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Cloning and analysis of the genes during the induction of mouse embryongenesis

小鼠胚胎发生诱导过程中基因的克隆与分析

基本信息

批准号：
08457037
负责人：
NAKANO Toru
金额：
$ 4.74万
依托单位：
Osaka University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
1996
资助国家：
日本
起止时间：
1996 至 1997
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-08457037/
关键词：
cDNA cloning hematopoiesis mice secretory proteins

项目摘要

When mouse embryonic stem cells (ES cells) are co-cultured on macrophage colony stimulating factor deficient OP9 stromal cells, hematopoietic cells can be differentiated via mesodermal cells. In order to clone the genes of extraccellular signaling proteins functioning during this process, cDNA cloning was carried out by using the day 5 differentiation induced cells. We utilized Signal Sequence Trap (SST) method which is an efficient cloning strategy for secretary proteins and type I transmembrane proteins and the method was innovated by our group.As a result, one cDNA fragment which must contain authentic signal sequence. Full length cDNA was cloned by using the SST cDNA fragment as aprobe, this candidate gene turns out to be a murine homologue of the chicken gene whose expression is controlled by c-Myb but whose function is unknown yet. Now, the expression and the function of this gene is investigated.Meanwhile, we consider it possible to clone the genes involved in the induction of early embryogenesis of mice by examining the similarities and the differences of the gene expression between ES cells and primordial germ cells. And we start to collect mRNA from purified primordial germ cells. We are planning to carry out differential display or SAGE (serial analysis of gene expression) to clone the genes.

当小鼠胚胎干细胞（ES细胞）在巨噬细胞集落刺激因子缺陷型OP 9基质细胞上共培养时，造血细胞可经由中胚层细胞分化。为了克隆在此过程中起作用的细胞外信号蛋白的基因，通过使用第5天分化诱导的细胞进行cDNA克隆。本课题组利用本课题组创新的一种高效克隆分泌蛋白和I型跨膜蛋白的方法--信号序列捕获法（Signal Sequence Trap，SST），得到了一个必须含有真实信号序列的cDNA片段。以SST cDNA片段为探针克隆了全长cDNA，结果表明该候选基因是一个与鸡基因同源的小鼠同源基因，其表达受c-Myb调控，但功能尚不清楚。目前，我们对该基因的表达和功能进行了研究，并通过比较胚胎干细胞和原始生殖细胞基因表达的异同，认为克隆小鼠早期胚胎发生相关基因是可能的。我们开始从纯化的原始生殖细胞中收集mRNA。我们计划进行差异显示或SAGE（基因表达系列分析）来克隆基因。