小鼠生发泡完整卵母细胞一个特异基因的克隆及其在胚胎的表达

Cloning of a GV-oocyte specific gene and its expression in preimplantation embryos of mice

目的 :克隆和筛选小鼠生发泡完整卵母细胞 (GV卵 )特异基因 ,并检测其一在胚胎中的表达。方法 :应用抑制性消减杂交技术 (SSH) ,建立GV卵特异的cDNA文库。通过斑点杂交法进一步筛选并对阳性克隆进行序列测定和同源性分析 ;采用RT PCR方法观察其一在着床前胚胎的表达。结果 :克隆发现 1 8个基因和 8个EST序列在GV卵中特异表达。RT PCR显示该阳性克隆的基因在GV卵、MII卵、1 细胞胚胎和 2 细胞胚胎有表达 ,其短片段是预期要扩增者 ,从MII卵开始表达下降 ;长片段在GV卵、MII卵、1 细胞胚胎和 2 细胞胚胎表达未见明显变化。长、短片段两端序列一致。结论 :该基因是GV卵特异cDNA文库中的一个基因 ,且是母源性基因 。

Objective: To clone mouse GV oocyte specific genes and examine the expression of one gene of unknown function in preimplantation embryos. Methods: Suppression subtractive hybridization(SSH) was performed to construct GV oocyte specific cDNA library. Dot blot was carried out to further screen the GV oocyte specific cDNA library. The positive colonies were recovered, sequenced and compared with known genes in Genebank. RT PCR was employed to observe the differential expression of one gene(Clone 25) from the subtractive cDNA library in preimplantation embryos. Results: Eighteen genes and 8 ESTs were identified being expressed specifically in GV oocytes. RT PCR showed there were 2 bands in GV oocytes, MII eggs, 1 cell embryos and 2 cell embryos; no band was detectable in 4 cell embryos,8 cell embryos, morula and blastocysts. One band was 391bp ,which was amplified as we expected; the other was 621bp. The 2 bands were cut from the agarose gel in GV oocyte lane, purified and sequenced. The sequencing results indicated the 2 ends of the 2 fragmens were the same. Conclusion: The gene is one of specific genes in the GV oocyte specific cDNA library,and it is a maternal gene. It might be involved in oocyte maturation and zygotic genome activation.