Sorourian Soofia, Behzad-Behbahani Abbas, Forouzanfar Mohsen, Jafarinia Mojtaba, Safari Fatemeh
Department of Biology, Marvdasht Branch, Islamic Azad University, Marvdasht, Iran.
Diagnostic Laboratory Sciences and Technology Research Center, School of Paramedical Sciences, Shiraz University of Medical Sciences, Shiraz, Iran.
Mol Biol Res Commun. 2025;14(1):15-26. doi: 10.22099/mbrc.2024.50513.2000.
Chinese Hamster Ovary (CHO) cells are essential in biopharmaceutical manufacturing. Scientists use CRISPR to enhance productivity. mRNAs contain UTRs that regulate gene expression, affecting protein abundance. Targeting these regions creates desirable knockout cells. The Caspase 8 Associated Protein 2 () gene is a promising target for improving host cell viability. This study used the CRISPR-Homology-Independent Targeted Integration (HITI) strategy to modify the 3'UTR region of the gene in CHO cells. The aim was to evaluate the effects of silencing on cell proliferation, viability, apoptosis, and the cell cycle. silencing was assessed post-modification by extracting genomic DNA from modified and unmodified CHO cells, followed by PCR and sequencing to confirm deletions. Cell proliferation and viability were measured using MTT assays, and cell cycle analysis was performed via flow cytometry. Apoptosis was evaluated through Annexin V/PE staining and flow cytometry, with apoptosis resistance assessed by determining the IC of sodium butyrate. Results showed deletion did not affect cell proliferation or the cell cycle but improved CHO cell viability and increased resistance to apoptosis. The IC for sodium butyrate was higher in knockout cells (7.84 mM) compared to native cells (3.43 mM), indicating enhanced apoptosis resistance. This study highlights 's role in apoptosis regulation without impacting cell proliferation or the cell cycle. deletion enhances viability and resistance to apoptosis, suggesting it as a target for improving recombinant protein production. Further research is needed to elucidate the molecular mechanisms and develop therapeutic strategies based on this approach.
中国仓鼠卵巢（CHO）细胞在生物制药生产中至关重要。科学家们使用CRISPR来提高其生产力。信使核糖核酸（mRNA）包含调控基因表达、影响蛋白质丰度的非翻译区（UTR）。靶向这些区域可产生理想的基因敲除细胞。半胱天冬酶8相关蛋白2（）基因是提高宿主细胞活力的一个有前景的靶点。本研究采用CRISPR-同源性独立靶向整合（HITI）策略对CHO细胞中的基因3'UTR区域进行修饰。目的是评估基因沉默对细胞增殖、活力、凋亡和细胞周期的影响。修饰后，通过从修饰和未修饰的CHO细胞中提取基因组DNA，然后进行聚合酶链反应（PCR）和测序以确认缺失，来评估基因沉默情况。使用噻唑蓝（MTT）法测量细胞增殖和活力，并通过流式细胞术进行细胞周期分析。通过膜联蛋白V/藻红蛋白（Annexin V/PE）染色和流式细胞术评估凋亡情况，通过测定丁酸钠的半数抑制浓度（IC）来评估抗凋亡能力。结果显示，基因缺失不影响细胞增殖或细胞周期，但提高了CHO细胞的活力并增强了抗凋亡能力。与天然细胞（3.43 mM）相比，基因敲除细胞中丁酸钠的IC更高（7.84 mM），表明抗凋亡能力增强。本研究突出了基因在不影响细胞增殖或细胞周期的情况下对凋亡调控的作用。基因缺失增强了活力和抗凋亡能力，表明它是改善重组蛋白生产的一个靶点。需要进一步研究以阐明分子机制并基于此方法开发治疗策略。
