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. 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).