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

在哺乳动物中，体细胞克隆并不总是导致个体发育，发育经常与各种异常和胚胎丢失相关，频率很高。这被认为是由于表观遗传重新编程错误导致的基因表达异常所致。在本研究中，我们进行了探索与体细胞克隆小鼠胚胎发育缺陷相关的基因。为了解决这个问题，我们对候选基因进行了全球筛选，方法是使用ES克隆的囊胚进行消减分析，并使用2细胞胚胎进行差异显示分析。从消减分析中，发现218个基因是囊胚期差异表达的基因。在这198个克隆中，测序成功，通过BLAST搜索分析，检测到158个基因是已知基因。为了确认差异表达，我们用实时荧光定量聚合酶链式反应进行了基因表达定量分析。结果表明，在ES克隆的囊胚中，10个基因中有5个是高表达的。此外，我们利用差异显示RT-PCR(DDRT-PCR)技术比较了2-细胞期NT和IVF胚胎的基因表达谱，以检测影响NT胚胎后期发育的一些异常。与体外受精胚胎相比，NT胚胎中基因表达异常，MERV-L和DNAJA2基因表达下调，Inpp5b和Chst12基因表达上调。进一步的分析表明，在NT胚胎中，干扰素-1、Dub-1、Spz1、DD2106和DD2111等合子激活基因的表达没有得到适当的激活，这表明参与合子基因组激活调控的细胞过程没有得到适当的调控。这些结果表明，在植入前发育的早期阶段就已经出现了基因表达异常，这是核重新编程的失败。

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.