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    Generation HIF-1α knockout of MKN45 gastric cancer cell line by CRISPR CAS9 system

  • Hengameh Sharifi,1 Hossein Safarpour,2 Mohsen Khorashadizadeh,3,*
    1. Department of Molecular Medicine, Faculty of Medicine, Birjand University of Medical Sciences, Birjand, Iran
    2. Cellular & Molecular Research Center, Birjand University of Medical Sciences, Birjand, Iran
    3. Department of Medical Biotechnology, Faculty of Medicine, Birjand University of Medical Sciences, Birjand, Iran


  • Introduction: Today, the knockout cell line can accelerate cancer research by providing a model for evaluating tumorigenic mechanisms, studying cancer cell drug resistance, identifying drug development targets and assessing cell therapy. Hypoxia is an in common feature of solid tumor microenvironment especially at its core site. This chronic hypoxia resulted in the development of resistance to drug and apoptosis, elevated angiogenesis and metastasis. Hypoxia-Inducible Factor (HIF)-1 plays a critical role in the response to low oxygen concentration in tumor cells, so it may be a viable candidate for use in the development of knock-out cells for cancer research. Thus, in the present study, HIF-1α knock-out was introduced to the human gastric cancer MKN45 cells using a lentivirus-mediated CRISPR/Cas9 system (TLCV2-Cas9).
  • Methods: The target HIF-1α sgRNA was initially designed and cloned into the TLCV2-Cas9 plasmid and confirmed by sequencing. Then, the resulted plasmid was transected into the human epithelial cell line HEK-293. The virus particles were harvested and used for transduction of MKN45 cells. After puromycin selection, genomic DNA was analyzed using the T7E1 for evaluating knock-out induction. clonal selection was performed to reach a pure knock-out clones and validated by sequencing.
  • Results: The TLCV2-Cas9 plasmid was constructed correctly based on the results of the sequencing. The HEK-293 cell line was successfully transfected with the plasmid that was obtained. The MKN45 cells were also infected with viral particles. The T7E1 test results demonstrate that the knockout induction was successfully completed. Finally, the sequencing results validated the creation of a pure knockout cell clone and the HIF-1α gene was successfully knocked out in MKN45 gastric cancer cells.
  • Conclusion: As a result of this research, a gastric cancer cell model that can be used for therapeutic purposes was created using the CRISPR CAS9 technology.
  • Keywords: CRISPR/Cas9 system, Gastric cancer, HIF-1α, knockout cell lines