Abstract
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.
Highlights
Therapeutic effects of in vivo gene therapy depend on the spacio-temporal distribution of the transgene products
On average, about 95% of cytosines were methylated in the SssI-treated plasmid DNA, whereas no cytosines were found to be methylated in the untreated one, supporting the validity of the bisulfite sequencing method to estimate the methylation of plasmids
The pCMV-Luc or pCpG-Luc in either unmethylated or methylated form was injected into mice by the hydrodynamic injection method, and a large volume of saline was injected into the mice 3 days later to reactivate the silenced transgene expression (Figure 2)
Summary
Therapeutic effects of in vivo gene therapy depend on the spacio-temporal distribution of the transgene products. We reported that a plasmid expressing murine interferon (IFN)-γ under the control of CpG-free human elongation factor (EF)-1 promoter produced a sustained level of IFN-γ compared with that with a CMV promoter after hydrodynamic injection into mice [13,14,15,16,17,18]. Another suggested mechanism includes the interaction of histones with plasmid DNA. The methylation status of the plasmids has been evaluated, and the involvement of histone deacetylation and the integration into the genome have been examined in mice
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
