Functional analysis of novel testis-expressed secreted and transmembrane proteins

项目说明 我们建议定义14种新的睾丸特异性分泌或跨膜蛋白的生殖功能。 使用新开发的快速CRISPR/Cas9基因操作策略。在过去的二十年里， 贝勒医学院的Matzuk实验室以及大阪大学的Ikawa和Okabe实验室， CRISPR/Cas9技术领域的世界领先专家已成功生产出可供研究的&gt；200小鼠模型 活体内的生殖过程。在这项提案中，我们将把我们在生物信息学和 操纵小鼠基因组以表征14个新化合物的体内功能和作用机制 睾丸特异的蛋白质。利用生物信息学策略，Matzuk实验室确定了100多个基因 在小鼠睾丸中特异表达，以前没有在体外或在 活体，并且有人类的同源基因。与此同时，池川和冈部的实验室已经开发出 CRISPR/Cas9系统可在体内快速高效地突变基因，从而避免胚胎干细胞 (ES)细胞和嵌合体阶段，已被证明是传统生物最麻烦和最劳动密集的阶段 一代基因敲除小鼠。使用我们的协同方法，我们的实验室专注于功能 2个新的分泌型配体基因和12个潜在的新基因的特征 跨膜蛋白。之所以选择这14种蛋白质，是因为它们与不孕症的潜在相关性 男性及其作为男性避孕药物靶标的可能性；事实上，FDA批准的药物中有70% 靶向跨膜蛋白或分泌蛋白。因此，我们精心选择了我们的基因，不仅是为了 与男性不育症有潜在的相关性，但也是未来小分子的潜在靶点 特别抑制精子发生、精子形态发生、精子活力和/或受精。到目前为止，我们有 评估了14个新基因中有12个突变的小鼠的生育状况，发现Null 6个基因的突变导致继发于精子形态发生、运动能力或受精缺陷的男性不育， 1个零突变导致严重的不育，1个零突变导致体外受精缺陷，4个零突变导致体外受精缺陷 基因突变并没有改变生育能力。因此，我们发现的大多数新基因的突变会导致基因缺陷 精子形成或功能，我们的团队在功能分析和 我们已经发表了100多篇论文。共同努力，我们的团队将致力于我们的第一年 提议确定突变小鼠模型的生育表型，并将致力于剩余的四个 几年来只对被认为对雄性生殖至关重要的蛋白质进行机械表征 与避孕有关的。这些原则证明研究具有重要的翻译意义 人类生殖遗传学和避孕发展。

PROJECT DESCRIPTION We propose to define the reproductive functions of 14 novel testis-specific secreted or transmembrane proteins using newly developed and rapid CRISPR/Cas9 gene manipulation strategies. Over the last two decades, the Matzuk laboratory at Baylor College of Medicine and the Ikawa and Okabe laboratories at Osaka University, world leading experts in CRISPR/Cas9 technology, have succeeded in producing >200 mouse models to study reproductive processes in vivo. In this proposal, we will bring together our expertise in bioinformatics and manipulation of the mouse genome to characterize the in vivo functions and mechanisms of action of 14 novel testis-specific proteins. Using bioinformatics strategies, the Matzuk laboratory identified over 100 genes that are specifically expressed in mouse testis, that previously had not been functionally characterized in vitro or in vivo, and that have human orthologs. In parallel, the Ikawa and Okabe laboratories have developed the CRISPR/Cas9 system to rapidly and efficiently mutate genes in vivo, thereby avoiding the embryonic stem (ES) cell and chimera stages that have proved the most troublesome and labor-intensive for the traditional generation of knockout mice. Using our synergistic approaches, our laboratories are focusing on the functional characterization of 2 novel genes that encode secreted ligands and 12 novel genes that encode potential transmembrane proteins. These 14 proteins were chosen because of their potential relevance to infertility in men and their likelihood as druggable targets for male contraception; indeed, 70% of the FDA-approved drugs target either transmembrane or secreted proteins. Therefore, we have carefully selected our genes to not only have potential relevance to infertility in men but also as future potential targets for small molecules that specifically inhibit spermiogenesis, sperm morphogenesis, sperm motility, and/or fertilization. To date, we have evaluated the fertility status of mice with mutations in 12 of the 14 novel genes and discovered that null mutations in 6 genes lead to male sterility secondary to sperm morphogenesis, motility, or fertilization defects, 1 null mutation results in severe subfertility, 1 null mutation leads to an in vitro fertilization defect, and 4 null mutations did not alter fertility. Thus, mutations in the majority of our identified novel genes lead to defects in sperm formation or function, areas in which our groups have abundant skills in functional analysis and for which we have published over 100 papers. Working together, our groups will dedicate the first year of our proposal to defining the fertility phenotypes of the mutant mouse models and will devote the remaining four years to mechanistically characterizing only the proteins deemed to be essential for male reproduction and having relevance to contraception. These proof-of-principle studies have important translational implications for human reproductive genetics and contraceptive development.