title: Cloning and Characterization of a Human Genomic Sequence that Alleviates Repeat-Induced Gene Silencing.
abstract: 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.
authors.affiliation: Graduate School of Biosphere Science, Hiroshima University, Higashi-hiroshima, Hiroshima, Japan.
authors.forename: Miki
authors.initials: M
authors.lastname: Fukuma
authors.affiliation: Graduate School of Biosphere Science, Hiroshima University, Higashi-hiroshima, Hiroshima, Japan.
authors.forename: Yuto
authors.initials: Y
authors.lastname: Ganmyo
authors.affiliation: Graduate School of Advanced Science and Engineering, Waseda University, Shinjuku-ku, Tokyo, Japan.
authors.forename: Osamu
authors.initials: O
authors.lastname: Miura
authors.affiliation: Graduate School of Advanced Science and Engineering, Waseda University, Shinjuku-ku, Tokyo, Japan.
authors.forename: Takashi
authors.initials: T
authors.lastname: Ohyama
authors.affiliation: Graduate School of Biosphere Science, Hiroshima University, Higashi-hiroshima, Hiroshima, Japan.
authors.forename: Noriaki
authors.initials: N
authors.lastname: Shimizu
mesh_terms: D000818:Animals; D001483:Base Sequence; D016466:CHO Cells; D003001:Cloning, Molecular; D006224:Cricetinae; D003412:Cricetulus; D015723:Gene Library; D020868:Gene Silencing; D015894:Genome, Human; D006801:Humans; D045170:Matrix Attachment Regions / Q000235:genetics*; D010957:Plasmids / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics; D014162:Transfection
chemical_list: D011994:Recombinant Proteins