MSc.Thesis Defense:Ahsen Ozcan
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  • MSc.Thesis Defense:Ahsen Ozcan

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MODELLING OF THE MECHANISM OF THE GLUCOCORTICOID RESISTANCE USING CRISPR/CAS9 MEDIATED GENOME EDITING

 

Ahsen Ozcan

Molecular Biology Genetics and Bioengineering, MSc Thesis Defense, 2015

 

Thesis Jury

Prof. Dr Batu Erman(Thesis Advisor), Prof.Dr. Selim Cetiner

 Prof.Dr. Uygar Tazebay

 

 

Date & Time: 30th, December 2015 –  09:30-11:30 am

Place: L048

Keywords : Glucocorticoid Receptor, Glucocorticoid Receptor Related Drug Resistance,CRISPR-Cas9, Genome Editing

 

Abstract

 

 

Glucocorticoid related drugs are widely used as anti-inflammatory agents and against hematologic malignancies. Glucocorticoid related drug 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 genome editing. Single cell cloning followed by 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 related cell death.