RNAi-mediated knockdown of Xist can rescue the impaired postimplantation development of cloned mouse embryos
RNAi-mediated knockdown of Xist can rescue the impaired postimplantation development of cloned mouse embryos
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DOI：
10.1073/pnas.1112664108
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发表时间：
2011-12-20
影响因子：
11.1
通讯作者：
Ogura, Atsuo
中科院分区：
文献类型：
--
作者：
Matoba, Shogo;Inoue, Kimiko;Ogura, Atsuo
通过体细胞核移植（SCNT）克隆哺乳动物是非常低效的。大多数SCNT产生的胚胎在植入后死亡，因为植入后胚胎发育过程中未识别的复杂表观遗传错误。在这里，我们确定了最上游水平的功能障碍，导致受损的发展克隆使用RNAi对Xist，一个基因负责X染色体失活（XCI）。将Xist特异性siRNA预先注射到重建的卵母细胞中，有效地纠正了桑椹胚阶段SCNT特异性异常Xist表达，但此后在囊胚阶段未能做到这一点。然而，我们发现，植入后不久，这种异常的XCI状态在克隆胚胎已被纠正自主在胚胎和胚外组织，可能是通过一个新建立的XCI控制植入后胚胎。胚胎移植实验表明，siRNA处理的胚胎早在胚胎第5.5天就显示出比对照高10倍的存活率，这种高存活率一直持续到足月，导致克隆效率显著提高（12%对对照的1%）。重要的是，与对照克隆不同，这些Xist-siRNA克隆在出生时仅显示出其基因表达的有限失调，表明植入前胚胎中Xist表达的校正对其出生后的正常性有长期影响。因此，与一般的假设相反，我们的研究结果表明，克隆胚胎的命运几乎完全取决于植入前的XCI状态。此外，我们的策略为哺乳动物SCNT克隆提供了一个有希望的突破，因为卵母细胞的RNAi治疗很容易适用于大多数哺乳动物物种。
Cloning mammals by somatic cell nuclear transfer (SCNT) is highly inefficient. Most SCNT-generated embryos die after implantation because of unidentified, complex epigenetic errors in the process of postimplantation embryonic development. Here we identify the most upstream level of dysfunction leading to impaired development of clones by using RNAi against Xist, a gene responsible for X chromosome inactivation (XCI). A prior injection of Xist-specific siRNA into reconstructed oocytes efficiently corrected SCNT-specific aberrant Xist expression at the morula stage, but failed to do so thereafter at the blastocyst stage. However, we found that shortly after implantation, this aberrant XCI status in cloned embryos had been corrected autonomously in both embryonic and extraembryonic tissues, probably through a newly established XCI control for postimplantation embryos. Embryo transfer experiments revealed that siRNA-treated embryos showed 10 times higher survival than controls as early as embryonic day 5.5 and this high survival persisted until term, resulting in a remarkable improvement in cloning efficiency (12% vs. 1% in controls). Importantly, unlike control clones, these Xist-siRNA clones at birth showed only a limited dysregulation of their gene expression, indicating that correction of Xist expression in preimplantation embryos had a long-term effect on their postnatal normality. Thus, contrary to the general assumption, our results suggest that the fate of cloned embryos is determined almost exclusively before implantation by their XCI status. Furthermore, our strategy provides a promising breakthrough for mammalian SCNT cloning, because RNAi treatment of oocytes is readily applicable to most mammal species.