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

描述（由申请人提供）：人类不孕症是一个全球性问题，胚胎植入失败占自然妊娠和体外受精过程中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、生长因子信号和细胞间的接触，这些接触协调了受感囊胚和受感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，大大提高了我们在这方面的知识。然而，我们对植入的理解仍处于初级阶段。植入研究，包括子宫容受性和去个体化的研究，很大程度上得益于Cre/LoxP技术，该技术允许以组织特定的方式对许多基因进行功能研究。虽然可诱导的Cre/LoxP系统已广泛应用于其他研究领域，但由于大多数诱导剂的甾体性质会干扰植入过程，它们在植入研究中的应用受到限制。目前使用的Pgr-Cre和Amhr2-Cre系是不可诱导的，在植入研究中各有其局限性。因此，迫切需要开发一种可诱导的组织特异性Cre系统，用于植入期间子宫内的条件性基因缺失。在本研究中，我们提出将rtTA敲入内源性Gli2位点，产生一个四环素诱导系，该系与tetO-Cre一起在植入过程中驱动子宫间质诱导Cre的表达。在Aim I中，我们将使用BAC重组来产生一个敲入结构，该结构将用于胚胎干细胞中的基因靶向，并最终用于种系嵌合体的生产。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用性。该小鼠品系对于研究胚胎植入、胚胎或出生后器官发生和癌变的研究人员来说是一个有价值的工具。

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.