RNAi-mediated knockdown of Xist does not rescue the impaired development of female cloned mouse embryos.
RNAi-mediated knockdown of Xist does not rescue the impaired development of female cloned mouse embryos.
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DOI：
10.1262/jrd.2012-195
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发表时间：
2013
期刊：
影响因子：
--
通讯作者：
Ogura A
中科院分区：
文献类型：
--
作者：
Oikawa M;Matoba S;Inoue K;Kamimura S;Hirose M;Ogonuki N;Shiura H;Sugimoto M;Abe K;Ishino F;Ogura A
在小鼠中，与体细胞核移植（SCNT）相关的主要表观遗传错误之一是在着床前两性中Xist的异位表达。我们发现这种异常的Xist表达可以通过从供体细胞中假定的活性X染色体上删除Xist而受到阻碍。在雄性克隆中，也发现预先注射Xist特异性siRNA可以显著改善克隆胚胎的植入后发育，这是由于桑葚胚期Xist的显著抑制。在这项研究中，我们检查了相同的敲除策略是否在雌性scnt衍生胚胎中也能起作用。卵母细胞激活后6-7 h，用卵丘细胞核重建胚胎，注射xist特异性siRNA。RNA FISH分析显示，siRNA处理成功抑制了桑葚胚期的Xist RNA，这表明卵裂球核中云型Xist信号的数量显著减少。然而，不同大小的卵裂球（从“针状”到“云状”）和Xist RNA信号的数量仍然存在于单个胚胎中。植入后，失调的Xist表达自主正常化，如在雄性克隆中，在胚胎和胚胎外组织中均为单等位基因表达状态。然而，在足月，注射了sirna的克隆胚胎的存活率没有显著提高。因此，siRNA注射在很大程度上有效地抑制了Xist在雌性克隆胚胎中的过度表达，但未能拯救它们，可能是因为无法模仿这些胚胎中一致的单等位基因Xist表达。这只能通过应用基因敲除策略而不是siRNA方法在女性胚胎中实现。
In mice, one of the major epigenetic errors associated with somatic cell nuclear transfer (SCNT) is ectopic expression of Xist during the preimplantation period in both sexes. We found that this aberrant Xist expression could be impeded by deletion of Xist from the putative active X chromosome in donor cells. In male clones, it was also found that prior injection of Xist-specific siRNA could significantly improve the postimplantation development of cloned embryos as a result of a significant repression of Xist at the morula stage. In this study, we examined whether the same knockdown strategy could work as well in female SCNT-derived embryos. Embryos were reconstructed with cumulus cell nuclei and injected with Xist-specific siRNA at 6–7 h after oocyte activation. RNA FISH analysis revealed that siRNA treatment successfully repressed Xist RNA at the morula stage, as shown by the significant decrease in the number of cloud-type Xist signals in the blastomere nuclei. However, blastomeres with different sizes (from “pinpoint” to “cloud”) and numbers of Xist RNA signals remained within single embryos. After implantation, the dysregulated Xist expression was normalized autonomously, as in male clones, to a state of monoallelic expression in both embryonic and extraembryonic tissues. However, at term there was no significant improvement in the survival of the siRNA-injected cloned embryos. Thus, siRNA injection was largely effective in repressing the Xist overexpression in female cloned embryos but failed to rescue them, probably because of an inability to mimic consistent monoallelic Xist expression in these embryos. This could only be achieved in female embryos by applying a gene knockout strategy rather than an siRNA approach.
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