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Iranian Journal of Biotechnology، جلد ۲۱، شماره ۲، صفحات ۷۵-۸۶
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عنوان انگلیسی Efficient CRISPR/Cas9-Mediated BAX Gene Ablation in CHO Cells To Impair Apoptosis and Enhance Recombinant Protein Production
چکیده انگلیسی مقاله Background: Despite recent advances in recombinant biotherapeutics production using CHO cells, their productivity remains lower than industrial needs, mainly due to apoptosis.
Objectives: Present study aimed to exploit CRISPR/Cas9 technology to specifically disrupt the BAX gene to attenuate apoptosis in recombinant Chinese hamster′s ovary cells producing erythropoietin.
Materials and Methods: The STRING database was used to identify the key pro-apoptotic genes to be modified by CRISPR/Cas9 technique. The single guide RNAs (sgRNAs) targeting identified gene (BAX) were designed, and CHO cells were then transfected with vectors. Afterward, changes in the expression of the Bax gene and consequent production rates of erythropoietin were investigated in manipulated cells, even in the presence of an apoptosis inducer agent, oleuropein.
Results: BAX disruption significantly prolonged cell viability and increased proliferation rate in manipulated clones (152%, P-value = 0.0002). This strategy reduced the levels of Bax protein expression in manipulated cells by more than 4.3-fold (P-value <0.0001). The Bax-8 manipulated cells displayed higher threshold tolerance to the stress and consequence apoptosis compared to the control group. Also, they exhibited a higher IC50 compared to the control in the presence of oleuropein (5095 μM.ml-1 Vs. 2505 μM.ml-1). We found a significant increase in recombinant protein production levels in manipulated cells, even in the presence of 1,000 μM oleuropein compared to the control cell line (p-value=0.0002).
Conclusions: CRISPR/Cas9 assisted BAX gene ablation is promising to improve erythropoietin production in CHO cells via engineering anti-apoptotic genes. Therefore, exploiting genome editing tools such as CRISPR/Cas9 has been proposed to develop host cells that result in a safe, feasible, and robust manufacturing operation with a yield that meets the industrial requirements.
کلیدواژه‌های انگلیسی مقاله Apoptosis,Bax gene,CRISPR/Cas9,Cell longevity,Gene Editing,Oleuropein
نویسندگان مقاله Amirabbsa Rahimi |
Laboratory of regenerative medicine and biomedical innovations, National Cell Bank Department, Pasteur Institute of Iran, Tehran, Iran

Morteza Karimipour |
Molecular Medicine Department, Pasteur Institute of Iran, Tehran, Iran

Reza Mahdian |
Molecular Medicine Department, Pasteur Institute of Iran, Tehran, Iran

Atefeh Alipour |
Department of Nano-Biotechnology, Pasteur Institute of Iran, Tehran, Iran

Saadi Hosseini |
Laboratory of regenerative medicine and biomedical innovations, National Cell Bank Department, Pasteur Institute of Iran, Tehran, Iran

Marzieh Mohammadi |
Molecular Medicine Department, Pasteur Institute of Iran, Tehran, Iran

Hooman Kaghazian |
Department of Research & Development, Production & Research Complex, Pasteur Institute of Iran, Tehran, Iran.

Abdolrahim Abbasi |
Institute of Human Virology, University of Maryland School of Medicine, Baltimore, MD

Hosein Shahsavarani |
Department of Cell and Molecular Biology, Shahid Beheshti University, Tehran, Iran.

Mohammad Ali Shokrgozar |
Laboratory of regenerative medicine and biomedical innovations, National Cell Bank Department, Pasteur Institute of Iran, Tehran, Iran

نشانی اینترنتی https://www.ijbiotech.com/article_167568_81233f3a2b05caeee153baa41295dc29.pdf
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