Retroviral expression in embryonic stem cells and hematopoietic stem cells
Retroviral expression in embryonic stem cells and hematopoietic stem cells
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DOI：
10.1128/mcb.20.20.7419-7426.2000
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发表时间：
2000-10-01
影响因子：
5.3
通讯作者：
Jaenisch, R
中科院分区：
文献类型：
--
作者：
Cherry, SR;Biniszkiewicz, D;Jaenisch, R
在原代细胞中实现逆转录病毒的长期表达一直是个问题。感染前病毒的从头DNA甲基化被认为是这种转录抑制的主要原因。在这里，我们报道了一种基于小鼠干细胞病毒(MSCV)长末端重复序列的逆转录病毒载体的研制，该载体在胚胎干细胞(ES)和造血干细胞(HS)中都能表达。感染的HS细胞及其分化的后代在连续过继转移后保持了长期稳定的逆转录病毒表达。此外，逆转录病毒感染的ES细胞显示出可检测到的绿色荧光蛋白(GFP)表达水平。此外，整合的前病毒在感染的ES细胞体外分化后仍能保持GFP的表达。感染ES细胞的长期传代导致甲基化介导的沉默，而短期表达不依赖于甲基化。我们从携带基于MSCV的前病毒的ES细胞中提取的转基因动物的组织不表达GFP。然而，用去甲基化试剂5-氮杂脱氧胞苷重新激活沉默的前病毒，证明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和甲基化非依赖机制的抑制。
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.