Modeling of the mechanism of the glucocorticoid resistance using CRISPR/Cas9 mediated genome editing
Özcan, Ahsen (2015) Modeling of the mechanism of the glucocorticoid resistance using CRISPR/Cas9 mediated genome editing. [Thesis]
Glucocorticoid related drugs are widely used as anti-inflammatory agents and against hematologic malignancies. Glucocorticoid resistance is a clinical problem which may result from the mutation of the glucocorticoid receptor (GR) gene. In the first part of the study we identify GR gene mutations in the human T lymphocyte Jurkat cell line. We sequenced the genomic locus encoding the GR and confirmed the presence of a heterozygous mutation in the fourth exon which encodes the DNA binding domain of this transcription factor. This single point missense mutation changes the 477th amino acid of this domain from arginine to histidine. Further, we confirmed that Jurkat cells are glucocorticoid resistant by applying high doses of dexamethasone. In the second part of the study, we targeted the GR exon 4 with CRISPR/Cas9 mediated genome editing. Single cell cloning followed by restriction fragment length polymorphism (RFLP) analysis of CRISPR/Cas9 treated cells confirmed the generation of various clones of novel GR mutant Jurkat cells. We analyzed the survival of these cells and found that they have even higher resistance to cell death induced by the glucocorticoid analog, dexamethasone. We demonstrate that the CRISPR/Cas9 genome editing tool is an efficient tool for genome modification and that the GR exon 4 region is critical for GR mediated cell death.
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