RNA interference is responsible for reduction of transgene expression after Sleeping Beauty transposase mediated somatic integration.
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 S...
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| Format: | Article |
| Language: | English |
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Public Library of Science (PLoS)
2012-01-01
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| Series: | PLoS ONE |
| Online Access: | http://europepmc.org/articles/PMC3343047?pdf=render |
| _version_ | 1819077296641802240 |
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| author | Christina Rauschhuber Anja Ehrhardt |
| author_facet | Christina Rauschhuber Anja Ehrhardt |
| author_sort | Christina Rauschhuber |
| collection | DOAJ |
| description | 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 transposon system, that displays only negligible transcriptional activity, similar numbers of CFUs were obtained. CONCLUSION: In summary, we provide evidence for the first time that after somatic integration transposon derived transgene expression is regulated by the endogenous RNAi machinery. In the future this finding will help to further improve the molecular design of the SB transposase vector system. |
| first_indexed | 2024-12-21T18:54:57Z |
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| id | doaj.art-4a281a2bc3c64f25b7ee0fc406eb8b8e |
| institution | Directory Open Access Journal |
| issn | 1932-6203 |
| language | English |
| last_indexed | 2024-12-21T18:54:57Z |
| publishDate | 2012-01-01 |
| publisher | Public Library of Science (PLoS) |
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| series | PLoS ONE |
| spelling | doaj.art-4a281a2bc3c64f25b7ee0fc406eb8b8e2022-12-21T18:53:39ZengPublic Library of Science (PLoS)PLoS ONE1932-62032012-01-0175e3538910.1371/journal.pone.0035389RNA interference is responsible for reduction of transgene expression after Sleeping Beauty transposase mediated somatic integration.Christina RauschhuberAnja EhrhardtBACKGROUND: 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 transposon system, that displays only negligible transcriptional activity, similar numbers of CFUs were obtained. CONCLUSION: In summary, we provide evidence for the first time that after somatic integration transposon derived transgene expression is regulated by the endogenous RNAi machinery. In the future this finding will help to further improve the molecular design of the SB transposase vector system.http://europepmc.org/articles/PMC3343047?pdf=render |
| spellingShingle | Christina Rauschhuber Anja Ehrhardt RNA interference is responsible for reduction of transgene expression after Sleeping Beauty transposase mediated somatic integration. PLoS ONE |
| title | RNA interference is responsible for reduction of transgene expression after Sleeping Beauty transposase mediated somatic integration. |
| title_full | RNA interference is responsible for reduction of transgene expression after Sleeping Beauty transposase mediated somatic integration. |
| title_fullStr | RNA interference is responsible for reduction of transgene expression after Sleeping Beauty transposase mediated somatic integration. |
| title_full_unstemmed | RNA interference is responsible for reduction of transgene expression after Sleeping Beauty transposase mediated somatic integration. |
| title_short | RNA interference is responsible for reduction of transgene expression after Sleeping Beauty transposase mediated somatic integration. |
| title_sort | rna interference is responsible for reduction of transgene expression after sleeping beauty transposase mediated somatic integration |
| url | http://europepmc.org/articles/PMC3343047?pdf=render |
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