Fukuma Miki, Ganmyo Yuto, Miura Osamu, Ohyama Takashi, Shimizu Noriaki
Graduate School of Biosphere Science, Hiroshima University, Higashi-hiroshima, Hiroshima, Japan.
Graduate School of Advanced Science and Engineering, Waseda University, Shinjuku-ku, Tokyo, Japan.
PLoS One. 2016 Apr 14;11(4):e0153338. doi: 10.1371/journal.pone.0153338. eCollection 2016.
Plasmids bearing a mammalian replication initiation region (IR) and a nuclear matrix attachment region (MAR) are spontaneously amplified in transfected mammalian cells, and such amplification generates chromosomal homogeneously staining regions (HSRs) or extrachromosomal double minutes (DMs). This method provides a novel, efficient, and rapid way to establish cells that stably produce high levels of recombinant proteins. However, because IR/MAR plasmids are amplified as repeats, they are frequently targeted by repeat-induced gene silencing (RIGS), which silences a variety of repeated sequences in transgenes and the genome. To address this problem, we developed a novel screening system using the IR/MAR plasmid to isolate human genome sequences that alleviate RIGS. The screen identified a 3,271 bp sequence (B-3-31) that elevated transgene expression without affecting the amplification process. Neither non-B structure (i.e., the inverted repeats or bending) nor known epigenetic modifier elements such as MARs, insulators, UCOEs, or STARs could explain the anti-silencing activity of B-3-31. Instead, the activity was distributed throughout the entire B-3-31 sequence, which was extremely A/T-rich and CpG-poor. Because B-3-31 effectively and reproducibly alleviated RIGS of repeated genes, it could be used to increase recombinant protein production.
携带哺乳动物复制起始区域（IR）和核基质附着区域（MAR）的质粒在转染的哺乳动物细胞中会自发扩增，这种扩增会产生染色体均匀染色区（HSR）或染色体外双微体（DM）。该方法为建立稳定产生高水平重组蛋白的细胞提供了一种新颖、高效且快速的途径。然而，由于IR/MAR质粒以重复序列的形式扩增，它们经常成为重复序列诱导基因沉默（RIGS）的靶点，RIGS会使转基因和基因组中的各种重复序列沉默。为了解决这个问题，我们开发了一种新型筛选系统，利用IR/MAR质粒分离能够减轻RIGS的人类基因组序列。该筛选鉴定出一个3271 bp的序列（B-3-31），它能提高转基因表达而不影响扩增过程。非B结构（即反向重复或弯曲）以及已知的表观遗传修饰元件，如MAR、绝缘子、非甲基化保守元件（UCOE）或支架/基质附着区域（STAR），都无法解释B-3-31的抗沉默活性。相反，这种活性分布在整个B-3-31序列中，该序列富含A/T且CpG含量低。由于B-3-31有效且可重复地减轻了重复基因的RIGS，它可用于提高重组蛋白的产量。
