Transcriptional reprogramming of somatic cells after nuclear transfer to mouse 4-cell embryos

We have developed a nuclear transfer (NT) system in which somatic nuclei are transplanted into mouse embryos arrested at the 4-cell stage. The transplanted somatic nuclei show swelling and epigenetic reprogramming towards 4-cell-like nuclei. To assess genome-wide transcriptional reprogramming of the injected nuclei, the newly transcribed genes in NT embryos were examined by RNA-seq analyses. As a control, NT was also performed using mouse embryos at the 2-cell stage. Overall design: Mouse C2C12 cells or primary Oryx dammah cells were fused with mouse 4-cell embryos arrested at G2/M phase with 0.1 µg/ml demecolcine. The reconstructed NT embryos were incubated in mouse embryo culture medium containing 0.1 µg/ml demecolcine with or without alpha-amanitin. After 24 hours of culture, the NT embryos were subjected to RNA-seq analyses to detect newly transcribed genes in a RNA polymerase II-dependent manner by comparing embryos without alpha-amanitin and those with alpha-amanitin. Furthermore, to examine the effect of DNA replication, nuclear transfer of C2C12 cells to 4-cell embryos was performed before and after DNA replication at the 4-cell stage (40 hours post insemination [hpi] and 46 hpi, respecitively, followed by 24 hours of culture). Then, transcriptomes of these embryos with or without DNA replication were compared. Reprogramming ability of 2-cell embryos was also examined by transplanting C2C12 cells to early (21 hpi) or late 2-cell embryos 30 hpi).

本研究构建了一套核移植（Nuclear Transfer, NT）系统，将体细胞核移植至停滞于4细胞期的小鼠胚胎中。移植的体细胞核会发生肿胀，并向类4细胞期细胞核方向发生表观遗传重编程。为评估注射细胞核的全基因组转录重编程情况，本研究通过RNA-seq分析对核移植胚胎中的新转录基因进行了检测。作为对照，本研究同时以2细胞期小鼠胚胎为受体开展核移植实验。
实验整体设计如下：
将小鼠C2C12细胞或原代旋角羚（Oryx dammah）细胞与经0.1 μg/ml秋水仙胺处理、停滞于G2/M期的小鼠4细胞胚胎进行融合。将重构得到的核移植胚胎置于添加有0.1 μg/ml秋水仙胺、并含有或不含α-鹅膏蕈碱的小鼠胚胎培养液中培养。培养24小时后，通过对比添加与未添加α-鹅膏蕈碱的胚胎，以RNA聚合酶II依赖的方式检测新转录基因，随后对核移植胚胎开展RNA-seq分析。
此外，为探究DNA复制对重编程的影响，本研究分别在4细胞期DNA复制前后（即受精后40小时[hpi]与46小时[hpi]），将C2C12细胞移植至小鼠4细胞胚胎中构建核移植体系，随后继续培养24小时。随后对经历与未经历DNA复制的胚胎的转录组进行比较分析。
此外，本研究还通过将C2C12细胞移植至早期（受精后21小时[hpi]）或晚期（受精后30小时[hpi]）的2细胞期小鼠胚胎中，检测了2细胞期胚胎的重编程能力。