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Investigation of ERCC2 Mutations Through Generation and Analysis of Genetically Modified Cell Lines

Lindelöw, Wilmer LU (2024) KBKM05 20241
Pure and Applied Biochemistry
Computational Chemistry
Abstract
This thesis aims to investigate the impact of ERCC2 mutations and their role in
the Nucleotide Excision Repair (NER) DNA repair mechanism. Using genetically
modified MCF10A cell lines with an inducible Cas9, the study explores how these
mutations affect cellular sensitivity to cisplatin, a chemotherapy drug. CRISPR-Cas9
technology was employed in an attempt to generate monoallelic cell lines with specific
oncogenic mutations targeting ERCC2 function, but a successful clone production
was not achieved. Nonetheless, the methodology shows promise, warranting further
attempts to establish these cell lines.
Popular Abstract
This thesis investigates the effects of mutations in the ERCC2 gene on the DNA repair process called Nucleotide Excision Repair (NER). NER is crucial for fixing damaged DNA, and mutations in ERCC2 can compromise this repair mechanism, potentially leading to an increased risk of cancer and affecting how cells respond to chemotherapy. The study specifically looks at how these mutations change the cell’s sensitivity to cisplatin, a chemotherapy drug that works by damaging DNA, making it a valuable tool for understanding the implications of impaired DNA repair.

To explore these effects, MCF10A cell lines, which are a type of non-cancerous immortalized breast cells, were attempted to be produced. CRISPR-Cas9 technology was applied to... (More)
This thesis investigates the effects of mutations in the ERCC2 gene on the DNA repair process called Nucleotide Excision Repair (NER). NER is crucial for fixing damaged DNA, and mutations in ERCC2 can compromise this repair mechanism, potentially leading to an increased risk of cancer and affecting how cells respond to chemotherapy. The study specifically looks at how these mutations change the cell’s sensitivity to cisplatin, a chemotherapy drug that works by damaging DNA, making it a valuable tool for understanding the implications of impaired DNA repair.

To explore these effects, MCF10A cell lines, which are a type of non-cancerous immortalized breast cells, were attempted to be produced. CRISPR-Cas9 technology was applied to introduce specific mutations into one allele of the ERCC2 gene in these cells. This advanced gene-editing technique allows for precise alterations in the DNA, aiming to mimic the oncogenic mutations found commonly in bladder cancer.

Although the experiment did not successfully produce the desired mutated cell lines, the methodology demonstrated significant potential. The challenges encountered highlight areas for refinement in the cell sorting process and clonal production, suggesting that further attempts could eventually achieve the goal. The findings from this study provide a proof of concept for future research. By continuing to develop these techniques, there is potential to enhance our understanding of genetic mutations in cancer and improve therapeutic strategies (Less)
Please use this url to cite or link to this publication:
author
Lindelöw, Wilmer LU
supervisor
organization
course
KBKM05 20241
year
type
H2 - Master's Degree (Two Years)
subject
keywords
ERCC2 gene, Nucleotide Excision Repair (NER), DNA repair, mutations, cancer risk, chemotherapy sensitivity, cisplatin, MCF10A cell lines, non-cancerous breast cells, CRISPR-Cas9 technology, gene editing, oncogenic mutations, bladder cancer, cell sorting, clonal production, genetic mutations, therapeutic strategies, proof of concept, DNA damage, methodology refinement, applied biochemistry
language
English
id
9159006
date added to LUP
2024-06-18 11:41:53
date last changed
2024-06-18 11:41:53
@misc{9159006,
  abstract     = {{This thesis aims to investigate the impact of ERCC2 mutations and their role in
 the Nucleotide Excision Repair (NER) DNA repair mechanism. Using genetically
 modified MCF10A cell lines with an inducible Cas9, the study explores how these
 mutations affect cellular sensitivity to cisplatin, a chemotherapy drug. CRISPR-Cas9
 technology was employed in an attempt to generate monoallelic cell lines with specific
 oncogenic mutations targeting ERCC2 function, but a successful clone production
 was not achieved. Nonetheless, the methodology shows promise, warranting further
 attempts to establish these cell lines.}},
  author       = {{Lindelöw, Wilmer}},
  language     = {{eng}},
  note         = {{Student Paper}},
  title        = {{Investigation of ERCC2 Mutations Through Generation and Analysis of Genetically Modified Cell Lines}},
  year         = {{2024}},
}