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

我们已经建立了从小鼠胚胎中特异性标记和纯化全能干细胞以及原始生殖细胞（PGC）的技术。利用纯化的PGCs和来自16个不同发育阶段的胚胎细胞，构建具有代表性的cDNA文库，并对cDNA克隆进行一遍测序，获得EST序列。在GenBank非冗余数据库中对PGC表达基因的EST进行同源性检索，发现大约三分之一的cDNA代表新序列，并且在我们的EST集合中发现了许多PGC中不知道表达的已知基因。我们对大约100个pgc表达基因进行了大规模的RT-PCR表达分析，以检测这些基因在生殖细胞发育过程中的表达变化。我们可以确定在种系发育的特定阶段显示共表达的不同基因簇。为了分析pgc表达基因的组织，大约2000个基因已被定位到小鼠基因组中。有趣的是，有一种趋势是pgc表达的基因聚集在基因组上。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表达的基因在胚胎干细胞中大量表达。综上所述，PGC可能具有与胚胎干细胞或囊胚等早期胚胎相似的表达谱，这意味着这些细胞具有一些共同的基因表达调控机制。

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.