Efficient Somatic Cell Nuclear Transfer by Overcoming Both Pre‐ and Post‐Implantation Epigenetic Barriers

Abstract Mammalian cloning through somatic cell nuclear transfer (SCNT) involves reprogramming terminally differentiated cells into totipotent embryos. Epigenetic barriers inherited from somatic cells impede reprogramming efficiency and lead to low SCNT embryo development rates. Recent studies have identified two primary types of epigenetic barriers in mouse SCNT embryos, defective pre‐implantation development due to abnormal gene expression around zygotic genome activation, associated with aberrant H3K9me3, H3K4me3, and histone acetylation, and defective post‐implantation development due to loss of H3K27me3‐mediated non‐canonical imprinting. Despite these findings, effective strategies to overcome these barriers in a single embryo have not been established. Here, Kdm4d and Kdm5b overexpression are combined with TSA treatment to overcome epigenetic barriers in pre‐implantation development, while using tetraploid complementation to replace extraembryonic lineage cells, thereby overcoming imprinting defects critical for post‐implantation development. This approach resulted in ≈30% full‐term development efficiency of reconstructed embryos. The strategy not only represents the highest SCNT efficiency achieved in mammals but also enhances the feasibility of efficient mammal cloning.