Epigenetic regulation of lentiviral transgene vectors in a large animal model
Epigenetic regulation of lentiviral transgene vectors in a large animal model
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DOI：
10.1016/j.ymthe.2005.07.685
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发表时间：
2006-01-01
影响因子：
12.4
通讯作者：
Pfeifer, A
中科院分区：
文献类型：
--
作者：
Hofmann, A;Kessler, B;Pfeifer, A
转基因动物对于遗传学研究和开发人类疾病的新疗法具有突出的相关性。最近的发展是通过慢病毒基因转移产生转基因动物。到目前为止，关于慢病毒转基因的研究主要集中在第一代（创始人或F0）动物上，其中大多数携带多个整合体。在这里，我们分析了分离到F1代后慢病毒转基因猪中单个整合体的转基因表达和表观遗传调控。出乎意料的是，三分之一的慢病毒整合体表现出低表达水平和高甲基化，如甲基化敏感的Southern印迹和亚硫酸氢盐测序所示。前病毒甲基化密度与表达水平呈负相关。此外，用DNA甲基化酶抑制剂5-azacyticline处理分离的转基因成纤维细胞诱导平均荧光强度（MFI）从8增加到26.1，增加了三倍。用组蛋白去乙酰化酶抑制剂阿司他丁A治疗使MFI仅增加到11.1。总之，慢病毒整合体在高等哺乳动物中的表达受表观遗传修饰的调节。与先前的预期相反，DNA甲基化在慢病毒表达中起重要作用。
Transgenic animals are of outstanding relevance for genetic studies and the development of novel therapies for human diseases. A recent development is the generation of transgenic animals by lentiviral gene transfer. So far, studies on lentiviral transgenesis focused on first-generation (founder or F0) animals-most of which carry multiple integrants. Here, we analyze transgene expression and epigenetic regulation of individual integrants in lentiviral transgenic pigs after segregation to the F1 generation. Unexpectedly, one-third of lentiviral integrants exhibited low expression levels and were hypermethylated, as demonstrated by methylation-sensitive Southern blotting and bisulfite sequencing. Proviral methylation density correlated inversely with expression levels. In addition, treatment of isolated transgenic fibroblasts with the DNA methylase inhibitor 5-azacyticline induced a threefold increase in mean fluorescence intensity (MFI) from 8 to 26.1. Treatment with the histone deacetylase inhibitor trichostatin A enhanced MFI to only 11.1. Taken together, expression of lentiviral integrants in higher mammals is regulated by epigenetic modifications. In contrast to previous expectations, DNA methylation plays an important role in lentiviral expression.