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目的 克隆和筛选小鼠生发泡完整卵母细胞 (GV卵 )中母源基因 ,并检测其中一个基因在植入前不同发育阶段胚中的表达 ,初步了解该基因在卵母细胞发育成熟及早期胚发育中的作用。 方法 应用抑制性消减杂交技术 (SSH) ,以生发泡完整的卵母细胞为检测子 (tester) ,8 细胞胚为驱赶子 (driver) ,建立GV卵中母源基因的cDNA文库。通过斑点杂交法进一步筛选GV卵中母源基因的cDNA文库 ,对阳性克隆进行序列测定和同源性分析 ;并采用RT PCR方法观察其中 1个阳性克隆的基因在着床前胚的表达。 结果 克隆得到 18个基因的cDNA序列和 8个同源EST序列在GV卵中特异表达 ,包括PeroxiredoxinⅡ基因。RT PCR显示PeroxiredoxinⅡ基因呈阶段性差异表达 ,在GV卵、MⅡ卵、1 细胞胚和 2 细胞胚有表达 ,但从MⅡ卵开始表达下降。而在 4 细胞胚、8 细胞胚、桑椹胚和囊胚中不表达。 结论 PeroxiredoxinⅡ基因是GV卵中母源基因cDNA文库中的一个基因 ,提示该基因在卵母细胞发育成熟和合子基因激活中起一定作用。
Objective To clone and screen the maternal genes of mouse embryogenesis-specific oocytes (GV ova) and to detect the expression of one of the genes in embryos at different developmental stages before implantation. To understand the gene maturation and early stage The role of embryo development. Methods SSH was used to generate complete cDNA of oocytes from GV eggs by using the tester to generate complete oocytes and 8-cell embryos as driver. The cDNA library of maternal genes in GV eggs was further screened by dot blot hybridization, and the positive clones were sequenced and analyzed for homology. The expression of one of the positive clones in pre-implantation embryos was observed by RT-PCR. Results The cDNA sequences of 18 genes and 8 homologous ESTs were cloned and expressed specifically in GV eggs, including Peroxiredoxin Ⅱ gene. RT-PCR showed that PeroxiredoxinⅡgene was differentially expressed in stages and expressed in GV eggs, MⅡ eggs, 1-cell embryos and 2-cell embryos, but decreased from MⅡ eggs. But not in 4-cell embryos, 8-cell embryos, morula and blastocysts. Conclusion Peroxiredoxin Ⅱ gene is a gene in the maternal cDNA library of GV eggs, suggesting that this gene plays a role in oocyte maturation and zygotic gene activation.