Iranian Biomedical Journal

en Engineering of the Caspase-3 Gene in Recombinant CHO Cells Caused More Apoptosis Resistance and enhanced Recombinant Protein Production Than the BAX Gene Related Fields Related Fields مقاله کامل Full Length/Original Article <div style="text-align: justify;">Background: BAX and caspase-3 are essential genes in the apoptotic pathway of cells, promoting the apoptotic cascade through different mechanisms. Inhibition of these genes can increase the longevity of cells in cell culture. This study aimed to compare the effects of clustered regularly interspaced short palindromic repeats-CRISPR-associated protein 9 (CRISPR-Cas9)-mediated knockdown of BAX and caspase-3 genes on apoptosis inhibition, cell lifespan, and erythropoietin (EPO) production in CHO cell lines. Methods: The BAX and caspase-3 gene expression was evaluated in the recombinant Chinese hamster ovary (rCHO) cell lines producing EPO using the CRISPR-Cas9 method. Their anti-apoptotic effects and the level of EPO expression were also compared. In addition, oleuropein (OP) as an apoptosis inducer, was introduced to the manipulated cell line to assess the stability and viability of the manipulated cell lines. Results: The rCHO cells with the manipulated BAX gene exhibited a higher cell density than those with the manipulated caspase-3 gene (152% vs. 142%). Despite the increased cell density in the cells with the BAX gene manipulation, EPO production was higher in the cells with the manipulated caspase-3 gene (1.58-fold increase in the BAX-manipulated cells compared to a 1.70-fold increase in the caspase-3-manipulated cells). Conclusion: Our observations suggest that the downregulation of the BAX and caspase-3 genes using the CRISPR method, inhibits apoptosis and enhances the yield of recombinant EPO, even in the presence of an apoptosis inducer. Additionally, reduction of caspase-3 expression was proved to be more effective than BAX in extending the lifespan of cells and producing heterologous recombinant proteins. </div> Apoptosis, BAX, Caspase-3, CRISPR-associated protein 9 149 158 http://ibj.pasteur.ac.ir/browse.php?a_code=A-10-797-3&slc_lang=en&sid=1 Amirabbas Rahimi sokrgozar1967@gmail.com 0000-0002-1982-8081 No Molecular Medicine Department, Pasteur Institute of Iran, Tehran, Iran Morteza Karimipoor shkrgozar1967@gmail.com 0000-0002-2406-5963 No Molecular Medicine Department, Pasteur Institute of Iran, Tehran, Iran Reza Mahdian shorgozar1967@gmail.com 0000-0003-1458-4767 No Molecular Medicine Department, Pasteur Institute of Iran, Tehran, Iran Atefeh Alipour shokgozar1967@gmail.com 0000-0003-1837-4150 No Department of Nano-Biotechnology, Pasteur Institute of Iran, Tehran, Iran Saadi Hosseini shokrozar1967@gmail.com 0000-0002-2411-7903 No Laboratory of Regenerative Medicine and Biomedical Innovations, National Cell Bank Department, Pasteur Institute of Iran, Tehran, Iran Marzieh Mohammadi shokrgzar1967@gmail.com 0009-0002-2518-7433 No Molecular Medicine Department, Pasteur Institute of Iran, Tehran, Iran Hooman Kaghazian shokrgoar1967@gmail.com 0000-0003-4301-3381 No Department of Research and Development, Production and Research Complex, Pasteur Institute of Iran, Tehran, Iran Hosein Shahsavarani shokrgozr1967@gmail.com 0000-0002-8123-0857 No Department of Cell and Molecular Biology, Shahid Beheshti University, Tehran, Iran Mohammad Ali Shokrgozar shokrgozar1967@gmail.com 0000-0001-5123-4155 Yes Laboratory of Regenerative Medicine and Biomedical Innovations, National Cell Bank Department, Pasteur Institute of Iran, Tehran, Iran