GENERATION OF AN INDUCIBLE SYSTEM IN THE UTERINE STROMA FOR IMPLANTATION STUDIES

用于植入研究的子宫间质中诱导系统的生成

• 批准号: 8358586
• 负责人: Liang Ma
• 金额: $ 23.64万
• 依托单位: WASHINGTON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-08-03 至 2014-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8358586
• 关键词: Accounting; Alleles; Binding; Chimera organism; Complex; Decidual Cell Reactions; Development; Doxycycline; Embryo; Embryo Loss; Embryonic Development; Event; Failure; Fertilization in Vitro; Gene Deletion; Gene Expression; Gene Targeting; Generations; Genes; Genetic; Genetic Recombination; Growth Factor; Hormonal; Human; Implant; Infertility; Knock-in Mouse; Knock-out; Knowledge; Lead; Mediating; Molecular; Mouse Strains; Mus; Nature; Organogenesis; Partner in relationship; Pathway interactions; Pharmaceutical Preparations; Phase; Placentation; Pregnancy; Procedures; Process; Production; Reporter; Research; Research Personnel; Southern Blotting; System; Technology; Tetracyclines; Tissues; Uterus; Validation; blastocyst; carcinogenesis; design; embryonic stem cell; failure Implantation; field study; homologous recombination; implantation; improved; intercellular communication; interest; natural Blastocyst Implantation; overexpression; postnatal; reproductive; tool; uterine receptivity

DESCRIPTION (provided by applicant): Human infertility is a global problem and failure of embryo implantation accounts for a significant percentage of pregnancy failure during both natural pregnancy and in vitro fertilization procedures. Implantation is an extremely complicated process requiring precisely controlled hormonal, growth factor signaling and cell-cell contacts which coordinate interactions between the competent blastocysts and the receptive uterus. In the past two decades we have greatly improved our knowledge on this subject by using sophisticated mouse genetics. However, our understanding of implantation is still rudimentary. Implantation research including the study of uterine receptivity and decidualization has benefited greatly from the Cre/LoxP technology which allows functional study of many genes in a tissue specific manner. Although inducible Cre/LoxP systems have been widely used in other fields of studies, their use in implantation studies has been limited by the steroidal nature of most of the inducers which interferes with the implantation process. The currently used Pgr-Cre and Amhr2-Cre lines are not inducible and each has their own limitations for implantation studies. Thus there is urgent need to develop an inducible tissue-specific Cre system for conditional deletion of genes in the uterus during implantation. In this proposal, we propose to knock rtTA into the endogenous Gli2 locus to generate a tetracycline-inducible line which in corporation with tetO-Cre can drive inducible Cre expression in the uterine stroma during implantation. In Aim I we will use BAC recombineering to generate a knock-in construct which will be used for gene targeting in ES cells and eventually germline chimera production. In Aim II, we will assess the usefulness of this knock-in allele in implantation studies. This mouse strain should be a valuable tool for researchers studying implantation as well as embryonic or postnatal organogenesis and carcinogenesis. PUBLIC HEALTH RELEVANCE: This project proposes to generate a tissue-specific inducible Cre system in the uterine stroma which can be used to knockout or overexpress gene of interest in the peri-implantation uterine stroma. This strain of mice will be an invaluable tool for reproductive biologists studying uterine receptivity, decidualization and placentation.

描述（由申请人提供）：人类不孕症是一个全球性问题，在自然妊娠和体外受精过程中，胚胎植入失败占妊娠失败的很大比例。着床是一个极其复杂的过程，需要精确控制激素、生长因子信号传导和细胞间接触，以协调有能力的囊胚和接受子宫之间的相互作用。在过去的二十年中，我们通过使用复杂的小鼠遗传学极大地提高了对这一主题的了解。然而，我们对植入的理解仍然很初级。包括子宫容受性和蜕膜化研究在内的着床研究​​极大地受益于 Cre/LoxP 技术，该技术允许以组织特异性方式对许多基因进行功能研究。尽管诱导型 Cre/LoxP 系统已广泛用于其他研究领域，但它们在植入研究中的使用受到大多数诱导剂的类固醇性质的限制，这些类固醇性质会干扰植入过程。目前使用的 Pgr-Cre 和 Amhr2-Cre 系不可诱导，并且每种系对于植入研究都有其自身的局限性。因此，迫切需要开发一种可诱导的组织特异性 Cre 系统，用于在植入过程中条件性地删除子宫中的基因。在本提案中，我们建议将 rtTA 敲入内源性 Gli2 位点以生成四环素诱导型细胞系，该细胞系与 tetO-Cre 结合可以在着床过程中驱动子宫基质中诱导型 Cre 表达。在目标 I 中，我们将使用 BAC 重组工程来生成敲入构建体，该构建体将用于 ES 细胞中的基因靶向并最终产生种系嵌合体。在目标 II 中，我们将评估该敲入等位基因在植入研究中的有用性。这种小鼠品系对于研究植入以及胚胎或出生后器官发生和癌变的研究人员来说应该是一个有价值的工具。 公共健康相关性：该项目提议在子宫基质中生成组织特异性诱导型 Cre 系统，该系统可用于敲除或过度表达围植入期子宫基质中的感兴趣基因。这种小鼠品系将成为生殖生物学家研究子宫容受性、蜕膜化和胎盘形成的宝贵工具。