Silencing of episomal transgene expression in liver by plasmid bacterial backbone DNA is independent of CpG methylation
Silencing of episomal transgene expression in liver by plasmid bacterial backbone DNA is independent of CpG methylation
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DOI：
10.1038/sj.mt.6300399
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发表时间：
2008-03-01
影响因子：
12.4
通讯作者：
Kay, Mark A.
中科院分区：
文献类型：
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作者：
Chen, Zhi-Ying;Riu, Efren;Kay, Mark A.
缺乏质粒细菌骨架（BB）DNA的小环DNA载体在转录上是持续的，而它们的亲本质粒对应物在肝脏中是沉默的。在本研究中，我们确定了反式提供的环状质粒BB在体内不沉默转基因表达盒，进一步证实了我们先前的结论，即质粒BB与表达盒的共价连接是DNA沉默所需的。考虑到质粒BB中CpG二核苷酸的高浓度，我们研究了DNA甲基化对体内转基因沉默的作用。常规质粒BB中CpG基序甲基化的存在或不存在对转录沉默没有显著影响。此外，从BB中去除CpG基序并没有改善转录沉默。当使用常规质粒载体两端的鸡cHS 4绝缘子的两个串联拷贝时，转基因沉默被部分抑制。这些结果与以下观点一致，即在肝脏中用质粒DNA载体观察到的转录抑制是由质粒DNA骨架上形成抑制性异染色质引起的，其然后顺式扩散并灭活转基因，并且CpG含量或甲基化在该过程中几乎没有影响。
Minicircle DNA vectors devoid of plasmid bacterial backbone, ( BB) DNAs, are transcriptionally persistent, whereas their parent plasmid counterparts are silenced in the liver. In this study we establish that circular plasmid BB provided in trans did not silence a transgene expression cassette in vivo, further confirming our previous conclusions that the covalent attachment of the plasmid BB to the expression cassette is required for DNA silencing. Given the high concentration of CpG dinucleotides in the plasmid BB, we investigated the role of DNA methylation on transgene silencing in vivo. The presence or absence of methylation in CpG motifs in routine plasmid BBs had no significant effect on transcriptional silencing. Furthermore, the removal of the CpG motifs from the BB did not ameliorate transcriptional silencing. Transgene silencing was partially inhibited when two tandem copies of the chicken cHS4 insulator at each end of a routine plasmid vector were used. These results are consistent with the idea that the transcriptional repression observed with plasmid DNA vectors in the liver is caused by formation of repressive heterochromatin on the plasmid DNA backbone, which then spreads and inactivates the transgene in cis, and that CpG content or methylation has little or no influence in the process.