AN IN VITRO SCREENING TOOL FOR DECIDUALIZATION

 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。蜕膜化是着床过程的一部分，涉及成纤维细胞样子宫内膜间质细胞的快速增殖，然后分化为上皮样蜕膜细胞，然后蜕膜细胞成为围绕着床胚胎的蜕膜组织的一部分。蜕膜化缺陷可直接导致植入失败。此外，早期蜕膜素缺陷还可能导致其他妊娠缺陷，如胎盘、胎儿宫内发育和分娩。到目前为止，由于缺乏合适的高通量筛选工具，蜕膜化过程还没有得到系统的研究。在这个提案中，我们建议使用最近开发的最先进的CRISPR/CAS基因组编辑技术来生成这种急需的工具。在目标I中，我们将使用CRISPR通过将两个荧光报告基因敲击到蜕膜标志物的内源性位点来产生用于蜕膜分化的双报告细胞系。在AIM II中，我们将使用这一新工具对可能干扰蜕膜化的化合物进行小规模高通量筛选。该项目的成功完成将在包括植入生物学在内的多个研究领域产生长期影响， 避孕、体外受精和环境毒理学。。

 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. Decidualization is part of the implantation process and involves rapid proliferation then differentiation of fibroblast-like endometrial stromal cells into epitheloid-like decidual cells whch later become part of the decidual tissue that surrounds the implanting conceptus. Decidualization defects can directly lead to implantation failure. In addition, early decidualizatin defects can also lead to other pregnancy defects such as placentation, intrauterine fetal growth, and parturition. Up to now, the process of decidualization has not been systematically studied due to the lack of a suitable high throughput screening tool. In this proposal, we propose to generate such much-needed tool using the recently developed state-of-the-art CRISPR/Cas genome editing technology. In Aim I we will use CRISPR to generate a dual-reporter cell line for decidualization by knocking two fluorescent reporters into endogenous loci of decidual markers. In Aim II, we will use this new tool to perform a small scale high throughput screening for chemical compounds that can interfere with decidualization. Successful completion of this project will have a long-lasting impact in multiple research fields including implantation biology, contraception, in vitro fertilization and environmental toxicology. .