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Development of a conditional gene knockout system to investigate the functional importance of regulatory genes in Plasmodium falciparum
Dissertation (MSC (Biochemistry))--University of Pretoria, 2023.
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| Format: | Thesis |
| Language: | English |
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University of Pretoria
2024
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| _version_ | 1867613717064056832 |
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| access_status_str | Open Access |
| author2 | Birkholtz, Lyn-Marie |
| author_browse | Birkholtz, Lyn-Marie |
| author_facet | Birkholtz, Lyn-Marie |
| collection | Thesis |
| dc_rights_str_mv | © 2023 University of Pretoria. All rights reserved. The copyright in this work vests in the University of Pretoria. No part of this work may be reproduced or transmitted in any form or by any means, without the prior written permission of the University of Pretoria. |
| description | Dissertation (MSC (Biochemistry))--University of Pretoria, 2023. |
| format | Thesis |
| id | oai:repository.up.ac.za:2263/94449 |
| institution | University of Pretoria (South Africa) |
| language | English |
| last_indexed | 2026-06-10T12:40:34.602Z |
| license_str | Other — see source repository |
| provenance_str_mv | Harvested via OAI-PMH from UPSpace — University of Pretoria Institutional Repository |
| publishDate | 2024 |
| publishDateRange | 2024 |
| publishDateSort | 2024 |
| publisher | University of Pretoria |
| publisherStr | University of Pretoria |
| record_format | dspace |
| source_str | UPSpace — University of Pretoria Institutional Repository |
| spelling | oai:repository.up.ac.za:2263/94449 Development of a conditional gene knockout system to investigate the functional importance of regulatory genes in Plasmodium falciparum Birkholtz, Lyn-Marie u18001638@tuks.co.za Niemand, Jandeli Van Zyl, Christea UCTD Sustainable Development Goals (SDGs) Plasmodium falciparum Dimerizable cre recombinase Genetic modification SDG-03: Good health and well-being Natural and agricultural sciences theses SDG-03 Dissertation (MSC (Biochemistry))--University of Pretoria, 2023. Malaria cases have surged in recent years, with the World Health Organization reporting 619 000 deaths in 2021. A recently approved malaria vaccine has been recommended for children younger than 5 years; this vaccine, however, does not prevent cases amongst older children and adults. This stresses the necessity to allocate resources wisely for sustainable malaria control and treatment, emphasized by the ongoing threat of antimalarial resistance. Advancements in knowledge of the most lethal malaria-causing species, Plasmodium falciparum, offer hope for malaria elimination. P. falciparum genetic modification strategies have been a growing field, allowing for the targeting of specific components of this deadly parasite's genome. Controlled genetic interrogation at specific time-points within the highly dynamic and complex life cycle of the parasite has become an increasingly popular method for establishing the essentiality of genes in this parasite. However, non-inducible genetic knockout (KO) interrogation strategies do not facilitate the genetic probing of regulatory genes, due to the immediate death phenotype observed. The Dimerisable Cre recombinase (DiCre) system is a KO genetic interrogation tool used to flox any nucleotide sequence flanked with the cre-specific loxP sites, but only when induced with rapamycin. A drawback of this system is the current cloning approach employed for this system, which relies on continuous cloning of selection markers used for screening, and these critical loxP sites. This highlights the demand to bridge the gap and establish a more regulated cloning approach to generate transgenic parasite lines. In this study, we developed a new DiCre cloning strategy. Specifically, we created a universal DiCre repair plasmid containing these loxP sites, together with a selection marker cassette and multiple cloning sites for homology regions and a recodonised gene insert. This will facilitate genomic integration through either the CRISPR-Cas9 or SLI genetic modification systems. The application of this technology was explored using two proxy regulatory genes, gcn5 and set7, which play a role in histone post-translational modifications across the parasite's epigenome. This study presents a unique cloning approach for conditional gene knockout and supports future research to help expand our knowledge of gene essentiality in the P. falciparum parasite. National Research Foundation SARChi Linked Biochemistry MSc (Biochemistry) Unrestricted Faculty of Natural and Agricultural Sciences 2024-02-12T07:57:50Z 2024-02-12T07:57:50Z 2024-05 2023-12-14 Dissertation * A2024 http://hdl.handle.net/2263/94449 10.25403/UPresearchdata.25163687 en © 2023 University of Pretoria. All rights reserved. The copyright in this work vests in the University of Pretoria. No part of this work may be reproduced or transmitted in any form or by any means, without the prior written permission of the University of Pretoria. application/pdf University of Pretoria |
| spellingShingle | UCTD Sustainable Development Goals (SDGs) Plasmodium falciparum Dimerizable cre recombinase Genetic modification SDG-03: Good health and well-being Natural and agricultural sciences theses SDG-03 Development of a conditional gene knockout system to investigate the functional importance of regulatory genes in Plasmodium falciparum |
| title | Development of a conditional gene knockout system to investigate the functional importance of regulatory genes in Plasmodium falciparum |
| title_full | Development of a conditional gene knockout system to investigate the functional importance of regulatory genes in Plasmodium falciparum |
| title_fullStr | Development of a conditional gene knockout system to investigate the functional importance of regulatory genes in Plasmodium falciparum |
| title_full_unstemmed | Development of a conditional gene knockout system to investigate the functional importance of regulatory genes in Plasmodium falciparum |
| title_short | Development of a conditional gene knockout system to investigate the functional importance of regulatory genes in Plasmodium falciparum |
| title_sort | development of a conditional gene knockout system to investigate the functional importance of regulatory genes in plasmodium falciparum |
| topic | UCTD Sustainable Development Goals (SDGs) Plasmodium falciparum Dimerizable cre recombinase Genetic modification SDG-03: Good health and well-being Natural and agricultural sciences theses SDG-03 |
| url | http://hdl.handle.net/2263/94449 |