密闭培养条件下小鼠早期胚胎Igf2/H19印迹调控区甲基化状态分析

胚胎密闭培养是空间胚胎发育研究的基本条件.本文主要研究密闭培养条件对小鼠早期胚胎发育过程中印迹基因Igf2/H19的印迹调控区(ICR)甲基化水平的影响.应用亚硫酸氢盐测序法(BSP)分析小鼠2-细胞胚胎在密闭条件下分别培养24h、48h和72h后,相对应的发育阶段胚胎Igf2/H19的ICR甲基化水平,以常规体外培养和体内发育的各阶段胚胎为对照.结果显示,密闭培养条件下,小鼠8-细胞胚胎、桑葚胚和囊胚的Igf2/H19的ICR甲基化水平都低于常规体外培养的结果,且更明显低于体内发育的结果;同时发现,小鼠8-细胞胚胎密闭培养时,Igf2/H19的ICR甲基化水平最低.由此可见,密闭培养会引起小鼠植入前各发育阶段胚胎Igf2/H19的ICR甲基化水平降低,并证明Igf2/H19的ICR甲基化水平可以作为监测哺乳动物早期胚胎发育状况的分子指标.

Methylation of Igf2/H19 Imprinting Control Region in Mouse Early Embryos Cultured in Sealed Conditions

The sealed-culture of embryo is the basic condition for the embryo development studies in space. The study was mainly the impaction of the methylation levels of Igf2/H19 imprinting control region (ICR) in the development process of the mouse early embryos under sealed-culture, with general-culture in vitro and in vivo development as control. Using the bisulfite sequencing PCR (BSP) to analyze the different methylation levels of Igf2/H19 ICR in every stage of embryo development, 2-cell mouse embryos were cultured for 24, 48, and 72 hours in sealed-culture. The result showed that the methylation levels of Igf2/H19 ICR in mouse 8-cell embryos, morula, blastocyst cultured in sealed-culture all were lower than those of general cultured in vitro, and significantly lower than those of development in vivo while the methylation level of mouse 8-cell embryos cultured in sealed-culture was the lowest. We concluded that sealed-culture made the methylation levels of Igf2/H19 imprinting control region of mouse preimplantation embryos decreased, and the study also had confirmed that the methylation level of Igf2/H19 imprinting control region could be as a molecular target to detect the development status of the mammal early embryos.