A Nonhuman Primate Model of Fragile X Associated Primary Ovarian Insufficiency

脆性 X 相关原发性卵巢功能不全的非人类灵长类动物模型

• 批准号: 8476134
• 负责人: Thomas M Burbacher
• 金额: $ 31.15万
• 依托单位: UNIVERSITY OF WASHINGTON
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-06-15 至 2015-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8476134
• 关键词: Address; Affect; Age-Months; Alleles; Animal Model; Animals; Area; Biological; Biological Models; Birth; Blood specimen; CGG repeat; CGG repeat expansion; Cell model; Cell physiology; Cells; Cellular Structures; Chimera organism; Chimerism; Communities; Derivation procedure; Development; Disease; ES Cell Line; Embryo; Ensure; Exhibits; FMR1; FMR1 Gene; Female; Fertility; Fragile X Syndrome; Future; Gene Transfer Techniques; Generations; Genes; Genetic; Goals; Growth; Hereditary Disease; Human; In Vitro; Individual; Infant; Laboratory Research; Lead; Length; Link; Mediating; Menopause; Messenger RNA; Modeling; Molecular; Monitor; Morphology; Mutation; Neurologic; Newborn Infant; Outcome; Ovarian; Ovarian Tissue; Ovariectomy; Phase; Phenotype; Pregnancy; Primates; Procedures; Production; Property; Proteins; Research; Risk; Site; Structure of primordial sex cell; Study models; Syndrome; Techniques; Technology; Testing; Time; Tissue Sample; Tissues; Transcript; Transgenic Organisms; Trinucleotide Repeats; Turner' s Syndrome; Woman; abstracting; animal model development; base; clinically relevant; design; early onset; embryonic stem cell; genetic manipulation; in vivo; in vivo Model; model development; neurobehavior; neurobehavioral; neurobehavioral test; nonhuman primate; novel; offspring; pluripotency; premature; protein expression; psychologic; public health relevance; self-renewal; stem cell technology; tool

DESCRIPTION (provided by applicant): A Nonhuman Primate Model of Fragile X Associated Primary Ovarian Insufficiency Abstract Fragile X encompasses a range of genetic conditions, all of which result as a function of changes within the FMR1 gene and abnormal production and/or expression of the FMR1 gene products. The normal allele (5-54 CGG repeats) is stably transmitted to offspring and FMR protein (FMRP) expression is normal. In individuals that carry the pre-mutation sequence (55-200 CGG repeat), the alleles remain unmethylated and are associated with increasing levels of FMR1 transcripts and decreasing levels of FMRP. Females with the Fragile X pre-mutation (FXPM) are at risk of developing FXPOI and early onset menopause, the only non- neurological/psychological condition attributed to the pre-mutation range of CGG repeat lengths. To fully investigate the molecular mechanisms and biological consequences of the human pre-mutation CGG repeat expansion responsible for FXPOI it is necessary to design and characterize appropriate in-vitro and in-vivo model systems. Embryonic stem cells (ESC) are an ideal tool with which to study developmentally regulated cell function due to their pluripotency, unlimited capacity for self-renewal, ability to accept targeted genetic manipulations and undergo directed differentiation. This proposal will seek to create for the first time in-vitro and in-vivo NHP models of FXPOI. Three specific aims will be addressed; (1) Generation of a NHP ESC-based model of FXPOI. Using site-specific transgenesis the human FXPM CGG repeat sequence is introduced into the endogenous FMR1 gene of a currently existing female NHP ESC line. The FXPM transgenic ESC line (ESCPOI) will then be used for the in-vitro differentiation and characterization of FXPM primordial germ cells. (2) Generation of female ESCPOI-chimeric NHP infants. Using the ESCPOI line from Aim 1, in conjunction with optimized techniques for the in-vitro production of ESC-chimeric embryos, we will generate female NHP infants that show expression of the FXPM within ovarian tissue (germline ESCPOI-chimeras). This specific aim represents a "proof-of-principle" step that will be fundamental to future full-scale expansion of an in-vivo NHP FXPOI model system. (3) For model characterization, ESCPOI expression in both somatic and germline tissues of liveborn infants will be assessed. Physical growth and neurobehavioral assessment in infants up to 6 months of age will be monitored using specialized testing procedures in our state-of-the-art Infant Primate Research Laboratory to determine if infants display any neurological deficits associated with the FXPM. Our proposal is focused on the use of ESC-based technologies in the NHP in an effort to provide renewable, defined and clinically relevant translational in-vitro and in-vivo NHP model systems of FXPOI to the broader Fragile X research community.

描述（由申请人提供）：脆弱X相关的原发性卵巢功能不全的非人类灵长类动物模型包含一系列遗传条件，所有这些都取决于FMR1基因内的变化以及FMR1基因产物的变化和异常产生和/或表达。正常的等位基因（5-54 CGG重复序列）稳定地传播到后代，FMR蛋白（FMRP）表达是正常的。在携带突变前序列的个体中（55-200 CGG重复），等位基因保持未甲基化，并且与FMR1转录物水平升高和FMRP水平降低有关。具有脆弱X前女性（FXPM）的女性有发展FXPOI和早期发作更年期的风险，这是唯一归因于CGG重复长度的未经神经/心理状况。为了充分研究人类预签的CGG重复扩张的分子机制和生物学后果，导致FXPOI的膨胀有必要设计和表征适当的体内和体内模型系统。胚胎干细胞（ESC）是一个理想的工具，它由于其多能性，无限制的自我更新能力，接受有针对性的遗传操作的能力并受到定向分化的能力，研究了开发调节的细胞功能。该建议将寻求为第一个创建 FXPOI的时间内和体内NHP模型。将解决三个具体目标； （1）生成基于NHP ESC的FXPOI模型。使用位点特异性转基因，将人FXPM CGG重复序列引入了当前现有的女性NHP ESC系的内源性FMR1基因中。然后，FXPM转基因ESC系（ESCPOI）将用于FXPM原始生殖细胞的体外分化和表征。 （2）女性Escpoi-Chimeric NHP婴儿的产生。使用AIM 1的ESCPOI系列，并结合优化的Esc-Chimeric胚胎体外产生的技术，我们将产生雌性NHP婴儿，以显示FXPM在卵巢组织中表达的表达（生殖线Escpoi-Chimeras）。这个具体的目标是“原则证明”步骤，这将是Vivo In-Vivo NHP FXPOI模型系统的未来全尺度扩展至关重要的。 （3）对于模型表征，将评估活生生婴儿的体细胞和生殖组织中的ESCPOI表达。将使用我们最先进的婴儿灵长类动物研究实验室中的专门测试程序来监测至6个月大的婴儿的身体生长和神经行为评估，以确定婴儿是否显示与FXPM相关的任何神经缺陷。我们的建议集中于在NHP中使用基于ESC的技术，以提供可再生，定义和临床相关的FXPOI的Vitro和Vivo In-Vivo NHP模型系统，以向更广泛的脆弱X研究社区提供。