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

描述(申请人提供)：人类不孕不育是一个全球性的问题，胚胎植入失败是自然妊娠和体外受精过程中妊娠失败的重要原因。着床是一个极其复杂的过程，需要精确控制激素、生长因子信号和细胞-细胞接触，以协调合格胚泡和接受子宫之间的相互作用。在过去的二十年里，我们通过使用复杂的小鼠遗传学极大地提高了我们对这一主题的了解。然而，我们对植入的理解仍然是初级的。着床研究，包括子宫容受性和蜕膜化的研究，极大地得益于CRE/loxP技术，它允许以组织特异性的方式研究许多基因的功能。尽管可诱导的Cre/loxP系统在其他领域的研究中得到了广泛的应用，但它们在植入研究中的应用受到了大多数干扰植入过程的诱导剂的类固醇性质的限制。目前使用的PGR-Cre和Amhr2-Cre系是不可诱导的，它们在植入研究中都有自己的局限性。因此，迫切需要开发一种可诱导的组织特异性Cre系统，用于在植入过程中有条件地删除子宫中的基因。在这个方案中，我们建议将RTTA敲击到内源Gli2基因座上，以产生一个四环素诱导的株系，与Teto-Cre一起，可以在着床过程中驱动子宫间质中可诱导的Cre表达。在目标I中，我们将使用BAC重组工程来产生敲入结构，该结构将用于在ES细胞中进行基因靶向，并最终产生生殖系嵌合体。在AIM II中，我们将评估这种敲入等位基因在植入研究中的有效性。这种小鼠品系应该是研究植入以及胚胎或出生后器官发生和癌症的研究人员的有价值的工具。 公共卫生相关性：该项目建议在子宫间质中产生一个组织特异性的可诱导Cre系统，该系统可用于敲除或过度表达植入周围子宫间质中感兴趣的基因。这种品系的小鼠将是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。

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.