Silencing of episomal transgene expression by plasmid bacterial DNA elements in vivo
Silencing of episomal transgene expression by plasmid bacterial DNA elements in vivo
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DOI：
10.1038/sj.gt.3302231
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发表时间：
2004-05-01
期刊：
影响因子：
5.1
通讯作者：
Kay, MA
中科院分区：
文献类型：
--
作者：
Chen, ZY;He, CY;Kay, MA
我们之前已经证明，通过细菌无dna表达盒可以持续提高转基因产物的表达水平，这种表达盒可以由小鼠肝脏中的碎片质粒形成，也可以作为微环载体传递。这表明细菌DNA序列在体外转基因沉默中发挥了作用。为了进一步了解沉默机制，我们系统地改变了表达盒和细菌骨干中的DNA成分，并比较了接受不同DNA形式的小鼠的基因表达谱。在测试的9个载体中，与2fDNA组相比，单独接受纯化表达盒的动物总是持续表达更高水平的转基因。相比之下，通过在细菌主干上进行一次切割而获得线性化DNA的动物具有与完整质粒组相似的表达谱。这三种线性dna在小鼠肝脏中形成大串联体和小圆圈，而ccDNA保持完整。在所有组中，肝脏中载体DNA的相对量保持相似。总之，这些结果进一步证实了DNA沉默效应是由表达盒和细菌DNA元件的共价键介导的。
We previously demonstrated that sustainable enhanced levels of transgene products could be expressed from a bacterial DNA-free expression cassette either formed from a fragmented plasmid in mouse liver or delivered as a minicircle vector. This suggested that bacterial DNA sequences played a role in episomal transgene silencing. To further understand the silencing mechanism, we systematically altered the DNA components in both the expression cassette and the bacterial backbone, and compared the gene expression profiles from mice receiving different DNA forms. In nine vectors tested, animals that received the purified expression cassette alone always expressed persistently higher levels of transgene compared to 2fDNA groups. In contrast, animals that received linearized DNA by a single cut in the bacterial backbone had similar expression profiles to that of intact plasmid groups. All three linear DNAs formed large concatemers and small circles in mouse liver, while ccDNA remained intact. In all groups, the relative amount of vector DNA in liver remained similar. Together, these results further established that the DNA silencing effect was mediated by a covalent linkage of the expression cassette and the bacteria DNA elements.