小鼠胚胎成纤维细胞差异表达基因的筛选  

SCREENING OF DIFFERENTIALLY EXPRESSED GENES IN THE CULTURED MOUSE EMBRYONIC FIBROBLASTS

作　　者：杨滔[1] 张咏[1] 崔春萍[1] 鱼咏涛[1] 尉承泽[1] 任爱辉[1] 赵士富[1] 

机构地区：[1]军事医学科学院放射研究所

出　　处：《解放军医学杂志》2002年第11期965-968,共4页Medical Journal of Chinese People's Liberation Army

基　　金："973"项目资助课题 (编号G1 9990 5390 3)

摘　　要：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。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.

关 键 词：胚胎学 胚胎干细胞 纤维母细胞 核酸杂交 微阵列杂交法 细胞增殖 细胞分化 

分 类 号：Q813[生物学—生物工程]