SCNT embryos are defective for H3K27me3 imprinting [RNA-seq]. SCNT embryos are defective for H3K27me3 imprinting [RNA-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. Overall design: RNA-seq comparing mouse blastocyst-stage embryos generated by in vitro fertilization (IVF) and somatic cell nuclear transfer

体细胞克隆（somatic cell nuclear transfer, SCNT）可实现动物克隆，但其成功率始终极低。已有研究揭示了两类阻碍体细胞克隆重编程的表观遗传障碍：供体细胞中的H3K9me3修饰与异常的Xist激活。本研究通过联合使用Xist敲除供体细胞与过表达Kdm4d，成功突破了上述两类障碍，实现了目前最高的小鼠克隆效率。但研究中仍观察到与体细胞克隆相关的发育缺陷与胎盘异常，提示此类克隆胚胎中仍存在其他表观遗传缺陷。尽管体细胞克隆胚胎已完成全局甲基化组重编程，但对体外受精（in vitro fertilization, IVF）与体细胞克隆囊胚的DNA甲基化组比较分析显示，克隆胚胎中仍存在大量异常甲基化区域。值得注意的是，对体细胞克隆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，这或可解释克隆胚胎着床后的发育缺陷。因此，本研究不仅为小鼠克隆提供了目前最为高效的技术方案，也为后续体细胞克隆技术的优化指明了方向。实验整体设计：对通过体外受精（IVF）与体细胞克隆制备的小鼠囊胚阶段胚胎开展RNA测序（RNA-seq）比较分析。