Effects of DNA methyltransferase inhibitor RG108 on methylation in buffalo adult fibroblasts and subsequent embryonic development following somatic cell nuclear transfer.

Buffalo are characteristic livestock of the Guangxi Zhuang Autonomous Region of China, but their low reproductive capacity necessitates the use of somatic cell nuclear transfer (SCNT). We investigated the effects of RG108 on DNA methylation in buffalo adult fibroblasts, and on subsequent SCNT embryo development. RG108 treatment (0, 5, 10, 20, and 100 mM) had no effect on cell morphology, viability, or karyotype (2n = 48), and cell growth followed a typical "S" curve. Immunohistochemistry showed that relative DNA methylation gradually decreased as RG108 concentration increased, and was significantly lower in the 20 and 100 mM groups compared to the 0, 5, and 10 mM treatments (0.94 ± 0.03 and 0.92 ± 0.05 vs 1.0 ± 0.02, 0.98 ± 0.05, and 0.98 ± 0.09, respectively; P < 0.05). Quantitative polymerase chain reaction revealed that DNMT1 gene expression of fibroblasts administered 10, 20, and 100 mM RG108 was significantly lower than those in the 0 and 5 mM groups (0.2 ± 0.05, 0.18 ± 0.07, and 0.3 ± 0.09 vs 1.0 ± 0.12 and 1.4 ± 0.12, respectively; P < 0.05). Treatment with 20 mM RG108 resulted in the lowest expression levels. Fibroblasts incubated with 20 mM RG108 for 72 h were used as donor cells to generate SCNT embryos. A greater number of such embryos developed into blastocysts compared to the non-treated group (28.9 ± 3.9 vs 15.3 ± 3.4%; P < 0.05). RG108 treatment can modify DNA methylation in buffalo adult fibroblasts and promote development of subsequent SCNT embryos.