Established a Gene Editing System Targeting to HIF-1α in Epithelial Cells
| Autor: | Debangana Sankhla |
|---|---|
| Rok vydání: | 2018 |
| Druh dokumentu: | 學位論文 ; thesis |
| Popis: | 106 Hypoxia inducible factors (HIFs) are well established mediators of tumor growth, epithelial to mesenchymal transition and metastasis. Most of the solid tumors overexpress HIF1-alpha which might be related to metastasis. In this study, the CRISPR- Cas9 technique was used to establish a gene editing system which targets HIF-1α in epithelial cells. The gene knock-out efficiency has been checked using Human Embryonic Kidney cells, HEK293 which contains a mutant version of the SV40 T-antigen, commonly called as 293T cells. Transient transfection method of the Knock out clone in these cells and subsequently the protein and mRNA levels of HIF-1α were detected by Western blotting and RT-PCR respectively. Later, a limiting dilution was performed from the mixed population of cells to select the survival clones and protein and mRNA levels of HIF-1α was detected to confirm the efficiency of the CRISPR/Cas9 mediated gene knock out. This study was confirmed using animal model to determine the effect of the knock out clone in-vivo. Some experiments were performed using nasopharyngeal carcinoma cells, as our lab predominantly focuses on head and neck cancers to check if this system can be applied to nasopharyngeal carcinoma cells. |
| Databáze: | Networked Digital Library of Theses & Dissertations |
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