Epi-CHO, an episomal expression system for recombinant protein production in CHO cells
Epi-CHO, an episomal expression system for recombinant protein production in CHO cells
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DOI：
10.1002/bit.20534
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发表时间：
2005-09-20
影响因子：
3.8
通讯作者：
Sunstrom, NA
中科院分区：
文献类型：
--
作者：
Kunaparaju, R;Liao, M;Sunstrom, NA
本研究描述了基于自主复制和转染质粒 DNA 保留的 CHO 细胞瞬时表达系统的开发。允许质粒染色体外扩增的瞬时表达系统允许更多的质粒拷贝在整个生产阶段持续存在于转染细胞中，从而导致转基因表达显着增加。该表达系统名为 Epi-CHO，包含 (1) 稳定转染多瘤病毒 (Py) 大 T (LT) 抗原基因 (PyLT) 的 CHO-K1 细胞系和 (2) DNA 表达载体 pPyEBV，其编码 Py 起点 (PyOri) 用于自主质粒扩增，并编码 Epstein-Barr 病毒 (EBV) 核抗原 1 (EBNA-1) 和 OriP 用于质粒保留。表达 PyLT 的 CHO-K1 细胞系（称为 CHO-T）适合在无血清培养基中悬浮生长，以促进大规模瞬时转染和重组基因表达。使用增强型绿色荧光蛋白（EGFP）和人生长激素（hGH）作为报告蛋白来证明转基因表达和生产力。用编码 hGH 的载体 pPyEBV 转染悬浮生长的 CHO-T 细胞，转染后 11 天培养物上清液中 hGH 的终浓度为 75 mg L-1。 (c) 2005 年 Wiley 期刊公司。
This study describes the development of a transient expression system for CHO cells based on autonomous replication and retention of transfected plasmid DNA. A transient expression system that allows extrachromosomal amplification of plasmids permits more plasmid copies to persist in the transfected cell throughout the production phase leading to a significant increase in transgene expression. The expression system, named Epi-CHO comprises (1) a CHO-K1 cell line stably transfected with the Polyomavirus (Py) large T (LT) antigen gene (PyLT) and (2) a DNA expression vector, pPyEBV encoding the Py origin (PyOri) for autonomous plasmid amplification and encoding Epstein-Barr Virus (EBV) nuclear antigen-1 (EBNA-1) and OriP for plasmid retention. The CHO-K1 cell line expressing PyLT, named CHO-T was adapted to suspension growth in serum-free media to facilitate large-scale transient transfection and recombinant gene expression. Enhanced green fluorescent protein (EGFP) and human growth hormone (hGH) were used as reporter proteins to demonstrate transgene expression and productivity. Transfection of suspension-growing CHO-T cells with the vector pPyEBV encoding hGH resulted in a final concentration of 75 mg L-1 of hGH in culture supernatants 11 days following transfection. (c) 2005 Wiley Periodicals, Inc.