宿主顺式介导的逆转录病毒在分化过程中于胚胎干细胞中表达的许可性的消除。
Host cis-mediated extinction of a retrovirus permissive for expression in embryonal stem cells during differentiation.
作者信息
Laker C, Meyer J, Schopen A, Friel J, Heberlein C, Ostertag W, Stocking C
机构信息
Abteilung für Zell- und Virusgenetik, Heinrich-Pette-Institut für Experimentelle Virologie und Immunologie an der Universität Hamburg, Germany.
出版信息
J Virol. 1998 Jan;72(1):339-48. doi: 10.1128/JVI.72.1.339-348.1998.
The use of retroviral vectors for gene transfer into animals has been severely hampered by the lack of provirus transcription in the early embryo and embryonic stem (ES) cells. This primary block in provirus expression is maintained in differentiated cells by a cis-acting mechanism that is not well characterized. Retroviral vectors based on the murine embryonal stem cell virus (MESV), which overcome the transcriptional block in ES cells, were constructed to investigate this secondary mechanism. These vectors transferred G418 resistance to ES cells with the same efficiency as to fibroblasts, but overall transcript levels were greatly reduced. A mosaic but stable expression pattern was observed when single cells from G418-resistant clones were replated in G418 or assayed for expression of LacZ or interleukin-3. The expression levels in independent clones were variable and correlated inversely with methylation. However, a second, more pronounced, block to transcription was found upon differentiation induction. Differentiation of the infected ES cells to cells permissive for retroviral expression resulted in repression and complete extinction of provirus expression. Extinction was not accompanied by increased levels of methylation. Provirus expression is thus regulated by two independent cis-acting mechanisms: (i) partial repression in the undifferentiated state, accompanied by increased methylation but compatible with long-term, low expression of retroviral genes, and (ii) total repression and extinction during early stages of differentiation, apparently independent of changes in methylation. These results indicate a time window early during the transition from an undifferentiated to a differentiated stage in which provirus expression is silenced. The mechanisms are presently unknown, but elucidation of these events will have an important impact on vector development for targeting stem cells and for gene therapy.
逆转录病毒载体用于将基因导入动物的过程，因早期胚胎和胚胎干细胞（ES细胞）中缺乏前病毒转录而受到严重阻碍。前病毒表达的这一主要障碍在分化细胞中通过一种尚未完全明确的顺式作用机制得以维持。构建了基于小鼠胚胎干细胞病毒（MESV）的逆转录病毒载体，以研究这一二级机制，该载体克服了ES细胞中的转录障碍。这些载体将G418抗性转移至ES细胞的效率与转移至成纤维细胞的效率相同，但总体转录水平大幅降低。当将来自G418抗性克隆的单细胞重新接种于含G418的培养基中，或检测其LacZ或白细胞介素-3的表达时，观察到一种镶嵌但稳定的表达模式。独立克隆中的表达水平各不相同，且与甲基化呈负相关。然而，在诱导分化时发现了对转录的第二个、更明显的障碍。受感染的ES细胞分化为允许逆转录病毒表达的细胞会导致前病毒表达受到抑制并完全消失。消失过程中甲基化水平并未升高。因此，前病毒表达受两种独立的顺式作用机制调控：（i）在未分化状态下部分抑制，伴随甲基化增加，但与逆转录病毒基因的长期低表达相容；（ii）在分化早期完全抑制并消失，显然与甲基化变化无关。这些结果表明，在从未分化阶段向分化阶段转变的早期存在一个时间窗口，在此期间前病毒表达被沉默。目前这些机制尚不清楚，但阐明这些事件将对靶向干细胞的载体开发和基因治疗产生重要影响。
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