Volume 13, Issue 3 (2023)
JMBS 2023, 13(3): 73-82 |
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Hamidi R, Ataei F, Hosseinkhani S. Cloning of Caspase 9 in eukaryotic vector, investigation of its expression and functional assay in cell. JMBS 2023; 13 (3) :73-82
URL: http://biot.modares.ac.ir/article-22-59629-en.html
URL: http://biot.modares.ac.ir/article-22-59629-en.html
1- Tarbiat Modares University
2- Tarbiat Modares University, دانشگاه تربیت مدرس , ataei_f@modares.ac.ir
2- Tarbiat Modares University, دانشگاه تربیت مدرس , ataei_f@modares.ac.ir
Abstract: (447 Views)
Aims: Programmed cell death is a vital cellular process that is highly conserved in evolution. Apoptosis, as a common mode of programmed cell death, is disturbed in the most human malignancies and leads the resistance of cancers to current treatment strategies. Caspase 9 is a key protein in mitochondrial apoptosis. Activated Caspase 9 leads to activation of Caspase 3/7, initiating a caspase cascade and killing cell. In this study, Caspase 9 gene was cloned into pcDNA3.1(+) and its expression and function evaluated in cell.
Methods: PCR amplification of Caspase 9 was performed by specific primers and ligated into pcDNA3.1(+) after double-digestion with KpnI and BamHI. After sequencing, pcDNA/Caspase 9 was transfected into SH-SY5Y cells and treated with doxorubicin. Caspase 9 function was determined by its effect on cell death level by trypan blue and PI staining, and Caspase 3 activity, and its expression in cells measured by western blotting.
Finding: Caspase 9 gene cloning was done and its expression in cell defined by western blot. Overexpression of Caspase 9 led to autoprocessing following homodimerization and induction of cell death and also increased cell sensitivity to doxorubicin treatment and declined cell viability.
Conclusion: The cloned Caspase 9 was functional in cell and enhanced apoptosis in the treated cells by doxorubicin through self-activation and subsequently amplification of Caspase 3 activation.
Methods: PCR amplification of Caspase 9 was performed by specific primers and ligated into pcDNA3.1(+) after double-digestion with KpnI and BamHI. After sequencing, pcDNA/Caspase 9 was transfected into SH-SY5Y cells and treated with doxorubicin. Caspase 9 function was determined by its effect on cell death level by trypan blue and PI staining, and Caspase 3 activity, and its expression in cells measured by western blotting.
Finding: Caspase 9 gene cloning was done and its expression in cell defined by western blot. Overexpression of Caspase 9 led to autoprocessing following homodimerization and induction of cell death and also increased cell sensitivity to doxorubicin treatment and declined cell viability.
Conclusion: The cloned Caspase 9 was functional in cell and enhanced apoptosis in the treated cells by doxorubicin through self-activation and subsequently amplification of Caspase 3 activation.
Article Type: Original Research |
Subject:
Molecular biotechnology
Received: 2022/02/15 | Accepted: 2022/04/12 | Published: 2023/10/22
Received: 2022/02/15 | Accepted: 2022/04/12 | Published: 2023/10/22
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