Contribution of Epigenetic Modifications to the Decline in Transgene Expression from Plasmid DNA in Mouse Liver
Short-term expression of transgenes is one of the problems frequently associated with non-viral in vivo gene transfer. To obtain experimental evidence for the design of sustainable transgene expression systems, the contribution of epigenetic modifications to the decline in transgene expression needs...
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MDPI AG
2015-08-01
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| Series: | Pharmaceutics |
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| Online Access: | http://www.mdpi.com/1999-4923/7/3/199 |
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| author | Lei Zang Makiya Nishikawa Mitsuru Ando Yuki Takahashi Yoshinobu Takakura |
| author_facet | Lei Zang Makiya Nishikawa Mitsuru Ando Yuki Takahashi Yoshinobu Takakura |
| author_sort | Lei Zang |
| collection | DOAJ |
| description | Short-term expression of transgenes is one of the problems frequently associated with non-viral in vivo gene transfer. To obtain experimental evidence for the design of sustainable transgene expression systems, the contribution of epigenetic modifications to the decline in transgene expression needs to be investigated. Bisulfite sequencing and reactivation by hydrodynamic injection of isotonic solution were employed to investigate methylation statues of CpG in transiently expressing plasmid, pCMV-Luc, in mouse liver after hydrodynamic delivery. The cytosines of CpGs in the promoter region of pCMV-Luc were methylated in mouse liver, but the methylation was much later than the decline in the expression. The expression from pre-methylated pCMV-Luc was insensitive to reactivation. Neither an inhibitor of DNA methylation nor an inhibitor of histone deacetylation had significant effects on transgene expression after hydrodynamic injection of pCMV-Luc. Partial hepatectomy, which reduces the transgene expression from the non-integrated vector into the genome, significantly reduced the transgene expression of human interferon γ from a long-term expressing plasmid pCpG-Huγ, suggesting that the CpG-reduced plasmid was not significantly integrated into the genomic DNA. These results indicate that the CpG-reduced plasmids achieve prolonged transgene expression without integration into the host genome, although the methylation status of CpG sequences in plasmids will not be associated with the prolonged expression. |
| first_indexed | 2024-04-11T12:59:22Z |
| format | Article |
| id | doaj.art-5ad0dfe1cab84b4d9431db122d12c98f |
| institution | Directory Open Access Journal |
| issn | 1999-4923 |
| language | English |
| last_indexed | 2024-04-11T12:59:22Z |
| publishDate | 2015-08-01 |
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| spelling | doaj.art-5ad0dfe1cab84b4d9431db122d12c98f2022-12-22T04:23:00ZengMDPI AGPharmaceutics1999-49232015-08-017319921210.3390/pharmaceutics7030199pharmaceutics7030199Contribution of Epigenetic Modifications to the Decline in Transgene Expression from Plasmid DNA in Mouse LiverLei Zang0Makiya Nishikawa1Mitsuru Ando2Yuki Takahashi3Yoshinobu Takakura4Department of Biopharmaceutics and Drug Metabolism, Graduate School of Pharmaceutical Sciences, Kyoto University, Sakyo-ku, Kyoto 606-8501, JapanDepartment of Biopharmaceutics and Drug Metabolism, Graduate School of Pharmaceutical Sciences, Kyoto University, Sakyo-ku, Kyoto 606-8501, JapanDepartment of Biopharmaceutics and Drug Metabolism, Graduate School of Pharmaceutical Sciences, Kyoto University, Sakyo-ku, Kyoto 606-8501, JapanDepartment of Biopharmaceutics and Drug Metabolism, Graduate School of Pharmaceutical Sciences, Kyoto University, Sakyo-ku, Kyoto 606-8501, JapanDepartment of Biopharmaceutics and Drug Metabolism, Graduate School of Pharmaceutical Sciences, Kyoto University, Sakyo-ku, Kyoto 606-8501, JapanShort-term expression of transgenes is one of the problems frequently associated with non-viral in vivo gene transfer. To obtain experimental evidence for the design of sustainable transgene expression systems, the contribution of epigenetic modifications to the decline in transgene expression needs to be investigated. Bisulfite sequencing and reactivation by hydrodynamic injection of isotonic solution were employed to investigate methylation statues of CpG in transiently expressing plasmid, pCMV-Luc, in mouse liver after hydrodynamic delivery. The cytosines of CpGs in the promoter region of pCMV-Luc were methylated in mouse liver, but the methylation was much later than the decline in the expression. The expression from pre-methylated pCMV-Luc was insensitive to reactivation. Neither an inhibitor of DNA methylation nor an inhibitor of histone deacetylation had significant effects on transgene expression after hydrodynamic injection of pCMV-Luc. Partial hepatectomy, which reduces the transgene expression from the non-integrated vector into the genome, significantly reduced the transgene expression of human interferon γ from a long-term expressing plasmid pCpG-Huγ, suggesting that the CpG-reduced plasmid was not significantly integrated into the genomic DNA. These results indicate that the CpG-reduced plasmids achieve prolonged transgene expression without integration into the host genome, although the methylation status of CpG sequences in plasmids will not be associated with the prolonged expression.http://www.mdpi.com/1999-4923/7/3/199transgene expressionCpGDNA methylationhistone deacetylationhydrodynamic injection |
| spellingShingle | Lei Zang Makiya Nishikawa Mitsuru Ando Yuki Takahashi Yoshinobu Takakura Contribution of Epigenetic Modifications to the Decline in Transgene Expression from Plasmid DNA in Mouse Liver Pharmaceutics transgene expression CpG DNA methylation histone deacetylation hydrodynamic injection |
| title | Contribution of Epigenetic Modifications to the Decline in Transgene Expression from Plasmid DNA in Mouse Liver |
| title_full | Contribution of Epigenetic Modifications to the Decline in Transgene Expression from Plasmid DNA in Mouse Liver |
| title_fullStr | Contribution of Epigenetic Modifications to the Decline in Transgene Expression from Plasmid DNA in Mouse Liver |
| title_full_unstemmed | Contribution of Epigenetic Modifications to the Decline in Transgene Expression from Plasmid DNA in Mouse Liver |
| title_short | Contribution of Epigenetic Modifications to the Decline in Transgene Expression from Plasmid DNA in Mouse Liver |
| title_sort | contribution of epigenetic modifications to the decline in transgene expression from plasmid dna in mouse liver |
| topic | transgene expression CpG DNA methylation histone deacetylation hydrodynamic injection |
| url | http://www.mdpi.com/1999-4923/7/3/199 |
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