Yamanaka Ken-ichi, Sakatani Miki, Kubota Kaiyu, Balboula Ahmed Zaky, Sawai Ken, Takahashi Masashi
National Agricultural Research Center for Kyushu Okinawa Region, Kumamoto, Japan.
J Reprod Dev. 2011 Jun;57(3):393-402. doi: 10.1262/jrd.10-181a. Epub 2011 Feb 18.
For the successful production of cloned animals by somatic cell nuclear transfer (NT), the epigenetic status of the differentiated donor cell is reversed to an embryonic totipotent status. However, in NT embryos, this process is aberrant, with genomic hypermethylation consistently observed. Here, we investigated the effects of silencing DNA methyltransferase 1 (DNMT1) mRNA by small interfering RNA (siRNA) on the DNA methylation status of the satellite I region and in vitro development of bovine NT embryos. First, the levels of DNMT1 expression were analyzed at 0, 24, 48, 72, 120 and 192 h after in vitro culture. Real-time PCR and western blotting analyses detected a significant decrease in DNMT1 mRNA in the siRNA-injected NT (siRNA-NT) group up to 72 h after in vitro culture. Next, the levels of DNA methylation of the satellite I region were analyzed at several time points after in vitro culture. The level of DNA methylation detected in siRNA-NT embryos was significantly less than those in NT embryos throughout in vitro development. Moreover, the developmental rate of embryos to blastocysts in the siRNA-NT group was significantly higher than that of NT embryos. Our data suggest that knockdown of DNMT1 mRNA in NT embryos can induce DNA demethylation, which may enhance reprogramming efficiency.
为了通过体细胞核移植（NT）成功生产克隆动物，分化的供体细胞的表观遗传状态需逆转至胚胎全能状态。然而，在NT胚胎中，这一过程是异常的，基因组高甲基化现象持续存在。在此，我们研究了利用小干扰RNA（siRNA）沉默DNA甲基转移酶1（DNMT1）mRNA对卫星I区域DNA甲基化状态以及牛NT胚胎体外发育的影响。首先，在体外培养0、24、48、72、120和192小时后分析DNMT1的表达水平。实时PCR和蛋白质印迹分析检测到，在体外培养至72小时后，注射siRNA的NT（siRNA-NT）组中DNMT1 mRNA显著减少。接下来，在体外培养后的几个时间点分析卫星I区域的DNA甲基化水平。在整个体外发育过程中，siRNA-NT胚胎中检测到的DNA甲基化水平显著低于NT胚胎。此外，siRNA-NT组胚胎发育至囊胚的比率显著高于NT胚胎。我们的数据表明，在NT胚胎中敲低DNMT1 mRNA可诱导DNA去甲基化，这可能提高重编程效率。
