Functional analysis of novel testis-expressed secreted and transmembrane proteins

项目描述 我们建议定义14个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实验室和大坂大学的Ikawa和Okabe实验室， 世界领先的CRISPR/Cas9技术专家，已经成功地生产了超过200个小鼠模型进行研究 体内的生殖过程。在这项提案中，我们将汇集我们在生物信息学方面的专业知识， 操纵小鼠基因组，以表征14种新的 睾丸特异性蛋白质利用生物信息学策略，Matzuk实验室确定了100多个基因， 在小鼠睾丸中特异性表达，以前没有在体外或体内进行功能表征。 体内，并且具有人类直系同源物。与此同时，Ikawa和Okabe实验室开发了 CRISPR/Cas9系统能够快速有效地在体内突变基因，从而避免胚胎干细胞 (ES)细胞和嵌合体阶段，已被证明是最麻烦和劳动密集型的传统 产生敲除小鼠。使用我们的协同方法，我们的实验室专注于功能 2个编码分泌型配体的新基因和12个编码潜在的 跨膜蛋白选择这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目标的可能性;事实上，70%的FDA批准的药物 靶向跨膜或分泌蛋白。因此，我们精心选择了我们的基因，不仅 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， 特异性抑制精子发生、精子形态发生、精子运动性和/或受精。到目前为止， 评估了14个新基因中12个突变的小鼠的生育能力， 6个基因的突变导致继发于精子形态发生、运动或受精缺陷的雄性不育， 1个无效突变导致严重的生育力低下，1个无效突变导致体外受精缺陷，4个无效突变导致体外受精缺陷。 突变并不改变生育能力。因此，我们鉴定的大多数新基因的突变导致了以下缺陷： 精子的形成或功能，在这些领域，我们的小组在功能分析和形成方面拥有丰富的技能。 我们已经发表了100多篇论文。我们的团队将共同努力， 建议定义突变小鼠模型的生育表型，并将致力于其余四个 多年来，只有被认为是男性生殖所必需的蛋白质才能机械地表征， 与避孕有关。这些原理证明研究对以下方面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.