Use this URL to cite or link to this record in EThOS: http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.597133
Title: Studies into the ablation of ISG64, ISG65 and ISG75 expression in Trypanosoma brucei using RNAi
Author: Burns, R. E. S.
Awarding Body: University of Cambridge
Current Institution: University of Cambridge
Date of Award: 2005
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Abstract:
ISG64, ISG65 are abundant surface proteins found distributed across the entire cell body and flagellum of bloodstream form Trypanosoma brucei. They are encoded by multiple genes arranged in tandem arrays. To date, their biological role remains unknown. The work presented here describes two RNAi strategies employed in an attempt to elucidate the function of ISG64, ISG65 and ISG75 through ablation of their expression. The strategies targeted the C- and N- terminal regions of the coding sequence, the both cloned and mixed cell populations were analysed. No ablation of ISG64 was seen using either strategy. With the exception of a single ISG75 RNAi cell line (R022, clone 1), ISG65 and ISG75 decreased to between 20-50% of wild-type levels 24 hours post induction. Upon induction of the R022 clone 1 cell line, the ISG75 decreased to 10% of wild-type levels. Moreover, the cells ceased proliferation and showed a abnormal morphology. This RNAi induced phenotype was subsequently found to be the result of the integration of the RNAi plasmid into an unexpected location and not due to the ablation of ISG75. The effectiveness of two tetracycline inducible RNAi vectors, P2T7Ti and p2T7Ti. 177, was also compared. The single copy, bloodstream specific GPI-PLC gene was targeted for ablation. The results revealed that in this case, under non-inducing conditions the repression of transcription from the T7 promoters were less stringent in the p2T7Ti vector than in the p2T7Ti.177 vector. However, no significant difference was observed between the p2T7Ti .177 vectors when either ISG65 or ISG75 were targeted for ablation. Because of this no firm conclusions about the effectiveness of either P2T7Ti and p2T7Ti.177 under non-inducing conditions could be drawn. Using the different RNAi it was possible to ablate ISG65 to ISG75 to between 10-50% of wild-type protein levels.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID: uk.bl.ethos.597133  DOI: Not available
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