Studies on developmental aberration of somatic clones by global gene expression analysis

体细胞克隆并不总是导致哺乳动物的个体发育，发育通常与各种异常和胚胎丢失有关。这被认为是由于表观遗传重编程错误导致的异常基因表达。本研究旨在探索与体细胞克隆小鼠胚胎发育缺陷相关的基因。为了解决这一问题，我们进行了全面的候选基因的筛选，使用ES克隆囊胚差减分析，并使用2-细胞胚胎的差异显示分析。差减分析共检测到218个差异表达基因。其中198个克隆成功测序，通过BLAST搜索分析，158个基因被检测为已知基因。为了确认差异表达，我们通过实时PCR进行了定量基因表达分析。结果表明，10个基因中有5个在ES克隆囊胚中高表达。此外，我们使用差异显示RT-PCR（DDRT-PCR）的NT和IVF胚胎在2-细胞期之间的基因表达模式进行比较，以检测一些影响NT胚胎后期发育的异常。与IVF胚胎相比，NT胚胎中MuERV-L和Dnaja 2基因表达下调，Inpp 5 b和Chst 12基因表达上调。进一步的分析表明合子激活基因如干扰素-β，Dub-1，Spz 1，DD 2106和DD 2111在NT胚胎中没有被正确激活，这表明参与合子基因组激活控制的细胞过程没有被适当调节。这些结果表明，异常的基因表达已经发生在早期阶段的植入前的发展作为一个失败的核重编程。

Somatic cloning does not always result in ontogeny in mammals, and development is often associated with various abnormalities and embryo loss with a high frequency. This is considered to be due to aberrant gene expression resulting from epigenetic reprogramming errors. In the present study we conducted to explore the genes that are involved in developmental defects of somatic cloned mouse embryos. In order to address this issue, we carried out global screening of candidate genes by subtraction analysis using ES-cloned blastocysts, and differential display analysis using 2-cell embryos. From subtraction analysis 218 genes were detected as differentially expressed genes at blastocysts. Of these 198 clones were successfully sequenced and by BLAST search analysis 158 genes are detected as known genes. To confirm differentially expression, we carried out quantitative gene expression analysis by real-time PCR. The results showed that 5 of 10 genes were actually highly expressed in ES-cloned blastocysts. Further, we compared gene expression patterns using differential display RT-PCR (DDRT-PCR) between the NT and IVF embryos at the 2-cell stage to detect some abnormalities affecting later development of NT embryos. Aberrant gene expression was detected in NT embryos compared with IVF embryos, and MuERV-L and Dnaja2 genes were down-regulated and Inpp5b and Chst12 genes were up-regulated in the NT embryos. Further analysis showed that the expression of zygotically activated genes such as Interferon-?, Dub-1, Spz1, DD2106 and DD2111 were not properly activated in NT embryos, suggesting that the cellular process involved in the control of the zygotic genome activation is not appropriately regulated. These results indicate that abnormal gene expression has already occurred at the early stage of preimplantation development as a failure of nuclear reprogramming.