Genetic Analysis of Germ Cell Formation

描述（由申请人提供）：尽管人类生殖细胞的形成和分化无疑与小鼠的生殖细胞形成和分化相似，但同样清楚的是，人类生殖细胞发育的遗传要求与小鼠的生殖细胞发育的遗传要求有很大不同。这一点可以通过对比人类和小鼠生殖细胞发育中性染色体的作用得到最清楚的说明。男性生殖细胞发育所需的几个Y染色体位点在小鼠中缺失。类似地，在X染色体的情况下，女性需要2条X染色体来进行卵母细胞发育，而小鼠只需要一条X染色体就可以生育。由于这些差异，我们探讨了ES细胞是否可能提供一个备受追捧的基于人类基因组的系统，利用该系统在体外操纵人类生殖细胞特化和分化的遗传学和表观遗传学。我们的初步数据表明，生殖细胞可以指定和分化的人ES细胞。因此，我们假设人ES细胞系统可用于特异性地探测人生殖细胞形成和分化的遗传学。此外，我们假设，下调的关键基因映射到一个区域的Y染色体，这是通常删除不育男性，AZFc区将废除的能力，形成和/或分化的人男性生殖细胞在体外和体内的移植系统。为了探索这一假设，我们建议：1）进一步表征NIH批准的人ES细胞系在体外和体内对生殖细胞谱系的贡献的能力。为此，我们将使用人ES细胞系H1、H14和HSF 1（NIH代码：WA 01、WA 14和UC 01）。2)沉默映射到AZFc缺失区间的Y染色体基因，并在体外评估生殖细胞形成。3)沉默映射到AZFc缺失区间的Y染色体基因，并评估生殖细胞在灵长类动物生精小管中定植并随后分化的能力。这项工作旨在开发一个强大的系统，专门探测人类生殖细胞发育的遗传学。因此，这项研究对我们了解人类基本生殖细胞发育具有重要意义，并有直接潜力增加10-15%不育夫妇的生殖细胞缺陷知识。

DESCRIPTION (provided by applicant): Although human germ cell formation and differentiation undoubtedly shares similarity with that of the mouse, it is also clear that the genetic requirements for human germ cell development differ substantially from those of mice. This is most-plainly illustrated by contrasting the roles of the sex chromosomes in germ cell development in humans and mice. Several Y chromosome loci that are required for germ cell development in men are absent in mice. Similarly, in the case of the X chromosome, women require 2 X chromosomes for oocyte development, whereas, mice are fertile with just a single X chromosome. Because of differences such as these, we explored whether ES cells might provide a much sought after human-genome based system with which to manipulate the genetics and epigenetics of human germ cell specification and differentiation in vitro. Our preliminary data indicates that germ cells can be specified and differentiated from human ES cells. Thus, we hypothesize that the human ES cell system can be used to specifically probe the genetics of human germ cell formation and differentiation. Moreover, we hypothesize that the downregulation of key genes that map to a region of the Y chromosome that is commonly deleted in infertile men, the AZFc region will abolish the ability to form and/or differentiate human male germ cells in vitro and in vivo in a transplant system. To explore this hypothesis, we propose to: 1) Characterize further the ability of NIH-approved human ES cell lines to contribute to the germ cell lineage in vitro and in vivo. For this purpose, we will use human ES cell lines H1, H14 and HSF1 (NIH codes: WA01, WA14 and UC01). 2) Silence the Y chromosome genes that map to the AZFc deletion interval and assess germ cell formation in vitro. 3) Silence the Y chromosome genes that map to the AZFc deletion interval and assess the ability of germ cells to colonize primate spermatogenic tubules and subsequently differentiate. This work aims to develop a robust system to specifically probe the genetics of human germ cell development. As such, this research is significant to our understanding of basic germ cell development in humans and has the direct potential to increase knowledge of germ cell defects in the 10-15% of couples who are infertile.