Genetic requirements for normal germ cell formation throughout development

整个发育过程中正常生殖细胞形成的遗传要求

• 批准号: 7495927
• 负责人: Shawn L. Chavez
• 金额: $ 4.48万
• 依托单位: STANFORD UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-09-30 至 2010-09-29
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7495927
• 关键词: AZF1 gene; Address; Age; Animal Model; Binding; Biological Assay; Bone Morphogenetic Proteins; Cell Differentiation process; Cell Line; Cell Lineage; Cells; Chromosomes, Human, Pair 3; Couples; Defect; Development; Diagnosis; Embryo; F Factor; Family; Female; Gene Expression; Gene Family; Gene Silencing; Genes; Genetic; Germ Cells; Germ Lines; Homologous Gene; Human; Human Development; Human Genetics; Human Genome; Immunohistochemistry; In Vitro; Infertility; Kidney; Laboratories; Link; Maintenance; Meiosis; Messenger RNA; Mus; Oligospermia; Population; Property; Proteins; RNA Interference; RNA-Binding Proteins; Reporter; Reverse Transcriptase Polymerase Chain Reaction; Role; Somatic Cell; Specific qualifier value; Stem cells; System; Testis; Tissue Grafts; Transplantation; Undifferentiated; Western Blotting; Woman; Work; Y Chromosome; Zona Pellucida; base; capsule; cell preparation; embryonic stem cell; human embryonic stem cell; human embryonic stem cell line; in vivo; mRNA Expression; male; men; novel therapeutics; prenatal; protein expression; reproductive; sex; sperm cell; stem

Defects in the development of human germ cells are common, however, relatively little is known about the genes required for normal germ cell formation and function in men and women. Previously, we demonstrated the ability of human embryonic stem (ES) cells to differentiate into human germ cells in vitro and identified germ line specific genes involved in male and female germ cell development. The studies outlined in this proposal aim to enhance the formation of human germ cells from NIH-approved human ES cell lines by differentiating ES cells in the presence of Bone Morphogenetic Proteins (BMPs) and enriching for VASA-postive germ cells via FACS. Further characterization of human germ cell differentiation will be performed throughout development by examining the expression of germ cell specific genes at the mRNA level by quantitative RT-PCR and at the protein level by Western Blot analysis and immunohistochemistry. Functional analysis of germ cells differentiated from human ES cells will be accomplished by evaluating the expression of late germ cell markers, sex specific meiotic genes and meiotic spindle proteins, and by zona pellucida binding assays. In addition, the functional properties of both male and female human-ES cell derived germ cells will be assessed in vivo using a mouse transplantation system. Moreover, we also propose to silence the DAZ/DAZL genes, a family of candidate fertility factors, by RNA interference (RNAi) in human ES cells and determine the effects on male and female germ cell formation and function in vitro. Approximately 10-15% of couples in our population who are of reproductive age are infertile. This work will not only contribute to our understanding of the genetic requirements for normal human germ cell development, but it may also provide new therapeutic approaches for the diagnosis and treatment of infertility in men and women.

人类生殖细胞发育中的缺陷是常见的，然而，对生殖细胞的发育缺陷知之甚少。 男性和女性正常生殖细胞形成和功能所需的基因。此前我们 证明了人胚胎干细胞（ES细胞）在体外分化为人生殖细胞的能力 并鉴定了涉及雄性和雌性生殖细胞发育的生殖系特异性基因。研究 该提案中概述的目的是促进NIH批准的人类ES形成人类生殖细胞 通过在骨形态发生蛋白（BMP）存在下分化ES细胞并富集 检测VASA阳性生殖细胞将进一步表征人生殖细胞分化 通过检测生殖细胞特异性基因在mRNA水平的表达， 通过定量RT-PCR和蛋白质印迹分析和免疫组织化学在蛋白质水平上检测。 从人ES细胞分化的生殖细胞的功能分析将通过评价 晚期生殖细胞标志物、性别特异性减数分裂基因和减数分裂纺锤体蛋白的表达， 透明质酸结合测定。此外，男性和女性人ES细胞的功能特性 使用小鼠移植系统在体内评估衍生的生殖细胞。而且我们也 提出通过RNA干扰（RNAi）沉默DAZ/DAZL基因，一个候选生育因子家族 在人ES细胞中，并确定对体外雄性和雌性生殖细胞形成和功能的影响。 在我国人口中，大约10-15%的育龄夫妇是不育的。这项工作将 不仅有助于我们理解正常人类生殖细胞的遗传要求， 发展，但它也可能提供新的治疗方法的诊断和治疗 男性和女性的不孕症。