原创力文档rna interference is responsible for reduction of transgene expression after sleeping beauty transposase mediated somatic integrationrna干扰负责减少转基因表达的睡美人后转座酶介导体集成.pdfVIP
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rna interference is responsible for reduction of transgene expression after sleeping beauty transposase mediated somatic integrationrna干扰负责减少转基因表达的睡美人后转座酶介导体集成
RNA Interference Is Responsible for Reduction of
Transgene Expression after Sleeping Beauty Transposase
Mediated Somatic Integration
1 1,2
Christina Rauschhuber , Anja Ehrhardt *
1 Max von Pettenkofer-Institute, Department of Virology, Ludwig-Maximilians-University Munich, Munich, Germany, 2 Institute of Virology and Microbiology, Center for
Biomedical Education and Research, Department of Human Medicine, Faculty of Health, University Witten/Herdecke, Witten, Germany
Abstract
Background: Integrating non-viral vectors based on transposable elements are widely used for genetically engineering
mammalian cells in functional genomics and therapeutic gene transfer. For the Sleeping Beauty (SB) transposase system it
was demonstrated that convergent transcription driven by the SB transposase inverted repeats (IRs) in eukaryotic cells
occurs after somatic integration. This could lead to formation of double-stranded RNAs potentially presenting targets for
the RNA interference (RNAi) machinery and subsequently resulting into silencing of the transgene. Therefore, we aimed at
investigating transgene expression upon transposition under RNA interference knockdown conditions.
Principal Findings: To establish RNAi knockdown cell lines we took advantage of the P19 protein, which is derived from the
tomato bushy stunt virus. P19 binds and inhibits 21 nucleotides long, small-interfering RNAs and was shown to sufficiently
suppress RNAi. We found that transgene expression upon SB mediated transposition was enhanced, resulting into a 3.2-fold
increased amount of colony forming units (CFU) after transposition. In contrast, if the transgene cassette is insulated from
the influence of chromosomal position effects by the chicken-derived cHS4 insulating sequences or when applying the Forg
Prince tra
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