Genome editing of the IL-7 receptor gene locus using talens
Yalçın, Gülperi (2014) Genome editing of the IL-7 receptor gene locus using talens. [Thesis]
IL-7 signaling is key to lymphocyte development and function in the mammalian immune system. In the first part of this study, we targeted the IL-7R alpha gene locus, encoding the IL-7 receptor protein, to identify its transcriptional control elements. We mutated two transcription factor binding sites in an evolutionarily conserved region containing a putative transcriptional enhancer by generating transcription activator like effector nucleases (TALENs) targeting these sites. We designed and constructed two pairs of TALENs targeting the glucocorticoid receptor (GR) and Notch binding sites in this region. We also targeted the exon 2 and exon 3 of IL-7R to delete a transcriptional control element in intron 2. We expressed these TALENs in the murine RLM11 (IL-7R positive) cell line and generated insertion and deletion mutations in the targeted sites. We used restriction fragment length polymorphism (RFLP) assays and DNA sequencing to detect the induced mutations and assessed their effects on IL-7R gene expression. We demonstrate that mutations induced in the GR transcription factor binding site do not reduce IL-7R gene expression, while mutations in the Notch binding site lower expression. In the second part of the study we directly targeted the gene encoding the GR transcription factor. We designed a TALEN pair targeting the translation start site to knockout gene expression. We introduced these TALENs into the human HCT116 cell line and performed RFLP assays to detect mutations. Our experiments demonstrate that TALENs can be used for genome editing to study gene transcription regulatory regions.
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