Optimizing treatment of DNA methyltransferase inhibitor RG108 on porcine fibroblasts for somatic cell nuclear transfer

Abstract

Aberration in DNA methylation is believed to be one of the major causes of abnormal gene expression and inefficiency of somatic cell nuclear transfer (SCNT). RG108, a non‐nucleoside DNA methyltransferase (DNMT) inhibitor, has been reported to facilitate somatic nuclear reprogramming and improved blastocyst formation. The aim of this study was to investigate interaction effect of RG108 treatment time (24–72 hr) and concentrations (0.05–50 µM) on donor cells, and further to optimize the treatment for porcine SCNT. Our results showed that RG108 treatment resulted in time‐dependent decrease of genome‐wide DNA methylation on foetal fibroblasts, which only happened after 72‐hr treatment in our experiments, and no interaction effect between treatment time and concentration. Remarkable decrease of methylation in imprinted gene H19 and increased apoptosis was observed in 5 and 50 µM RG108‐treated cells. Furthermore, the blastocyst rates of SCNT embryos were increased as the fibroblasts treated with RG108 at 5 and 50 µM, and additional treatment during cultivation of SCNT embryos would not provide any advantage for blastocyst formation. In conclusion, the RG108 treatment of 72 hr and 5 μM would be optimized time and concentration for porcine foetal fibroblasts to improve the SCNT embryonic development. In addition, combined treatment of RG108 on donor cells and SCNT embryos would not be beneficial for embryonic development.

Combined Chaetocin/Trichostatin A Treatment Improves the Epigenetic Modification and Developmental Competence of Porcine Somatic Cell Nuclear Transfer Embryos

“…However, SCNT embryos exhibited abnormal DNA methylation patterns with higher levels compared to fertilized embryos and subsequent low development efficiency. Thus, several inhibitors have been used to regulate DNA methylation in order to improve epigenetic reprogramming and developmental competence in SCNT embryos (Xu et al, 2013;Taweechaipaisankul et al, 2019;Wu et al, 2019). In this study, we show that chaetocin and TSA significantly reduced 5-mc levels, an indicator of DNA methylation, and subsequent increased embryonic development in porcine SCNT embryos, consistent with previous studies (Li X. et al, 2008;Cao et al, 2017;Jeong et al, 2020).…”

Aspergillus fumigatus, One Uninucleate Species with Disparate Offspring

“…Epigenetic inhibitors were selected from previous publications, some of them having already been shown to be active against A. fumigatus [29][30][31][32][33][34][35][36][37][38]. All inhibitors were added to G, GA, GAP and MM media with successive dilutions from the starting solution.…”

Role and mechanism of DNA methylation and its inhibitors in hepatic fibrosis

“…Although these drugs are chemically stable, present few toxic side effects, and can be administered orally, they require high doses to exert their demethylation effects, which exceed clinically safe doses (Uddin and Fandy, 2021). RG108 (N-phthalyl-L-tryptophan) is a selective DNMT1 inhibitor and the first small-molecule non-nucleoside DNMT inhibitor to induce demethylation effects in cancer cells (Wu et al, 2019). Using an indole moiety, RG108 binds to the SAM binding site of DNMT1, competitively occupying the SAM site and inhibiting DNA methylation progression.…”