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Transcript Profiling of Individual Twin Blastomeres Derived by Splitting Two-Cell Stage Murine Embryos

机译:分裂两个细胞阶段的小鼠胚胎衍生的单个双卵裂球的转录分析。

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摘要

In invertebrates and amphibians, informational macromolecules in egg cytoplasm are organized to provide direction to the formation of embryonic lineages, but it is unclear whether vestiges of such prepatterning exist in mammals. Here we examined whether twin blastomeres from 2-cell stage mouse embryos differ in mRNA content. mRNA from 26 blastomeres derived from 13 embryos approximately mid-way through their second cell cycle was subjected to amplification. Twenty amplified samples were hybridized to arrays. Of those samples that hybridized successfully, 12 samples in six pairs were used in the final analysis. Probes displaying normalized values >0.25 (n = 4573) were examined for consistent bias in expression within blastomere pairs. Although transcript content varied between both individual embryos and twin blastomeres, no consistent asymmetries were observed for the majority of genes, with only 178 genes displaying a >1.4-fold difference in expression across all six pairs. Although class discovery clustering showed that blastomere pairs separated into two distinct groups in terms of their differentially expressed genes, when the data were tested for significance of asymmetrical expression, only 39 genes with >1.4-fold change ratios in six of six blastomere pairs passed the two-sample t-test (P < 0.05). Transcripts encoding proteins implicated in RNA processing and cytoskeletal organization were among the most abundant, differentially distributed mRNA, suggesting that a stochastically based lack of synchrony in cell cycle progression between the two cells might explain at least some and possibly all of the asymmetries in transcript composition.
机译:在无脊椎动物和两栖动物中,卵细胞质中的信息大分子被组织起来,为胚胎谱系的形成提供了方向,但尚不清楚哺乳动物中是否存在这种预先构图的痕迹。在这里,我们检查了来自2细胞期小鼠胚胎的双卵裂球的mRNA含量是否不同。在第二个细胞周期的中期,对来自13个胚胎的26个卵裂球的mRNA进行扩增。将二十个扩增的样品杂交到阵列。在成功杂交的样品中,六对中的12个样品用于最终分析。检查显示归一化值> 0.25(n = 4573)的探针在卵裂球对中表达的一致偏倚。尽管转录物的含量在单个胚胎和双卵裂球之间都不同,但是大多数基因都没有观察到一致的不对称性,只有178个基因在所有六对中的表达差异均大于1.4倍。尽管类别发现聚类表明,卵裂球对根据其差异表达的基因分为两个不同的组,但是当测试数据的不对称表达的显着性时,六个卵裂球对中的六个中只有39个具有> 1.4倍变化率的基因通过了两样本t检验(P <0.05)。编码涉及RNA加工和细胞骨架组织的蛋白质的转录本是分布最丰富,差异最大的mRNA之一,这表明基于随机性的两细胞之间细胞周期进程缺乏同步可能解释了转录本组成中的至少一些甚至所有不对称性。

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