Harraghy Niamh, Buceta Montserrat, Regamey Alexandre, Girod Pierre-Alain, Mermod Nicolas
Laboratory of Molecular Biotechnology, University of Lausanne, Lausanne, Switzerland.
Methods Mol Biol. 2012;801:93-110. doi: 10.1007/978-1-61779-352-3_7.
Chinese hamster ovary (CHO) cells are the system of choice for the production of complex molecules, such as monoclonal antibodies. Despite significant progress in improving the yield from these cells, the process to the selection, identification, and maintenance of high-producing cell lines remains cumbersome, time consuming, and often of uncertain outcome. Matrix attachment regions (MARs) are DNA sequences that help generate and maintain an open chromatin domain that is favourable to transcription and may also facilitate the integration of several copies of the transgene. By incorporating MARs into expression vectors, an increase in the proportion of high-producer cells as well as an increase in protein production are seen, thereby reducing the number of clones to be screened and time to production by as much as 9 months. In this chapter, we describe how MARs can be used to increase transgene expression and provide protocols for the transfection of CHO cells in suspension and detection of high-producing antibody cell clones.
中国仓鼠卵巢（CHO）细胞是生产复杂分子（如单克隆抗体）的首选系统。尽管在提高这些细胞的产量方面取得了显著进展，但选择、鉴定和维持高产细胞系的过程仍然繁琐、耗时，而且结果往往不确定。基质附着区域（MARs）是有助于产生和维持有利于转录的开放染色质结构域的DNA序列，还可能促进转基因多个拷贝的整合。通过将MARs整合到表达载体中，可以看到高产细胞的比例增加以及蛋白质产量提高，从而将需要筛选的克隆数量和生产时间减少多达9个月。在本章中，我们描述了如何利用MARs提高转基因表达，并提供了悬浮CHO细胞转染及高产抗体细胞克隆检测的实验方案。
