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Novel DNAs for efficient and durable transgene expression

用于高效、持久转基因表达的新型 DNA

基本信息

批准号：
18390034
负责人：
KAMIYA Hiroyuki
金额：
$ 8.4万
依托单位：
Hokkaido University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
2006
资助国家：
日本
起止时间：
2006 至 2007
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-18390034/
关键词：
nonviral vector exogenous DNA gene therapy intranuclear disposition transgene expression

项目摘要

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 … More 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

通过基于流体动力学的注射将裸露的荧光素酶质粒 DNA 递送至小鼠肝脏中，通过染色质免疫沉淀 (ChIP) 分析来分析与质粒 DNA 结合的组蛋白的修饰。此外，还检查了第二次基于流体动力学的注射对质粒 DNA 表达的影响。ChIP 分析显示组蛋白 H3 的修饰状态从 4 小时到 4 周保持恒定。令人惊讶的是，注射不含DNA的盐水增强了4天和14天前施用的预先存在的DNA的荧光素酶表达。我们的结果表明组蛋白修饰在沉默中不起任何作用。相反，我们的数据表明转基因表达是通过基于流体动力学的注射操作激活的，并且从激活状态返回导致沉默。在体内检查了具有高组蛋白亲和力的左手弯曲序列（[CATGTTTTT]_n，n = 20-40）对质粒表达的影响。通过基于流体动力学的注射将裸露的荧光素酶质粒递送至小鼠肝脏，并在不同时间点对荧光素酶活性进行定量。含有弯曲序列的质粒显着增强了转基因表达，并且具有[CATGTTTTT]_<36>的质粒显示出最高的表达。这些结果表明，具有高组蛋白亲和力的序列可以控制体内质粒的转基因表达。我们还检查了数百个碱基的单链（ss）DNA片段以进行基因校正。基因校正效率各不相同（0.8-9.3%），具体取决于目标位置和有义/反义链。与反义片段相比，有义 ss DNA 片段以相同或更高的效率校正目标基因。由有义片段高效校正的目标位置也倾向于由反义片段高效校正。这些结果表明正义单链DNA片段对于突变基因的校正是有用的。校正效率的变化可能取决于双链DNA中目标位置的序列。较少的