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首页> 外文期刊>Molecular and Cellular Biology >Retroviral Expression in Embryonic Stem Cells and Hematopoietic Stem Cells
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Retroviral Expression in Embryonic Stem Cells and Hematopoietic Stem Cells

机译：逆转录病毒在胚胎干细胞和造血干细胞中的表达

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Achieving long-term retroviral expression in primary cells has been problematic. De novo DNA methylation of infecting proviruses has been proposed as a major cause of this transcriptional repression. Here we report the development of a mouse stem cell virus (MSCV) long terminal repeat-based retroviral vector that is expressed in both embryonic stem (ES) cells and hematopoietic stem (HS) cells. Infected HS cells and their differentiated descendants maintained long-term and stable retroviral expression after serial adoptive transfers. In addition, retrovirally infected ES cells showed detectable expression level of the green fluorescent protein (GFP). Moreover, GFP expression of integrated proviruses was maintained after in vitro differentiation of infected ES cells. Long-term passage of infected ES cells resulted in methylation-mediated silencing, while short-term expression was methylation independent. Tissues of transgenic animals, which we derived from ES cells carrying the MSCV-based provirus, did not express GFP. However, treatment with the demethylating agent 5-azadeoxycytidine reactivated the silent provirus, demonstrating that DNA methylation is involved in the maintenance of retroviral repression. Our results indicate that retroviral expression in ES cells is repressed by methylation-dependent as well as methylation-independent mechanisms.
机译：在原代细胞中实现长期逆转录病毒表达一直是有问题的。已经提出感染原病毒的从头DNA甲基化是这种转录抑制的主要原因。在这里，我们报告小鼠干细胞病毒（MSCV）基于长末端重复序列的逆转录病毒载体的发展，该载体在胚胎干（ES）细胞和造血干（HS）细胞中表达。在连续的过继转移后，感染的HS细胞及其分化后代保持长期稳定的逆转录病毒表达。此外，逆转录病毒感染的ES细胞显示出可检测的绿色荧光蛋白（GFP）表达水平。而且，在感染的ES细胞体外分化后，整合的原病毒的GFP表达得以维持。感染的ES细胞的长期传代会导致甲基化介导的沉默，而短期表达则独立于甲基化。我们从携带基于MSCV的原病毒的ES细胞衍生的转基因动物组织不表达GFP。然而，用去甲基化剂5-氮杂脱氧胞苷进行的处理重新激活了沉默的原病毒，表明DNA甲基化参与了逆转录病毒抑制的维持。我们的结果表明，逆转录病毒在ES细胞中的表达受到甲基化依赖性和甲基化依赖性机制的抑制。

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