in the assemblage A isolate. Furthermore, it was observed that the untransfected trophozoites outcompeted the transfected trophozoites, which hindered the selection process.
Therefore, to select the transfectants, a stable mammalian fluorescent vector, pRFPneo, and green fluorescent plasmid, pGFPneo, were constructed. The plasmid vectors were transfected into the assemblage A isolate separately. The transfected trophozoites were selected using drug G418. However, no stable cell line could be developed as all the transfected trophozoites were dead by day 4. The failure of stable transfection needs to be further investigated, particularly in vector construct.
As a conclusion, this study has demonstrated a transient expression in assemblage B was possible. The absence of fluorescent trophozoites in assemblage A suggested that both assemblages A and B require different transfection condition to express the exogenous gene. The improvements of transient and stable fluorescent system in this not only provide a platform to examine the possibility of sexual reproduction in future, but also assist in understanding the transcriptional activity of Giardia.
|Publication Type:||Thesis (Honours)|
|Murdoch Affiliation:||School of Veterinary and Biomedical Sciences|
|Supervisor:||Thompson, Andrew and Marano, Robert|
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