Improved technology of mouse somatic cell nuclear transfer that greatly increases cloning efficiency

克隆小鼠的繁殖率仍然很低，这被认为是由于体细胞核移植(SCNT)胚胎中异常的表观遗传修饰和X染色体失活(XCI)状态所致。在这里，我们开发了新的小鼠SCNT技术，如下所示；1)从SCNT胚胎的4细胞期开始用丙丙酸处理24 h，提高了SCNT囊胚XCI状态的标志物Oct4和三甲基化组蛋白H3赖氨酸27的表达；2)SCNT后胞质内注射去离子BSA，促进了原核期乙酰化组蛋白H3赖氨酸9和乙酰化组蛋白H4赖氨酸12的表达和克隆后代的产生；3)从SCNT卵母细胞激活开始，用三环素A处理8h，然后再用维生素C处理7h，促进SCNT胚胎原核DNA中5-细胞电影氧化为5-羟甲基胞嘧啶，从而显著增加克隆后代的产量。

Production rate of cloned mice still is very low, which is thought to be attributed to abnormal epigenetic modifications and X-chromosomal inactivation (XCI) status in somatic cell nuclear transfer (SCNT) embryos. Here, we developed novel mouse SCNT technologies as follows; 1) treatment with valproi acid for 24 h from the 4-cell stage of SCNT embryos, which improved expression of Oct4 and trimethylated histone H3 lysine 27, a marker of the XCI status in SCNT blastocysts, 2) intracytoplasmic injection of deionized BSA after SCNT, which promoted expression of acetylated histone H3 lysine 9 and acetylated histone H4 lysine 12 at the pronuclear stage and production of cloned offspring, and 3) treatments with tricostatine A for 8 h from the beginning of activation of SCNT oocytes followed by vitamin C for 7 h, which promoted the oxidation of 5-methylcytocine to 5-hydroxymethylcytosine in pronuclear DNA of SCNT embryos and led to greatly increased production of cloned offspring.