Helmholtz Centre for Infection Research, Braunschweig, Germany.
BMC Biotechnol. 2009 Dec 14;9:100. doi: 10.1186/1472-6750-9-100.
Recombinant protein expression in mammalian cells is mostly achieved by stable integration of transgenes into the chromosomal DNA of established cell lines. The chromosomal surroundings have strong influences on the expression of transgenes. The exploitation of defined loci by targeting expression constructs with different regulatory elements is an approach to design high level expression systems. Further, this allows to evaluate the impact of chromosomal surroundings on distinct vector constructs.
We explored antibody expression upon targeting diverse expression constructs into previously tagged loci in CHO-K1 and HEK293 cells that exhibit high reporter gene expression. These loci were selected by random transfer of reporter cassettes and subsequent screening. Both, retroviral infection and plasmid transfection with eGFP or antibody expression cassettes were employed for tagging. The tagged cell clones were screened for expression and single copy integration. Cell clones producing > 20 pg/cell in 24 hours could be identified. Selected integration sites that had been flanked with heterologous recombinase target sites (FRTs) were targeted by Flp recombinase mediated cassette exchange (RMCE). The results give proof of principle for consistent protein expression upon RMCE. Upon targeting antibody expression cassettes 90-100% of all resulting cell clones showed correct integration. Antibody production was found to be highly consistent within the individual cell clones as expected from their isogenic nature. However, the nature and orientation of expression control elements revealed to be critical. The impact of different promoters was examined with the tag-and-targeting approach. For each of the chosen promoters high expression sites were identified. However, each site supported the chosen promoters to a different extent, indicating that the strength of a particular promoter is dominantly defined by its chromosomal context.
RMCE provides a powerful method to specifically design vectors for optimized gene expression with high accuracy. Upon considering the specific requirements of chromosomal sites this method provides a unique tool to exploit such sites for predictable expression of biotechnologically relevant proteins such as antibodies.
在哺乳动物细胞中，重组蛋白的表达主要通过将转基因稳定整合到已建立的细胞系的染色体 DNA 中实现。染色体周围环境对转基因的表达有很强的影响。通过使用不同的调控元件靶向表达构建体来利用定义的基因座，是设计高表达系统的一种方法。此外，这允许评估染色体周围环境对不同载体构建体的影响。
我们探索了在 CHO-K1 和 HEK293 细胞中靶向不同表达构建体到先前标记的基因座时的抗体表达，这些细胞系表现出高报告基因表达。这些基因座是通过随机转移报告基因盒并随后筛选选择的。使用逆转录病毒感染和质粒转染 eGFP 或抗体表达盒来进行标记。对标记的细胞克隆进行表达和单拷贝整合筛选。可以鉴定出 24 小时内产生 >20pg/细胞的细胞克隆。选择侧翼带有异源重组酶靶位点（FRTs）的标记整合位点，通过 Flp 重组酶介导的盒交换（RMCE）进行靶向。结果为 RMCE 后一致的蛋白质表达提供了原理证明。在靶向抗体表达盒时，所有产生的细胞克隆中有 90-100%显示出正确的整合。如预期的那样，由于它们的同源性质，在单个细胞克隆中发现抗体产生非常一致。然而，表达控制元件的性质和方向被证明是关键的。通过标记和靶向方法检查了不同启动子的影响。对于所选的每个启动子，都确定了高表达位点。然而，每个位点都以不同的程度支持所选的启动子，表明特定启动子的强度主要由其染色体环境决定。
RMCE 为具有高准确性的优化基因表达提供了一种强大的方法。在考虑染色体位点的具体要求的情况下，该方法为利用这些位点预测表达生物技术相关蛋白（如抗体）提供了独特的工具。
