Generation of a Model to Study Uterine Gland Function

项目摘要 不孕症和流产是影响妇女的常见健康疾病。子宫腺有 在生育、发育不良和疾病中的重要生物学作用。流产是最常见的 在子宫内膜中观察到人类妊娠并发症、反复流产和不孕症。 腺体敲除动物模型。子宫腺上皮细胞（GE）及其分泌物 在子宫容受性、胚泡/孕体存活和着床中具有生物学作用， 基质细胞蜕膜化-建立和维持 怀孕然而，由于缺乏合适的小鼠，子宫腺生物学研究受到阻碍 模型有条件地删除或过度表达基因，特别是在GE的成年子宫。的 本提案的目的就是解决这一问题。Prss 29（蛋白酶、丝氨酸29或植入丝氨酸 蛋白酶基因2）是青春期后在成年人的GE中稳健且特异性表达的基因， 小鼠子宫，而不是在其他女性生殖道组织。目的1：改良Cre重组酶， 将使用CRISPR/Cas9基因组将iCre（iCre）插入Prss 29基因的3'非翻译区 工程. iCre的活性和细胞特异性表达将在发育中测定， 使用Rosa 26报告小鼠的成体组织。目的2是建立和验证Prss 29的有用性， iCre小鼠用于成年子宫腺功能研究。叉头框a2（Foxa 2）特异性表达 在成年小鼠和人类子宫的GE中，在调节 卵巢类固醇激素反应基因和GE功能在怀孕期间。Prss 29-iCre小鼠将 用于有条件地删除成年子宫中的Foxa 2并确定其对生育力的影响， 怀孕Prss 29-iCre小鼠将与RiboTag小鼠结合使用以主动分离 翻译的mRNA，将进行测序，以揭示GE活跃的妊娠转录组。 本申请特别针对NIH项目公告PA-13-303，标题为“NIH 探索性/发展性研究补助金计划（父R21）”，鼓励研究补助金 应用程序来支持项目的早期和概念阶段。在这些结束时， 研究中，我们希望已经产生了一种新的小鼠模型，用于子宫腺的研究 生物学，再生和疾病，填补了我们现有的子宫腺知识的重大空白 怀孕期间的功能。

PROJECT SUMMARY Infertility and pregnancy loss are common health disorders affecting women. Uterine glands have important biological roles in fertility, dysplasia and disease. Pregnancy loss is the most common complication of human gestation, and recurrent pregnancy loss and infertility are observed in uterine gland knockout animal models. Uterine glandular epithelia (GE) and, by inference, their secretions have biological roles in uterine receptivity, blastocyst/conceptus survival and implantation, and stromal cell decidualization- all essential processes for the establishment and maintenance of pregnancy. However, uterine gland biology research is hampered by the lack of suitable mouse models to conditionally delete or overexpress genes specifically in the GE of the adult uterus. The goal of this proposal is to address that problem. Prss29 (protease, serine 29 or implantation serine proteinase gene two) is a gene expressed robustly and specifically after puberty in the GE of the adult mouse uterus and not in other female reproductive tract tissues. In Aim 1, improved Cre recombinase (iCre) will be inserted into the 3’ untranslated region of the Prss29 gene using CRISPR/Cas9 genome engineering. Activity and cell-specific expression of iCre will be determined in the developing and adult tissues using Rosa26 reporter mice. Aim 2 is to establish and validate the usefulness of Prss29- iCre mice for study of adult uterine gland function. Forkhead box a2 (Foxa2) is specifically expressed in the GE of the adult mouse and human uterus and has a potential biological role in regulation of ovarian steroid hormone responsive genes and GE function during pregnancy. Prss29-iCre mice will be used to conditionally delete Foxa2 in the adult uterus and determine its impact on fertility and pregnancy. Prss29-iCre mice will be used in conjuction with RiboTag mice to isolate actively translated mRNAs, which will be sequenced to uncover the GE active transcriptome of pregnancy. This application specifically targets NIH program announcement PA-13-303 entitled “NIH Exploratory/Developmental Research Grant Program (Parent R21)” that encourages research grant applications to support the early and conceptual stages of a project. At the conclusion of these studies, we expect to have generated a novel mouse model useful for the study of uterine gland biology, regeneration, and disease and fill a significant gap in our existing knowledge of uterine gland function during pregnancy.