Auditioning of CHO host cell lines using the artificial chromosome expression (ACE) technology
Auditioning of CHO host cell lines using the artificial chromosome expression (ACE) technology
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使用人工染色体表达 (ACE) 技术试听 CHO 宿主细胞系
DOI：
10.1002/bit.22407
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发表时间：
2009
影响因子：
3.8
通讯作者：
J. Mott
中科院分区：
文献类型：
--
作者：
M. Kennard;D. Goosney;Diane Monteith;S. Roe;David Fischer;J. Mott
为了最大限度地提高重组蛋白在哺乳动物细胞中的表达，需要考虑许多因素，例如转染方法、载体构建、筛选技术和培养条件。此外，宿主细胞系可以对蛋白质表达产生深远的影响。然而，试听或直接比较宿主细胞系以获得最佳蛋白质表达可能很困难，因为大多数转染方法是基于将感兴趣的基因随机整合到宿主细胞基因组中。因此，不可能确定不同宿主细胞系之间的表达差异是否是由于宿主细胞本身的表型或遗传因素（例如基因拷贝数或基因位置）造成的。为了改善细胞系的生成，ACE 系统是基于具有多个重组受体位点的预工程人工染色体而开发的。该系统可实现靶向转染，并已有效用于快速生成表达高水平单克隆抗体的稳定 CHO 细胞系。 ACE 系统的一个关键特征是能够分离和纯化含有感兴趣基因的 ACE，并将相同的 ACE 转染到不同的宿主细胞系中。此功能允许直接试听宿主细胞，因为宿主细胞已被在相同遗传环境中包含相同数量基因拷贝的 ACE 转染。为了研究这一试听特征，使用含有人类单克隆 IgG1 抗体基因拷贝的相同 ACE 转染了三种 CHO 宿主细胞系（CHOK1SV、CHO-S 和 DG44）。生成克隆细胞系，可以直接比较 CHO 宿主细胞之间的抗体表达和稳定性。结果显示，CHOK1SV 宿主细胞系表达的抗体水平分别是 DG44 和 CHO-S 宿主细胞系的两到五倍以上。为了确认 ACE 本身不是造成 CHO-S 克隆中抗体表达低的原因，从这些细胞中分离和纯化 ACE，然后转染回新鲜的 CHOK1SV 细胞中。与 CHO-S 中的表达相比，新转染至 CHOK1SV 的 ACE 产生的抗体表达增加了五倍，并证实不同 CHO 宿主细胞之间的表达差异是由于细胞表型而不是基因拷贝数和/或位置的差异。这些结果证明了 ACE 系统在提供快速、直接的技术来试听宿主细胞系以获得最佳重组蛋白表达方面的实用性。生物技术。生物工程。 2009年； 104：526–539 © 2009 Wiley 期刊公司。
In order to maximize recombinant protein expression in mammalian cells many factors need to be considered such as transfection method, vector construction, screening techniques and culture conditions. In addition, the host cell line can have a profound effect on the protein expression. However, auditioning or directly comparing host cell lines for optimal protein expression may be difficult since most transfection methods are based on random integration of the gene of interest into the host cell genome. Thus it is not possible to determine whether differences in expression between various host cell lines are due to the phenotype of the host cell itself or genetic factors such as gene copy number or gene location. To improve cell line generation, the ACE System was developed based on pre‐engineered artificial chromosomes with multiple recombination acceptor sites. This system allows for targeted transfection and has been effectively used to rapidly generate stable CHO cell lines expressing high levels of monoclonal antibody. A key feature of the ACE System is the ability to isolate and purify ACEs containing the gene(s) of interest and transfect the same ACEs into different host cell lines. This feature allows the direct auditioning of host cells since the host cells have been transfected with ACEs that contain the same number of gene copies in the same genetic environment. To investigate this audition feature, three CHO host cell lines (CHOK1SV, CHO‐S and DG44) were transfected with the same ACE containing gene copies of a human monoclonal IgG1 antibody. Clonal cell lines were generated allowing a direct comparison of antibody expression and stability between the CHO host cells. Results showed that the CHOK1SV host cell line expressed antibody at levels of more than two to five times that for DG44 and CHO‐S host cell lines, respectively. To confirm that the ACE itself was not responsible for the low antibody expression seen in the CHO‐S based clones, the ACE was isolated and purified from these cells and transfected back into fresh CHOK1SV cells. The resulting expression of the antibody from the ACE newly transfected into CHOK1SV increased fivefold compared to its expression in CHO‐S and confirmed that the differences in expression between the different CHO host cells was due to the cell phenotype rather than differences in gene copy number and/or location. These results demonstrate the utility of the ACE System in providing a rapid and direct technique for auditioning host cell lines for optimal recombinant protein expression. Biotechnol. Bioeng. 2009; 104: 526–539 © 2009 Wiley Periodicals, Inc.
影响因子：
2.6
作者：
Vanderbyl, SL;Sullenbarger, B;Lasky, LC
通讯作者：
Lasky, LC