| Project/Area Number |
18390034
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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 University, Fac. Pharm. Sci, Associate Professor (10204629)
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| Co-Investigator(Kenkyū-buntansha) |
AKITA Hidetaka Hokkaido University, Fac Pharm. Sci, Associate Professor (80344472)
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| Project Period (FY) |
2006 – 2007
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| Project Status |
Completed (Fiscal Year 2007)
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| Budget Amount *help |
¥13,130,000 (Direct Cost: ¥11,600,000、Indirect Cost: ¥1,530,000)
Fiscal Year 2007: ¥6,630,000 (Direct Cost: ¥5,100,000、Indirect Cost: ¥1,530,000)
Fiscal Year 2006: ¥6,500,000 (Direct Cost: ¥6,500,000)
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| Keywords | nonviral vector / exogenous DNA / gene therapy / intranuclear disposition / transgene expression |
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| Research Abstract |
Naked luciferase-plasmid DNA was delivered into mouse liver by hydrodynamics-based injection, an modifications of the histones bound to the plasmid DNA were analyzed by a chromatin immunoprecipitation (ChIP) analysis. In addition, the effects of a second hydrodynamics-based injection on the expression from the plasmid DNA were examined The ChIP analysis revealed that the modification status of histone H3 remained constant from 4 hr t 4 weeks. Surprisingly, the injection of saline without DNA enhanced the luciferase expression from the preexisting DNA administered 4 and 14 days previously. Our results suggest that histone modification plays no role in the silencing. Instead, our data suggest that the transgene expression is activated by the hydrodynamics-based injection manipulation, and that the return from the activated status causes the silencing.
The effects of a left-handedly curved sequence ([CATGTTTTT]_n, n=20-40) with high histone affinity on plasmid expression were examined in
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vivo. A naked luciferase-plasmid was delivered into mouse liver by a hydrodynamics-based injection, and the luciferase activities were quantitated at various time points. Plasmids containing the curved sequence markedly enhanced transgene expression and one with [CATGTTTTT]_<36> showed the highest expression. These results suggest that sequences with high histone affinity could control transgene expression from plasmids in vivo.
We also examined several hundred-base single-stranded (ss) DNA fragments for gene correction. The gene correction efficiency varied (0.8-9.3%), depending on target positions and sense/antisense strands. Sense ss DNA fragments corrected the target gene with equal or higher efficiencies as compared to their antisense counterparts. The target positions corrected with high efficiency by the sense fragments also tended to be corrected efficiently by the antisense fragments. These results suggest that the sense ss DNA fragments are useful for the correction of mutated genes. The variation in the correction efficiency may depend on the sequence of the target position in double-stranded DNA. Less
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