Effect of the content of unmethylated CpG dinucleotides in plasmid DNA on the sustainability of transgene expression
Effect of the content of unmethylated CpG dinucleotides in plasmid DNA on the sustainability of transgene expression
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DOI：
10.1002/jgm.1317
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发表时间：
2009-05-01
影响因子：
3.5
通讯作者：
Takakura, Yoshinobu
中科院分区：
文献类型：
--
作者：
Mitsui, Masaru;Nishikawa, Makiya;Takakura, Yoshinobu
背景非病毒基因转移通常受到转基因短期表达的影响。我们先前已经证明，具有降低的CpG含量的质粒表现出更长的鼠表达。干扰素（IFN）-β或IFN-γ，其有效抑制转移性肿瘤生长。通过控制质粒DNA中CpG基序的数量和位置，可以进一步延长转基因表达的持续时间。方法将不同CpG含量的荧光素酶表达质粒通过流体动力学注射法注射到小鼠尾静脉。研究了CpG含量对转基因表达持续时间的影响，重点是胞嘧啶甲基化和促炎细胞因子。在此基础上，构建IFN-γ表达质粒，观察其转基因表达和对肺转移的抑制作用。结果含少量CpG基序的质粒在肝脏中表现出较长的荧光素酶活性。质粒中CpG基序的甲基化降低了表达，并且对于具有高CpG含量的质粒，这种降低的程度更大。促炎细胞因子对表达影响不大。IFN-γ表达质粒pCpG-Mu-γ仅在cDNA区域含有20个CpG基序，其IFN-γ浓度可维持在治疗水平，对Minor细胞的肺转移有明显的抑制作用。结论通过降低IFN-γ表达质粒载体的CpG含量，成功地延长了IFN-γ的转基因表达时间，这导致IFN基因转移的抗癌活性增加。版权所有（C）2009约翰威利父子有限公司
Background Nonviral gene transfer generally suffers from short-term expression of transgenes. We have previously demonstrated that plasmids with reduced CpG content exhibited a more prolonged expression of murine. interferon (IFN)-beta or IFN-gamma, which was effective in inhibiting metastatic tumor growth. A further extension of the duration of transgene expression could be achieved by controlling the number and location of CpG motifs in plasmid DNA.Methods Luciferase-expressing plasmids with differing CpG content were injected into the tail vein of mice by the hydrodynamic injection method. The effects of CpG content on the duration of transgene expression were examined, focusing on cytosine methylation and pro-inflammatory cytokines. Based on the findings, IFN-gamma-expressing plasmids were constructed and their transgene expression and inhibitory effect oil pulmonary metastasis were evaluated.Results Plasmids with a few CpG motifs showed a prolonged luciferase activity in the liver. Methylation of CpG motifs in plasmids reduced the expression and the extent of this reduction was greater for plasmids with a high CpG content. Pro-inflammatory cytokines hardly affected the expression. pCpG-Mu gamma, the IFN-gamma-expressing plasmid, which contains 20 CpG motifs only in the cDNA region, exhibited a sustained IFN-gamma concentration at therapeutic levels, and had a great inhibitory effect on the pulmonary metastasis of Minor cells.Conclusions The duration of transgene expression of IFN-gamma was successfully increased by reducing the CpG content of IFN-expressing plasmid vector, which resulted in art increased anticancer activity of IFN gene transfer. Copyright (C) 2009 John Wiley & Sons, Ltd.