| Project/Area Number |
15390034
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| Research Category |
Grant-in-Aid for Scientific Research (B)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
Drug development chemistry
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| Research Institution | HOKKAIDO UNIVERSITY |
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Principal Investigator |
KAMIYA Hiroyuki Hokkaido Univ., Grad.Sch.Pharm.Sci., Associate Professor, 大学院・薬学研究科, 助教授 (10204629)
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| Project Period (FY) |
2003 – 2005
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| Project Status |
Completed (Fiscal Year 2005)
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| Budget Amount *help |
¥9,400,000 (Direct Cost: ¥9,400,000)
Fiscal Year 2005: ¥2,700,000 (Direct Cost: ¥2,700,000)
Fiscal Year 2004: ¥2,700,000 (Direct Cost: ¥2,700,000)
Fiscal Year 2003: ¥4,000,000 (Direct Cost: ¥4,000,000)
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| Keywords | exogenous DNA / transgene expression / gene correction / controlled intranuclear disposition / 核内動態 / DNAダンベル / ループ配列 |
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| Research Abstract |
Naked luciferase-plasmid DNA was delivered into mouse liver by a hydrodynamics-based injection, and the amounts of intranuclear plasmid DNA, luciferase, and its mRNA were quantitated at various time points. Methylation of the promoter of the luciferase gene was also analyzed. Expression efficiency from one copy of the exogenous DNA dramatically decreased over time, and the DNA was methylated and degraded into fragments. Unexpectedly, methylation of the intact plasmid DNA was low and did not increase over time. Rather, the fragmented DNA was methylated more frequently than the intact plasmid. These results suggest that the CpG methylation and the degradation of exogenous DNA, and its 'silencing', occurred in parallel in the nucleus.
To control intranuclear disposition of plasmid DNA, replicating plasmid DNA was constructed. The replicating plasmid DNA was resistant to rapid decrease in its amount and expressed the luciferase gene more efficiently than the corresponding non-replicating plasmid. Plasmid DNAs containing a sequence that binds to the histone proteins were also constructed. The luciferase gene expression was enhanced when the histone-binding sequence was located in a proper position.
Additionally, single-stranded DNA fragment was prepared to control intranuclear disposition of gene correction devices. This novel DNA fragment corrected a target gene with more than 10-fold more efficiency as compared to the conventional double-stranded DNA fragment (PCR fragment). Further enhancement of the gene correction was achieved when another DNA fragment was designed based on in vitro experiments on a protein involved in homologous recombination. This would be results of improved availability of the gene correction device.
Importance of intranuclear disposition has been indicated in this study. We hope that the novel DNAs described here would be highly useful for gene therapy after further improvement.
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