An efficient and targeted gene integration system for high-level antibody expression
An efficient and targeted gene integration system for high-level antibody expression
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DOI：
10.1016/j.jim.2007.01.022
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发表时间：
2007-04-30
影响因子：
2.2
通讯作者：
Shen, Bei Fen
中科院分区：
文献类型：
--
作者：
Huang, Ying;Li, Yan;Shen, Bei Fen
连接基因组扩增的随机整合已被用来产生稳定和高水平表达重组抗体所需的细胞系。但这项技术费时费力，而且由于位置效应，表达水平是不可预测的。在这里，我们使用FRT/FLP策略构建了一个用于高水平抗体表达的细胞-载体系统，以克服位置效应。关键是将FRT序列定位于转录和基因扩增速度较快的染色体位置，并保持扩增的基因。为了筛选具有高转录活性和可扩增能力的目的基因座，将两个弱化标记(可选择的半乳糖苷酶和可扩增的二氢叶酸还原酶，DHFR)和FRT序列同步克隆到一个质粒中。将该表达载体导入中国仓鼠卵巢宿主细胞系，从721个克隆中筛选出20个候选细胞系。构建了携带FRT融合潮霉素基因的抗体基因打靶载体，通过FLP重组酶将抗体基因打靶到染色体FRT位点。20个细胞系中有3个可以作为定点重组的宿主细胞。采用Southern杂交和荧光原位杂交技术，筛选出37号候选工程细胞系。它的基因组中只有一个带有FRT标记的基因座。FISH分析表明，抗体基因均位于基因靶向和基因扩增细胞系基因组中FRT标记的原始位点。在候选细胞株37中成功表达了3种重组抗体。在转瓶中连续培养6天，最高产量可达200微克/毫升以上。(C)2007 Elsevier B.V.保留所有权利。
Random integration linking genomic amplification has been used to generate desired cell lines for stable and high-level expressing recombinant antibodies. But this technique is laborious, and the expression level is unpredictable due to position effects. Here, we have constructed a cell-vector system for high-level antibody expression using an FRT/FLP strategy to overcome position effects. The key is to target the FRT sequence to chromosomal locations where there is a high rate of transcription and gene amplification, and the amplified genes can be maintained. To screen desired loci with high transcriptional activity and amplifiable capacity, dual weakened markers (selectable galactosidase and amplifiable dihydrofolate reductase, DHFR) and the FRT sequence were synchronously cloned into a plasmid. After transfection of a Chinese hamster ovary host cell line with this plasmid, we selected 20 candidate cell lines from 721 individual clones. An antibody gene-targeting vector carrying an FRT-fused hygromycin gene was constructed to target antibody genes into the chromosomal FRT site by FLP recombinase. Three out of 20 cell lines can be used as host cells for site-specific recombination. By using southern blot and fluorescence in situ hybridization (FISH), a candidate engineered cell line, number 37, was chosen. It contains a single FRT-tagged locus in its genome. FISH analysis indicated that the antibody genes were all located at the original FRT-tagged locus in the genome of the gene-targeted and gene-amplified cell lines. Three kinds of recombinant antibodies were successfully expressed in candidate cell line 37. The highest producers produced more than 200 mu g/ml of the antibody in 6 days of continuous culture in a spinner flask. (C) 2007 Elsevier B.V. All rights reserved.