Overconfluent culture promotes cell cycle synchronization in mouse embryonic stem cells for efficient cloned embryo...

Mouse embryonic stem cell (ESC) cloning via nuclear transfer is a potential platform for direct generation of genetically modified mice. However, the low G1 cell population in ESCs hampers their broader use in nuclear transfer, as S-phase donors induce 1-cell arrest in reconstructed oocytes. This study aimed to enrich the G1-phase population of ESCs to improve ESC-based cloning efficiency. We demonstrated that EB3 ESCs derived from the 129/Ola strain and C57BL/6×129/Sv hybrid ESCs exhibited superior traits for nuclear transfer in terms of cleavage rate and blastocyst formation but showed reduced efficiency in development to term. Transcriptomic analyses revealed that this failure was likely due to aberrant imprinted gene expression in critical clusters, such as H19-Igf2 or Dlk1-Dio3. In contrast, although cloned offspring were successfully generated at a promising rate (approximately 6%) using EGR-R01 (BDF1×129/Sv) ESCs, early cleavage to the 2-cell embryo was inefficient. To address this, we induced cell cycle synchronization by culturing ESCs to overconfluency, which markedly increased the G1 proportion, as confirmed via FACS analysis. This alteration led to a significant improvement in first cleavage efficiency without compromising the developmental potential of ESC-derived cloned embryos to live birth. Overall, our study highlights the importance of both cell cycle synchronization and imprinting maintenance in improving ESC cloning efficiency to facilitate broad applications, including the direct generation of genetically modified mice.