Fragile X pre-mutation and ovarian insufficiency

描述(由申请人提供)：该项目直接响应了美国国立卫生研究院的号召，要求开发和表征专门适用于研究FMR1预突变对卵巢功能不全影响的模型。脆性X智力低下1基因(FMR1)包含一系列位于5‘非编码区的约30个CGG三联体重复序列。CGG重复数超过200个三联体的突变扩展会导致脆性X综合征。目前研究的重点是CGG重复序列的前突变扩增至~40~200之间，及其与卵巢功能不全的关系。重大数据描述了生殖功能不全，包括携带FMR1前突变扩展的妇女中的卵巢早衰。突变前的扩张和生殖不足之间的联系是最近才提出的，因此目前还不清楚这种前突变导致女性生殖能力改变的机制。我们的目标是确定对确定FMR1预突变在女性生殖中的作用有用的模型。拟议的项目有两个具体目标。目标1将利用现有的脆性X预突变小鼠模型，全面描述女性生育能力。目的是评估与妇女记录的生殖参数类似的生殖参数，以确定小鼠模型的意义及其与生殖的关系。在AIM 2上进行的研究将创造携带针对颗粒、垂体前叶和卵母细胞的预突变扩展CGG重复序列的条件性敲入。对女性的研究表明，这些细胞类型中的每一种都可能受到前突变的影响，从而在导致卵巢功能不全中发挥作用。颗粒、卵母细胞和垂体前叶构成了下丘脑-垂体-卵巢内分泌系统的关键组成部分，对正常生育至关重要。单独针对每种细胞类型将使我们能够仔细评估每种细胞类型中前突变的贡献，因为它与生殖过程有关。利用链霉菌噬菌体C31(？C31)位点特异性整合酶指导转基因整合的一种新的转基因方法将被用来产生有条件的转基因。在靶向预突变小鼠的生产中，我们将产生一种条件转基因小鼠，它将成为更大的研究社区的工具，允许将预突变扩展到任何组织或细胞类型。我们拥有一支在女性生殖生物学、小鼠遗传学和脆弱X患者群体方面拥有专业知识的研究团队，我们准备共同应对NIH的这一定向呼吁。随着这些研究的完成，我们将拥有专门适合于探索FMR1预突变影响女性生殖系统的机制的全球和定向预突变扩展小鼠模型。

DESCRIPTION (provided by applicant): This project directly addresses the call by NIH for the development and characterization of models specifically suited to study the impact of FMR1 premutation on ovarian insufficiency. The Fragile X Mental Retardation 1 gene (FMR1) contains a series of approximately 30 CGG triplet repeats located in the 5' UTR. Mutational expansion of the number of CGG repeats to greater than 200 triplets leads to Fragile X Syndrome. The focus of the present studies is the premutation expansion of the CGG repeats to between ~40 and 200, and its association with ovarian insufficiency. Significant data describe reproductive insufficiencies including premature ovarian failure in women carrying the FMR1 premutation expansion. The association between pre- mutation expansion and reproductive insufficiencies was made recently and thus there is currently no understanding of the mechanisms whereby this premutation result in altered reproductive capacity in women. Our objective is to identify models that will be useful in defining the role of FMR1 premutation in female reproduction. The proposed project has two specific objectives. Aim 1 will take advantage of an existing mouse model of fragile X premutation and fully characterize female fertility. The objective is to assess reproductive parameters analogous to those documented in women in order to establish the significance of the mouse model and its relationship to reproduction. The studies carried out in Aim 2 will create conditional knock-ins carrying premutation expanded CGG repeats targeted to the granulosa, anterior pituitary, and oocyte. Studies in women indicate each of these cell types may be impacted by the premutation and thus play a role in the resulting ovarian insufficiency. The granulosa, oocyte, and anterior pituitary form key components of the hypothalamic-pituitary-ovarian endocrine system important for normal fertility. Targeting to each cell type individually will allow us to carefully assess the contribution of the premutation in each cell type as it relats to the reproductive process. A novel transgenesis method relying on the use of the Streptomyces phage C31 (?C31) site specific integrase to direct transgene integration will be used to generate the conditional transgene. In production of the targeted premutation mice we will generate a conditional transgenic mouse that will be a tool for the greater research community allowing for targeting of the premutation expansion to any tissue or cell type. We have an assembled research team with expertise in female reproductive biology, mouse genetics, and the fragile X patient population and together we are poised to address this directed call by NIH. With the completion of these studies we will have both global and targeted premutation expansion mouse models specifically suited to explore the mechanisms whereby FMR1 premutation impacts the reproductive system in women.