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

当小鼠胚胎干细胞(ES细胞)与巨噬细胞集落刺激因子缺失的OP9基质细胞共培养时，造血细胞可以通过中胚层细胞分化。为了克隆在这一过程中起作用的细胞外信号蛋白的基因，我们利用第5天诱导分化的细胞进行了基因克隆。我们利用信号序列陷阱(SST)的方法对分泌型蛋白和I型跨膜蛋白进行了有效的克隆，并对该方法进行了创新，得到了一个必须包含真实信号序列的cdna片段。以SST基因片段为探针克隆了全长基因，该候选基因与鸡c-Myb基因同源，但其功能尚不清楚。现对该基因的表达和功能进行研究，并通过比较ES细胞和原始生殖细胞基因表达的相似性和差异性，为克隆小鼠早期胚胎发生相关基因奠定基础。我们开始从纯化的原始生殖细胞中收集信使核糖核酸。我们正计划进行差异显示或SAGE(基因表达序列分析)来克隆这些基因。

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.