SCNT embryos are defective for H3K27me3 imprinting [Bisulfite-seq]

Animal cloning can be achieved through somatic cell nuclear transfer (SCNT), yet the success rate remains very low. Recent studies have revealed two epigenetic barriers, H3K9me3 in donor cells and abnormal Xist activation, that impede SCNT reprogramming. Here we overcome both barriers by combining the use of Xist knockout donor cells and overexpressing Kdm4d, which allowed us to achieve the highest mouse cloning efficiency. However, SCNT-associated developmental defects and abnormal placenta were still observed, suggesting the existence of additional epigenetic defects in these SCNT embryos. Comparative DNA methylome analysis of IVF and SCNT blastocysts identified many abnormally methylated regions in SCNT embryos, despite successful global methylome reprogramming. Strikingly, allelic transcriptome analyses of SCNT blastocysts revealed a complete loss-of-imprinting at the H3K27me3-dependent imprinted genes, which may account for postimplantation developmental defects of SCNT embryos. This study thus not only provides the most efficient method for mouse cloning but also points the way for further improve SCNT cloning. WGBS comparing the methylation characteristics between mouse blastocyst-stage embryos generated by in vitro fertilization (IVF) and somatic cell nuclear transfer (SCNT).

动物克隆可通过体细胞核移植（Somatic Cell Nuclear Transfer, SCNT）实现，但其克隆成功率始终极低。近期研究已揭示两类阻碍体细胞核移植重编程的表观遗传障碍：供体细胞内的H3K9me3修饰与异常的Xist激活。本研究通过联合应用Xist基因敲除的供体细胞与Kdm4d过表达策略，成功克服了上述两类障碍，实现了迄今为止最高的小鼠克隆效率。然而，研究团队仍在克隆胚胎中观察到体细胞核移植相关的发育缺陷与胎盘异常，这表明此类克隆胚胎中仍存在其他未被探明的表观遗传缺陷。尽管全基因组甲基化组重编程已成功实现，但对体外受精（In Vitro Fertilization, IVF）与体细胞核移植囊胚进行的DNA甲基化组比较分析仍发现，克隆胚胎中存在大量异常甲基化区域。值得注意的是，对体细胞核移植囊胚的等位基因转录组分析显示，依赖H3K27me3的印记基因出现了完全的印记丢失现象，这或可解释体细胞核移植胚胎的植入后发育缺陷。因此，本研究不仅为小鼠克隆提供了迄今最高效的技术方案，同时也为后续优化体细胞核移植克隆技术指明了方向。本研究采用全基因组亚硫酸氢盐测序（Whole Genome Bisulfite Sequencing, WGBS），比较了体外受精（In Vitro Fertilization, IVF）与体细胞核移植（Somatic Cell Nuclear Transfer, SCNT）所产生的小鼠囊胚阶段胚胎的甲基化特征。