小鼠胚胎成纤维细胞差异表达基因的筛选

应用微阵列杂交法对培养前，后的小鼠胚胎成纤维细胞（MFF）mRNA进行检测，找到24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MEF mRNA进行抑制性消减杂交，产物与pGEM-T载体连接构建cDNA削减文库并转染JM109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145个克隆中，测得了128条EST序列，经生物信息学分析，它们代表了42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。

SCREENING OF DIFFERENTIALLY EXPRESSED GENES IN THE CULTURED MOUSE EMBRYONIC FIBROBLASTS

Mouse AtlasTM cDNA Arrays were used for comparative studies of gene expression in mouse embryonic fibroblasts (MEF) before and after in vitro cultivation and 24 types of differentially expressed genes were identified. To screen and identify the differentially expressed new genes of MEF, cDNA suppression subtractive hybridization (SSH) technique was used. The cDNAs derived from cultured MEF after SSH were subcloned into pGEM T easy vectors to set up the subtractive library, transfected into E. coli strain JM109, sequenced and analyzed with bio informatics methods after PCR amplification.Altogether 42 types differentially expressed genes and 3 novel ESTs were identified and categorized from total 128 ESTs derived from cultured MEF after SSH. It is suggested that the genes expressed by MEF were the important molecular basis supporting the survival, proliferation and self renewal capacities of embryonic stem cells.