用DDRT-PCR技术克隆小鼠早期胚胎发育相关基因

mRNA差异显示 (DDRT PCR)技术在哺乳动物早期胚胎发育相关基因研究中的应用 ,因获得足够量的早期胚胎材料困难而受到限制 .通过对DDRT PCR技术各种条件参数进行优化组合 ,并对某些环节进行改良 ,以小鼠的MⅡ卵、2 细胞胚胎和 4 细胞胚胎为材料进行差异显示 ,仅以相当于5 0个卵细胞的量为起始材料 ,便得到了理想的差示结果 .从差异条带中挑取感兴趣的差异条带进行回收、阳性鉴定、亚克隆、序列分析、并在反向Northern杂交基础上设计了鉴定实验 .结果发现 ,有一个片段差异显著且是阶段性特异表达 .经GenBank检索 ,发现该片段仅有同源的EST ,其全长及功能尚不清楚 ,是一个功能未知基因 ,将该片段命名为ed1.反向Northern杂交结果表明 ,ed1在 2 细胞期胚胎中有表达 ,而在MⅡ卵及 4 细胞胚胎中均不表达 .

Cloning of Early Mouse Embryo Development Related Genes Using Modified DDRT-PCR

The analysis of differential gene expression during preimplantation embryogenesis has been hindered by the paucity of biological material. Improved mRNA differential display RT PCR method was used for the total RNA extraction from mouse MⅡ eggs, two cell embryos,and four cell embryos. The starting material for each case was about fifty cells,and the desired experimental results were obtained. Seven differential amplicons were obtained and the most evident one was named ed 1, which was recovered, subcloned, sequenced, and identified by reverse Northern hybridization. The BLAST programs were used to search NCBI nr. A fragment ed1 with high similarity to many sequences in the existing database was found, but its full length and function are unknown. Reverse Northern hybridization experiments indicated that ed 1 was stage specific expressive gene in the two cell embryo, and no blot in MⅡ eggs, and four cell embryo.