Recombinant protein expression by targeting pre-selected chromosomal loci.
Recombinant protein expression by targeting pre-selected chromosomal loci.
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DOI：
10.1186/1472-6750-9-100
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发表时间：
2009-12-14
影响因子：
3.5
通讯作者：
Wirth D
中科院分区：
文献类型：
--
作者：
Nehlsen K;Schucht R;da Gama-Norton L;Krömer W;Baer A;Cayli A;Hauser H;Wirth D
重组蛋白在哺乳动物细胞中的表达主要是通过将转基因稳定整合到已建立的细胞系的染色体DNA中实现的。染色体环境对转基因的表达有很强的影响。通过靶向具有不同调控元件的表达结构来利用已定义的基因座是设计高水平表达系统的一种方法。此外，这允许评估染色体环境对不同载体构建的影响。我们探索了在CHO-K1和HEK293细胞中将不同的表达结构定位于先前标记的位点后抗体的表达情况，这些细胞表现出高报告基因表达。这些基因座是通过随机转移报告盒和随后的筛选来选择的。分别采用逆转录病毒感染和携带绿色荧光蛋白或抗体表达盒的质粒转染法进行标记。对标记的细胞克隆进行表达和单拷贝整合筛选。可鉴定出24小时内产生20 pg/cell的细胞克隆。通过FLP重组酶介导的盒式交换(RMCE)，筛选出与异源重组酶靶点(FRT)相邻的整合位点。这些结果为在RMCE上实现一致的蛋白质表达提供了原理依据。在靶向抗体表达盒后，所有得到的细胞克隆中有90%-100%显示出正确的整合。抗体的产生在单个细胞克隆中被发现是高度一致的，正如它们的同源性质所预期的那样。然而，表达调控元件的性质和方向被揭示为关键。不同启动子的影响通过标记和目标定位的方法进行了检验。对于每个选定的启动子，都确定了高表达部位。然而，每个位点对所选启动子的支持程度不同，这表明特定启动子的强度主要由其染色体背景决定。RMCE为高精度的基因优化表达载体的设计提供了一种强有力的方法。在考虑到染色体位点的特殊要求后，该方法提供了一种独特的工具来利用这些位点来预测抗体等与生物技术相关的蛋白质的表达。
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.
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