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应用微阵列杂交法对培养前、后的小鼠胚胎成纤维细胞 (MEF)mRNA进行检测 ,找到 2 4类差异表达的基因 ,为进一步筛选MEF可能表达的新基因 ,对培养前 (driver)、后 (tester)的MEFmRNA进行抑制性消减杂交 ,产物与pGEM T载体连接构建cDNA消减文库并转染JM10 9大肠杆菌。随机挑取阳性克隆 ,PCR扩增后对所得的差异表达基因进行测序及生物信息学分析。在随机挑取的 14 5个克隆中 ,测得了 12 8条EST序列 ,经生物信息学分析 ,它们代表了 4 2类不同基因和 3条新基因序列。结果表明 ,培养后MEF相对于培养前胚胎成纤维细胞确实存在差异表达基因群。提示 ,进一步的实验研究可能找出对ES细胞增殖分化有重要调控作用的基因或基因群。
The mRNA of mouse embryonic fibroblasts (MEF) before and after culture was detected by microarray hybridization method, and 24 differentially expressed genes were found. To further screen the new genes that may be expressed by MEF, The tester MEF mRNA was subjected to suppression subtractive hybridization and the product was ligated with the pGEM T vector to construct a cDNA subtractive library and transfected into JM109 Escherichia coli. The positive clones were picked at random and the differentially expressed genes were sequenced and analyzed by bioinformatics after PCR amplification. Among 14 5 randomly selected clones, 12 8 EST sequences were detected. Based on bioinformatics analysis, they represented 42 different types of genes and 3 new gene sequences. The results showed that after cultured MEF relative to pre-cultured embryonic fibroblasts do exist differentially expressed genes. It is suggested that further experimental studies may find out the gene or gene group that plays an important regulatory role in the proliferation and differentiation of ES cells.