CRISPR基因敲除及磷酸化对FOXL2蛋白功能调控机制研究

项目名称： CRISPR基因敲除及磷酸化对FOXL2蛋白功能调控机制研究

项目编号： No.81471880

项目类型： 面上项目

立项/批准年度： 2015

项目学科： 医药、卫生

项目作者： 唐胜建

作者单位： 潍坊医学院

项目金额： 72万元

中文摘要： FOXL2是位于3号染色体最重要的调控基因之一，其突变会导致小睑裂综合征及卵巢早衰、不孕不育等。目前已发现了FOXL2多个突变，但其导致疾病的分子机制至今尚不明确，后基因组时代蛋白质功能研究是最重要的方向，蛋白磷酸化调控是研究蛋白质功能的重要手段。课题前期应用质谱技术发现了FOXL2翻译后S33、S238两个重要的磷酸化修饰位点（国内外尚未见报道）。课题拟应用目前世界最先进的CRISPR介导的基因敲除技术对FOXL2磷酸化位点进行研究：1)筛选出稳定敲除FOXL2基因的细胞系；2)筛选出受FOXL2及其磷酸化调控的基因；3)生产出针对S33、S238的磷酸化特异抗体；4)阐明LAST1等激酶能否磷酸化FOXL2的S33、S238；5)阐明FOXL2及其磷酸化在小鼠眼睑、卵巢不同发育阶段的表达调控模式。通过研究，阐明CRISPR介导的基因敲除及磷酸化对FOXL2蛋白功能调控的分子生物学机制。

中文关键词： FOXL2；磷酸化；CRISPR；基因敲除；蛋白功能

英文摘要： The FOXL2 gene is one of the most important regulatory genes,located on chromosome 3，the mutations of which often lead to blepharophmiosis ptosis epicanthus inversus syndrome (BPES),and premature ovarian failure(POF), infertility. Many mutation sites were reported in the past 10 years, but the detailed molecular mechanism remained unclear. In the post-genomic era, protein function research is one of the most important direction, protein phosphorylation regulation is an important means of study on protein function.To further investigate the function of FOXL2, we performed a MASS analysis and found two new phosphorylation sites, S33 and S238(It has not been reported at home and abroad).In addition, CRISPR (clustered regularly interspaced short palindromic repeats) is the most advanced gene knock-out technique at cell line level. Based this technique and the aforementioned two phosphorylation sites, we plan to conduct the following research: 1) Screen the FOXL2 knock-out cell line using CRISPR; 2)Identify the genes regulated by FOXL2 and its phosphorylation; 3)Successfully get the site-specific and phosphorylation specific antibody; 4)Elucidate if LATS1 could phosphorylate FOXL2 at S33 and S238; 5)Delineate the expression and phosphorylation pattern of FOXL2 in mouse eyelid and ovary at different developmental stage. After accomplishment of above research, we will illustrate the biological mechanisms of CRISPR-mediated gene knockout and phosphorylation in the regulation of FOXL2 protein function.

英文关键词： FOXL2;phosphorylation;CRISPR;gene knockout;Protein function