Apoptosis and incomplete DNA methylation reprogramming in cloned embryos reduce cloning efficiency. 5-aza-2′-deoxycytidine (5-aza-dC) is proven to regulate apoptosis and DNA methylation reprogramming, however, the treatment method and potential role of 5-aza-dC during cloned embryo development are still not well studied. This study displayed that treating donor cells with 5-aza-dC (AN group) significantly reduced the blastocyst rate, while treating cloned embryos (NA group) or both donor cells and cloned embryos (ANA group) significantly promoted the blastocyst formation, and the ANA group was the best treatment of 5-aza-dC to enhance the development of cloned embryos. Then, compared with the NT group, the ANA group showed the significantly enhanced nuclear remodeling. The apoptotic cell numbers and rates of blastocysts were significantly reduced, and the expression levels of significantly upregulated anti-apoptosis gene Bcl2l1 and downregulated pro-apoptosis genes Bax, P53 and Caspase3 were observed in the ANA group. Further study demonstrated that the transcription levels of DNA methylation reprogramming genes Dnmt1, Dnmt3a, Tet1 and Tet3 were significantly upregulated, and, significant genomic DNA remethylation, DNA demethylation of pluripotency gene Oct4, and DNA remethylation of tissue specific gene Thy1 were observed at the blastocyst stage in the ANA group. Embryo development related genes including Igf2, H19, Oct4, Nanog, Sox2, Eif1a, Cdx2 and ATP1b1 were significantly upregulated, and Thy1 and Col5a2 were remarkably silenced at the 4-cell and blastocyst stages in the ANA group. In conclusion, the best 5-aza-dC treatment enhanced the development of cloned embryos by inhibiting apoptosis and improving DNA methylation reprogramming.

克隆胚胎中的凋亡和不完全的DNA甲基化重编程降低了克隆效率。已证明5-氮杂-2'-脱氧胞苷（5-氮杂-dC）可以调节细胞凋亡和DNA甲基化重编程，但是，对5-氮杂-dC在克隆胚胎发育过程中的治疗方法和潜在作用仍未进行很好的研究。这项研究表明，用5-氮杂-dC处理供体细胞（AN组）显着降低了胚泡率，而处理克隆胚胎（NA组）或同时处理供体细胞和克隆胚（ANA组）均显着促进了胚泡的形成，并且ANA组是5-aza-dC增强克隆胚胎发育的最佳治疗方法。然后，与NT组相比，ANA组显示出明显增强的核重构。凋亡细胞数量和囊胚发生率显着降低，在ANA组中观察到抗凋亡基因Bcl2l1和凋亡原基因Bax，P53和Caspase3的表达明显上调。进一步的研究表明，DNA甲基化重编程基因Dnmt1，Dnmt3a，Tet1和Tet3的转录水平显着上调，并且在胚泡中观察到了显着的基因组DNA重甲基化，多能性基因Oct4的DNA脱甲基以及组织特异性基因Thy1的DNA重甲基化。 ANA组的舞台。与胚胎发育相关的基因包括Igf2，H19，Oct4，Nanog，Sox2，Eif1a，Cdx2和ATP1b1显着上调，而Thy1和Col5a2在ANA组的4细胞和胚泡期显着沉默。结论，