Zahn-Zabal M, Kobr M, Girod P A, Imhof M, Chatellard P, de Jesus M, Wurm F, Mermod N
Laboratory of Molecular Biotechnology, Center for Biotechnology UNIL-EPFL, University of Lausanne, CBUE, DC-IGC, CH-1015, Lausanne, Switzerland.
J Biotechnol. 2001 Apr 27;87(1):29-42. doi: 10.1016/s0168-1656(00)00423-5.
One of the major hurdles of isolating stable, inducible or constitutive high-level producer cell lines is the time-consuming selection procedure. Given the variation in the expression levels of the same construct in individual clones, hundreds of clones must be isolated and tested to identify one or more with the desired characteristics. Various boundary elements (BEs), matrix attachment regions, and locus control regions (LCRs) were screened for their ability to augment the expression of heterologous genes in Chinese hamster ovary (CHO) cells. Of the chromatin elements assayed, the chicken lysozyme matrix-attachment region (MAR) was the only element to significantly increase stable reporter expression. We found that the use of the MAR increases the proportion of high-producing clones, thus reducing the number of clones that need to be screened. These benefits are observed both for constructs with MARs flanking the transgene expression cassette, as well as when constructs are co-transfected with the MAR on a separate plasmid. Moreover, the MAR was co-transfected with a multicomponent regulatable beta-galactosidase expression system in C2C12 cells and several clones exhibiting regulated expression were identified. Hence, MARs are useful in the development of stable cell lines for production or regulated expression.
分离稳定、可诱导或组成型高表达细胞系的主要障碍之一是耗时的筛选过程。鉴于同一构建体在各个克隆中的表达水平存在差异，必须分离并测试数百个克隆，以鉴定出一个或多个具有所需特性的克隆。对各种边界元件（BE）、基质附着区域和位点控制区域（LCR）增强中国仓鼠卵巢（CHO）细胞中异源基因表达的能力进行了筛选。在所检测的染色质元件中，鸡溶菌酶基质附着区域（MAR）是唯一能显著增加稳定报告基因表达的元件。我们发现，使用MAR可提高高表达克隆的比例，从而减少需要筛选的克隆数量。无论是转基因表达盒两侧带有MAR的构建体，还是构建体与MAR在单独质粒上共转染时，都能观察到这些益处。此外，MAR与C2C12细胞中的多组分可调节β-半乳糖苷酶表达系统共转染，并鉴定出了几个表现出调节表达的克隆。因此，MAR在用于生产或调节表达的稳定细胞系的开发中很有用。
