Damavandi Narges, Raigani Mozhgan, Joudaki Atefeh, Davami Fatemeh, Zeinali Sirous
Department of Biotechnology, College of Science, University of Tehran, Tehran, Iran; Biotechnology Research Center, Pasteur Institute of Iran, Tehran, Iran; Kawsar Genomics and Biotech Center, Tehran, Iran.
Biotechnology Research Center, Pasteur Institute of Iran, Tehran, Iran.
Protein Expr Purif. 2017 Dec;140:60-64. doi: 10.1016/j.pep.2017.08.002. Epub 2017 Aug 9.
The Chinese Hamster Ovary (CHO) cell lines, applicable to post-translational modifications, are preferred systems for biopharmaceutical protein production. In this study, by using the Jump-In™ TI™ technology which employs PhiC31 and R4 bacteriophage recombinases, a platform CHO-K1 cell line containing a R4-attP site was generated. Here, a combination of Quantitative Fluorescent-Polymerase Chain Reaction (QF-PCR) and semi-random, two-step PCR (ST-PCR), was performed to feature the platform cell clones. Our results show that QF-PCR and ST-PCR, can be utilized for efficient and accelerated cell line characterization. By applying these approaches, we were able to accurately identify the copy number of integrated R4-attP sites and the genomic position of recombination of many clones. Three novel PhiC31 pseudo-attP sites were identified on chromosomes 1, 3 and 6, and their genomic features were analyzed. The characterized platform cell lines are stable, and because of single-copy, site-specific R4 recognition attP site, the cell lines could be retargeted for recombinant protein production and drug discovery applications.
中国仓鼠卵巢（CHO）细胞系适用于翻译后修饰，是生物制药蛋白质生产的首选系统。在本研究中，通过使用采用PhiC31和R4噬菌体重组酶的Jump-In™ TI™技术，构建了一个含有R4-attP位点的平台CHO-K1细胞系。在此，采用定量荧光聚合酶链反应（QF-PCR）和半随机两步PCR（ST-PCR）相结合的方法对平台细胞克隆进行表征。我们的结果表明，QF-PCR和ST-PCR可用于高效、快速的细胞系鉴定。通过应用这些方法，我们能够准确鉴定许多克隆中整合的R4-attP位点的拷贝数和重组的基因组位置。在1号、3号和6号染色体上鉴定出三个新的PhiC31假attP位点，并对其基因组特征进行了分析。所表征的平台细胞系是稳定的，并且由于单拷贝、位点特异性的R4识别attP位点，这些细胞系可重新用于重组蛋白生产和药物发现应用。
