Cloning of Caspase 9 in eukaryotic vector, investigation of its expression and functional assay in cell

Hamidi, Roghaye; Ataei, Farangis; Hosseinkhani, Saman

Cloning of Caspase 9 in eukaryotic vector, investigation of its expression and functional assay in cell

Document Type : Original Research

Authors

Roghaye Hamidi

Farangis Ataei

Saman Hosseinkhani

Tarbiat Modares University

Abstract

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.

Keywords

Cloning

Caspase 9

Apoptosis

Doxorubicin

20.1001.1.23222115.1401.13.3.12.9

Subjects

Molecular biotechnology

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