Scriptaid improves the reprogramming of donor cells and enhances canine-porcine interspecies embryo development

Abstract

Histone methylation, histone acetylation, and DNA methylation are the important factors for somatic cell nuclear transfer (SCNT). Histone deacetylase inhibitors (HDACi) and DNA methyltransferase inhibitors (DNMTi) have been used to improve cloning efficiency. In particular, scriptaid, an HDACi, has been shown to improve SCNT efficiency. However, no studies have been performed on canines. Here, we evaluated the effects of scriptaid on histone modification in canine ear fibroblasts (cEFs) and cloned canine embryos derived from cEFs. The early development of cloned canine-porcine interspecies SCNT (iSCNT) embryos was also examined. cEFs were treated with scriptaid (0, 100, 250, 500, 750, and 1000nM) in a medium for 24h. Scriptaid treatment (all concentrations) did not significantly affect cell apoptosis. Treatment with 500nM scriptaid caused a significant increase in the acetylation of H3K9, H3K14, and H4K5. cEFs treated with 500nM scriptaid showed significantly decreased Gcn5, Hat1, Hdac6, and Bcl2 and increased Oct4 and Sox2 expression levels. After SCNT with canine oocytes, H3K14 acetylation was significantly increased in the one- and two-cell cloned embryos from scriptaid-treated cEFs. In iSCNT, the percentage of embryos in the 16-cell stage was significantly higher in the scriptaid-treated group (21.6±2.44%) than in the control (7.5±2.09%). The expression levels of Oct4, Sox2, and Bcl2 were significantly increased in 16-cell iSCNT embryos, whereas that of Hdac6 was decreased. These results demonstrated that scriptaid affected the reprogramming of canine donor and cloned embryos, as well as early embryo development in canine-porcine iSCNT, by regulating reprogramming and apoptotic genes.

Keywords: Canine somatic cell nuclear transfer; Donor nuclear reprogramming; Histone acetylation; Interspecies SCNT (iSCNT); Scriptaid.