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Molecular genetic analysis on the regulatory mechanism for germ-soma differentiation in mammals

哺乳动物生殖细胞分化调控机制的分子遗传学分析

基本信息

批准号：
11234204
负责人：
ABE Kuniya
金额：
$ 46.21万
依托单位：
The Institute of Physical and Chemical Research (2002)Kumamoto University (1999-2001)
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research on Priority Areas
财政年份：
1999
资助国家：
日本
起止时间：
1999 至 2002
项目状态：
已结题

项目摘要

We have established techniques to specifically mark and purify totipotent stem cells as well as primordial germ cells (PGC) from mouse embryos. Using purified PGCs and embryonic cells collected from 16 different developmental stages, representative cDNA libraries have been constructed, and cDNA clones were subjected to one pass sequencing to obtain EST sequences. Homology search of the EST from PGC-expressed genes against GenBank non-redundant database revealed that approximately one third of the cDNA represents novel sequences, and we found a number of known genes whose expression were not known in the PGC in our EST collection. Large-scale RT-PCR expression analysis of about 100 PGC-expressed genes was carried out to examine temporal changes in expression of those genes during germ cell development. We could define distinct gene clusters showing co-expression at specific stages of germ line development. In order to analyze organization of the PGC-expressed genes, 〜2,000 genes have been mapped onto mouse genome. Interestingly, there is a tendency that PGC-expressed genes are clustered on the genome. We have made custom cDNA array using PGC-EST sequences and have done expression analysis, and found that PGC-expressed genes are expressed abundantly in embryonic stem (ES) cells. Taken together, PGC may have expression profile similar to that of ES cell or early embryo like blastocyst implying that these cells share some common regulatory mechanisms for gene expression.

我们已经建立了特异性标记和纯化小鼠胚胎全能干细胞以及原始生殖细胞（PGC）的技术。利用纯化的PGCs和从16个不同发育阶段收集的胚胎细胞，构建了具有代表性的cDNA文库，并对cDNA克隆进行一次测序以获得EST序列。通过与GenBank非冗余数据库的同源性比较，发现约1/3的cDNA为新序列，并发现了一些在PGC中表达未知的已知基因。对约100个PGC表达基因进行了大规模RT-PCR表达分析，以研究生殖细胞发育过程中这些基因表达的时间变化。我们可以定义不同的基因簇，在生殖系发育的特定阶段显示共表达。为了分析PGC表达基因的组织，已经将102，000个基因定位到小鼠基因组上。有趣的是，存在PGC表达基因在基因组上聚集的趋势。我们利用PGC-EST序列制作了定制的cDNA阵列，并进行了表达分析，发现PGC-EST表达的基因在胚胎干细胞（ES）中大量表达。总之，PGC可能具有与ES细胞或早期胚胎样囊胚相似的表达谱，这意味着这些细胞具有一些共同的基因表达调控机制。