Comparison of Gene Expression and Genome-Wide DNA Methylation Profiling between Phenotypically Normal Cloned Pigs and Conventionally Bred Controls

Animal breeding via Somatic Cell Nuclear Transfer (SCNT) has enormous potential in agriculture and biomedicine. However, concerns about whether SCNT animals are as healthy or epigenetically normal as conventionally bred ones are raised as the efficiency of cloning by SCNT is much lower than natural breeding or In-vitro fertilization (IVF). Thus, we have conducted a genome-wide gene expression and DNA methylation profiling between phenotypically normal cloned pigs and control pigs in two tissues (muscle and liver), using Affymetrix Porcine expression array as well as modified methylation-specific digital karyotyping (MMSDK) and Solexa sequencing technology. Typical tissue-specific differences with respect to both gene expression and DNA methylation were observed in muscle and liver from cloned as well as control pigs. Gene expression profiles were highly similar between cloned pigs and controls, though a small set of genes showed altered expression. Cloned pigs presented a more different pattern of DNA methylation in unique sequences in both tissues. Especially a small set of genomic sites had different DNA methylation status with a trend towards slightly increased methylation levels in cloned pigs. Molecular network analysis of the genes that contained such differential methylation loci revealed a significant network related to tissue development. In conclusion, our study showed that phenotypically normal cloned pigs were highly similar with normal breeding pigs in their gene expression, but moderate alteration in DNA methylation aspects still exists, especially in certain unique genomic regions.通过体细胞核移植（Somatic Cell Nuclear Transfer, SCNT）开展动物育种，在农业与生物医药领域具备巨大应用潜力。然而，体细胞核移植的克隆效率远低于自然繁育或体外受精（In-vitro Fertilization, IVF），学界始终存在关于体细胞核移植动物是否与常规繁育动物同样健康、表观遗传状态是否正常的质疑。为此，本研究针对肌肉与肝脏两种组织中的表型正常克隆猪与对照猪，开展全基因组基因表达与DNA甲基化谱分析，所用技术包括Affymetrix猪基因表达芯片、改良甲基化特异性数字核型分析（modified methylation-specific digital karyotyping, MMSDK）以及Solexa测序技术。研究观察到，无论克隆猪还是对照猪，其肌肉与肝脏组织均呈现典型的组织特异性基因表达与DNA甲基化差异。克隆猪与对照猪的基因表达谱整体高度相似，但存在少量基因的表达发生改变。在两种组织的独特序列中，克隆猪的DNA甲基化模式均表现出更大差异。尤其有少量基因组位点的DNA甲基化状态存在差异，表现为克隆猪的甲基化水平略有升高。对携带这些差异甲基化位点的基因进行分子网络分析后发现，其显著富集于与组织发育相关的通路网络。综上，本研究表明，表型正常的克隆猪在基因表达层面与常规繁育猪高度相似，但在DNA甲基化层面仍存在一定程度的改变，尤其是在部分独特基因组区域。