Kawabe Yoshinori, Inao Takanori, Komatsu Shodai, Huang Guan, Ito Akira, Omasa Takeshi, Kamihira Masamichi
Department of Chemical Engineering, Faculty of Engineering, Kyushu University, 744 Motooka, Nishi-ku, Fukuoka 819-0395, Japan.
Graduate School of Systems Life Sciences, Kyushu University, 744 Motooka, Nishi-ku, Fukuoka 819-0395, Japan.
J Biosci Bioeng. 2017 Mar;123(3):390-397. doi: 10.1016/j.jbiosc.2016.10.011. Epub 2016 Nov 14.
Chinese hamster ovary (CHO) cells are one of the most useful host cell lines for the production of biopharmaceutical proteins. Although a series of production processes have been refined to improve protein productivity and cost performance, establishing producer cells is still time-consuming and labor-intensive. Recombinase-mediated site-specific gene integration into a predetermined chromosomal locus may enable predictable protein expression, reducing the laborious process of cell screening. We previously developed an accumulative site-specific gene integration system (AGIS) using Cre recombinase and mutated loxP sites for transgene integration and amplification in the CHO cell genome. Epigenetic modifier elements such as insulators are effective DNA cis-regulatory elements for stabilizing transgene expression. Here, we attempted to enhance transgene expression in recombinant CHO cells generated by AGIS using a production enhancer DNA element (PE) derived from the CHO genome. The PE was introduced into an expression unit for a recombinant scFv-Fc antibody. The effect on scFv-Fc productivity of PE position and orientation within the transgene was evaluated, while keeping the background chromosomal structure constant. For the optimal PE arrangement, scFv-Fc productivity was enhanced 2.6-fold compared with an expression unit without a PE. The enhancing effect of the PE on transgene expression was also observed when two or three PE-flanked expression units were inserted as tandem repeats. These results indicate that AGIS using the PE-flanked expression unit is a promising approach for establishing producer cell lines for biopharmaceutical protein production.
中国仓鼠卵巢（CHO）细胞是生产生物制药蛋白最有用的宿主细胞系之一。尽管一系列生产工艺已经得到优化以提高蛋白生产力和成本效益，但建立生产细胞仍然耗时且劳动强度大。重组酶介导的位点特异性基因整合到预定的染色体位点可能使蛋白表达具有可预测性，从而减少细胞筛选的繁琐过程。我们之前开发了一种累积位点特异性基因整合系统（AGIS），该系统利用Cre重组酶和突变的loxP位点在CHO细胞基因组中进行转基因整合和扩增。诸如绝缘子等表观遗传修饰元件是稳定转基因表达的有效的DNA顺式调控元件。在此，我们尝试使用源自CHO基因组的生产增强子DNA元件（PE）来增强由AGIS产生的重组CHO细胞中的转基因表达。将PE引入重组单链抗体-融合蛋白（scFv-Fc）抗体的表达单元中。在保持背景染色体结构不变的情况下，评估了PE在转基因内的位置和方向对scFv-Fc生产力的影响。对于最佳的PE排列，与没有PE的表达单元相比，scFv-Fc生产力提高了2.6倍。当两个或三个侧翼带有PE的表达单元串联重复插入时，也观察到了PE对转基因表达的增强作用。这些结果表明，使用侧翼带有PE的表达单元的AGIS是建立用于生物制药蛋白生产的生产细胞系的一种有前景的方法。
