Detection and functional analysis in the genes associated with the development and differentiation of germ cells

在之前的研究中，我们建立了一个可视化系统，用于使用敲入ES克隆来可视化小鼠胚胎干细胞在培养中的生殖细胞发育，在该克隆中，视觉报告基因来自小鼠Vasa同源基因MVH。在评估各种培养条件时，我们发现在低糖培养条件下，生殖细胞的形成明显受到抑制。使用重复聚合酶链式反应(PCR)差减方法，我们确定了在低糖和高糖介质中差异表达的基因。进一步研究了在高糖介质中主要表达的3个基因：硫氧还蛋白相互作用蛋白(TXNIP)、垂体肿瘤转化基因1(Pttg)和RuvB样蛋白2(RuvB12)。这些基因在成人和胚胎性腺中也有高表达，特别是RuvB12，它编码一种依赖于ATP的DNA解旋酶，在成人睾丸的精母细胞和精子细胞以及原始生殖细胞中特异地检测到。此外，利用绿色荧光蛋白(GFP)融合构建的方法，我们发现RuvBl2在睾丸生殖细胞的胞核和胞浆中都有表达。这些发现表明，葡萄糖代谢和生殖细胞发育之间可能存在某种关系。

In a previous study, we established a system for visualizing the development of germ cells from mouse embryonic stem (ES) cells in culture using knock-in ES clones in which visual reporter genes were expressed from the mouse vasa homolog, Mvh. While assessing various culture conditions, we found that germ-cell formation was markedly depressed in low glucose medium. Using a repeated polymerase chain reaction (PCR) subtraction method, we identified genes that were differentially expressed in low versus high glucose media. Three genes that were predominantly expressed in high glucose medium, thioredoxin-interacting protein (Txnip), pituitary tumor-trans forming gene 1 (Pttg), and RuvB-like protein 2 (RuvB12), were further investigated. These genes were also found to be highly expressed in adult and embryonic gonads, and RuvB12 in particular, which encodes an ATP-dependent DNA helicase, was specifically detected in the spermatocytes and spermatids of the adult testis as well as in primordial germ cells. Furthermore, using a green fluorescent protein (GFP) fusion construct, we found that RuvBl2 was expressed in both the nucleus and cytoplasm of testicular germ cells. These findings suggest a possible relationship between glucose metabolism and germ-cell development.