RS-1 enhanced the efficiency of somatic cell reprogramming via activation of RAD51 in somatic cell nuclear transfer

Abstract

Background & Aim Reprogramming efficiency of somatic cell nuclei is very low in both somatic cell nuclear transfer (SCNT) and iPSCs technologies. Recently, it was reported that the genetic instability and DNA damage arise prior to transcriptional activity during reprogramming. Subsequently, in the absence of mitotic nuclear remodeling, DNA replication is delayed and errors are exacerbated (Egli D et al., 2017). Methods, Results & Conclusion Here, in order to find a regulator of DNA damage during reprogramming events, we observed the effect of RAD51 stimulatory compound (RS-1) supplements in DNA repair process of mouse SCNT embryos in the early embryogenesis and compared with in vitro fertilization (IVF)-derived embryos. First, Rad51 mRNA expression in both embryos of SCNT and IVF groups was observed at 1-cell, 2-cell, and 4-cell stages. In IVF group, relative Rad51 mRNA expression was markedly decreased from 1-cell to 2-cell stages and then restored in 4 cell stage. However, in SCNT group, its expression decreased from 1-cell to 2-cell stages at a similar rate with IVF group, but not recovered at 4-cell stage. Based on these results, we have observed DNA damage detected by rH2AX and DNA repair of double strand breaks by Rad51 expressions. The rH2AX expression was highly localized in the nucleus of IVF embryo when compared with SCNT groups until 10 h after reconstruction of embryos. However Rad51 was not expressed in the nucleus until 10 h after SCNT moreover, at 12h after SCNT, rH2AX showed highly dotted in nuclear in both IVF and SCNT+RS-1 group. A large number of embryos were observed Rad51 expression only in IVF and SCNT+RS-1 groups. In this study, we found different expression of some cell cycle-related genes among IVF, SCNT and SCNT+RS-1 groups by RNA-sequencing. The SCNT+RS-1 group significantly enhanced blastocyst formation rate of the cloned embryos when compared to those of the RS-1-untreated group, and reduced the rate of DNA fragmentation. Furthermore SCNT+RS-1 group improved full term clone mice rates and significantly increased the derivation efficiency of pluripotent stem cells from cloned embryos (SCNT-PSCs). Based on these results, we suggest that DNA damage and Rad51-mediated DNA repair during DNA replication could complement each other and its regulation has plays an important role in somatic cell nuclei reprogramming process.