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，并且发育经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基因表达异常。本研究旨在探讨体细胞克隆小鼠胚胎发育缺陷的相关基因。为了解决这一问题，我们利用es克隆囊胚进行了减法分析，并利用2细胞胚胎进行了差异展示分析，对候选基因进行了全球筛选。从减法分析中，218个基因在囊胚中被检测到差异表达基因。198个克隆成功测序，BLAST检索分析发现158个基因为已知基因。为了证实差异表达，我们采用实时荧光定量PCR进行了基因表达分析。结果表明，在es克隆的囊胚中，10个基因中有5个实际上是高表达的。此外，我们使用差异显示RT-PCR （DDRT-PCR）比较了NT和IVF胚胎在2细胞期的基因表达模式，以检测影响NT胚胎后期发育的一些异常。与体外受精相比，NT胚中存在基因表达异常，MuERV-L和Dnaja2基因下调，Inpp5b和Chst12基因上调。进一步分析表明，合子激活基因如干扰素-？， Dub-1, Spz1， DD2106和DD2111在NT胚胎中未被正常激活，表明参与控制合子基因组激活的细胞过程未被适当调节。这些结果表明，异常基因表达已经发生在胚胎着床前发育的早期阶段，作为核重编程的失败。

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.