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Assessing the role of the KISS1 receptor in breast cancer cell behaviour with the use of CRISPR-mediated gene editing
Dissertation (MSc (Human Physiology))--University of Pretoria, 2023.
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| Format: | Thesis |
| Language: | English |
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University of Pretoria
2023
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| _version_ | 1867613634068217856 |
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| access_status_str | Open Access |
| author2 | Van den Bout, Jan Iman |
| author_browse | Van den Bout, Jan Iman |
| author_facet | Van den Bout, Jan Iman |
| collection | Thesis |
| dc_rights_str_mv | © 2022 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 (Human Physiology))--University of Pretoria, 2023. |
| format | Thesis |
| id | oai:repository.up.ac.za:2263/89957 |
| institution | University of Pretoria (South Africa) |
| language | English |
| last_indexed | 2026-06-10T12:39:15.557Z |
| license_str | Other — see source repository |
| provenance_str_mv | Harvested via OAI-PMH from UPSpace — University of Pretoria Institutional Repository |
| publishDate | 2023 |
| publishDateRange | 2023 |
| publishDateSort | 2023 |
| 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:2263/89957 Assessing the role of the KISS1 receptor in breast cancer cell behaviour with the use of CRISPR-mediated gene editing Van den Bout, Jan Iman alex13marais@gmail.com Anderson, Ross Marais, Alex Leah UCTD CRISPR-Cas9 Breast cancer Dissertation (MSc (Human Physiology))--University of Pretoria, 2023. The recent advancements in gene editing have provided scientists with a powerful tool for the investigation of the role of individual proteins in cellular physiology and disease. Gene editing was preceded by another powerful technology that changed cell biology, short hairpin ribonucleic acid (shRNA) dependent messenger RNA (mRNA) degradation. In this project, we sought to establish cell lines derived from the triple negative breast cancer (TNBC) cell line, MDA-MB-231, that expressed low or no Kisspeptin receptor (KISS1R) protein. KISS1R is the cognate receptor of a peptide called kisspeptin. Our previous studies have shown that KISS1R is expressed in different TNBC cell lines but responds differently to a kisspeptin derivative, kisspeptin-10 (KP-10). Using a cell line with low or no KISS1R expression, we can further investigate this phenomenon of alternative signalling to understand the potential role of KISS1R in breast cancer biology. Here, we first established an MDA-MB-231 cell line stably expressing KISS1R shRNA through lentiviral vector delivery and show that while we reduce KISS1R mRNA levels by more than 90%, enough protein remains to allow for a functional response to KP-10 in the form of calcium release. Subsequently, we established a Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-mediated gene editing strategy using HEK-293T cells as a model cell line after which we used electroporation and single cell sorting to establish clonal lines with insertion-deletion mutations (INDELs) for KISS1R. Our efforts have resulted in the establishment of several clones of HEK-293T and MDA-MB-231 cells with no wildtype alleles and frameshifts or large deletions in the KISS1R gene. These can now be used for direct functional studies of KISS1R in TNBC. Physiology MSc (Human Physiology) Unrestricted 2023-03-03T13:08:21Z 2023-03-03T13:08:21Z 2023-09 2023-03-03 Dissertation * S2023 https://repository.up.ac.za/handle/2263/89957 en © 2022 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 CRISPR-Cas9 Breast cancer Assessing the role of the KISS1 receptor in breast cancer cell behaviour with the use of CRISPR-mediated gene editing |
| title | Assessing the role of the KISS1 receptor in breast cancer cell behaviour with the use of CRISPR-mediated gene editing |
| title_full | Assessing the role of the KISS1 receptor in breast cancer cell behaviour with the use of CRISPR-mediated gene editing |
| title_fullStr | Assessing the role of the KISS1 receptor in breast cancer cell behaviour with the use of CRISPR-mediated gene editing |
| title_full_unstemmed | Assessing the role of the KISS1 receptor in breast cancer cell behaviour with the use of CRISPR-mediated gene editing |
| title_short | Assessing the role of the KISS1 receptor in breast cancer cell behaviour with the use of CRISPR-mediated gene editing |
| title_sort | assessing the role of the kiss1 receptor in breast cancer cell behaviour with the use of crispr mediated gene editing |
| topic | UCTD CRISPR-Cas9 Breast cancer |
| url | https://repository.up.ac.za/handle/2263/89957 |