RRC ID 67371
Author Fukuma M, Ganmyo Y, Miura O, Ohyama T, Shimizu N.
Title Cloning and Characterization of a Human Genomic Sequence that Alleviates Repeat-Induced Gene Silencing.
Journal PLoS One
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.
Volume 11(4)
Pages e0153338
Published 2016-1-1
DOI 10.1371/journal.pone.0153338
PII PONE-D-16-00549
PMID 27078685
PMC PMC4831671
MeSH Animals Base Sequence CHO Cells Cloning, Molecular Cricetinae Cricetulus Gene Library Gene Silencing* Genome, Human* Humans Matrix Attachment Regions / genetics* Plasmids / genetics Plasmids / metabolism Recombinant Proteins / biosynthesis Recombinant Proteins / genetics Transfection
IF 2.74
DNA material pB-3-31 (RDB18708)