Sun H L, Meng L N, Zhao X, Jiang J R, Liu Q Y, Shi D S, Lu F H
Animal Reproduction Institute, State Key Laboratory for Conservation and Utilization of Subtropical Agro-Bioresources, Guangxi University, Nanning, China.
Reproductive Medicine Department, Binzhou Medical University Hospital, Binzhou, China.
Genet Mol Res. 2016 Sep 2;15(3):gmr8455. doi: 10.4238/gmr.15038455.
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.
水牛是中国广西壮族自治区的特色家畜，但它们的低繁殖能力使得有必要采用体细胞核移植（SCNT）技术。我们研究了RG108对水牛成年成纤维细胞DNA甲基化以及后续SCNT胚胎发育的影响。RG108处理（0、5、10、20和100 mM）对细胞形态、活力或核型（2n = 48）没有影响，细胞生长遵循典型的“S”曲线。免疫组织化学显示，随着RG108浓度的增加，相对DNA甲基化逐渐降低，与0、5和10 mM处理组相比，20和100 mM组显著更低（分别为0.94±0.03和0.92±0.05，而0、5和10 mM处理组分别为1.0±0.02、0.98±0.05和0.98±0.09；P < 0.05）。定量聚合酶链反应显示，给予10、20和100 mM RG108的成纤维细胞中DNMT1基因表达显著低于0和5 mM组（分别为0.2±0.05、0.18±0.07和0.3±0.09，而0和5 mM组分别为1.0±0.12和1.4±0.12；P < 0.05）。20 mM RG108处理导致最低的表达水平。用20 mM RG108孵育72小时的成纤维细胞用作供体细胞来生成SCNT胚胎。与未处理组相比，更多此类胚胎发育成囊胚（28.9±3.9%对15.3±3.4%；P < 0.05）。RG108处理可以改变水牛成年成纤维细胞中的DNA甲基化并促进后续SCNT胚胎的发育。
