Novel DNAs for efficient and durable transgene expression

通过基于流体动力学的注射将裸DNA酶质粒DNA递送到小鼠肝脏中，通过染色质免疫沉淀（ChIP）分析结合到质粒DNA的组蛋白的修饰。此外，第二次基于流体动力学的注射对质粒DNA表达的影响进行了检查。ChIP分析显示，组蛋白H3的修饰状态从4小时到4周保持不变。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。我们的研究结果表明，组蛋白修饰在沉默中不起作用。相反，我们的数据表明，转基因表达被基于流体动力学的注射操作激活，并且从激活状态的返回导致沉默。 ...更多信息 vivo.通过基于流体动力学的注射将裸荧光素酶质粒递送到小鼠肝脏中，并在不同时间点定量荧光素酶活性。含有弯曲序列的质粒显着增强转基因表达，并且具有[CATGTTTTT]_的质粒<36>显示最高表达。这些结果表明，具有高组蛋白亲和力的序列可以控制转基因质粒在体内的表达。我们还检查了几个百碱基的单链（ss）DNA片段的基因校正。基因校正效率变化（0.8-9.3%），取决于靶位置和有义/反义链。正义ss DNA片段与它们的反义片段相比以相等或更高的效率校正靶基因。通过有义片段高效校正的靶位置也倾向于通过反义片段有效校正。这些结果表明，正义ss DNA片段是有用的突变基因的校正。校正效率的变化可能取决于双链DNA中靶位置的序列。少

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