Novel Tools for Detecting FGF8 for Developmental Biology Research

描述(申请人提供)：成纤维细胞生长因子8(FGF8)是一种在正常胚胎发育和组织动态平衡过程中具有关键功能的信号蛋白；异常的成纤维细胞生长因子信号导致人类出生缺陷和FGF8分泌失调，从而导致许多癌症的发生。尽管该蛋白在脊椎动物原肠形成、左右不对称、颅面、四肢、肾脏、心血管以及中枢神经系统发育中具有重要作用，但由于缺乏足够敏感的抗FGF8抗体，该蛋白在体内的分子生物学基本问题仍未得到解答。首席研究人员和该领域的许多其他人已经测试并试图优化商业产生的抗体，也产生了定制的抗体；尽管许多实验室在免疫组织化学方面具有重要的专业知识，但抗体不够敏感，并且具有令人望而却步的高背景，用于发育研究。基因标记策略是目前最好的选择，然而，她和其他人发现，产生报告等位基因所需的Fgf8基因座的改变可以扰乱正常的转录调控，也可能干扰翻译调控机制。此外，这样的策略不允许评估内源性蛋白质的位置和功能。这项应用的目标是开发高亲和力、FGF8特异的标记适配子，从而使首席研究人员和其他人能够解决重要的悬而未决的问题，如定位、信号容量和范围、受体相互作用以及小鼠体内胚胎发育过程中不同FGF8蛋白亚型的组织特异性生产和加工。在未来，这些适配子可以用于研究致癌机制，并可能测试治疗特性。 项目简介：成纤维细胞生长因子8(FGF8)是一种在正常胚胎发育和组织动态平衡过程中具有关键功能的信号蛋白；异常的成纤维细胞生长因子信号导致人类出生缺陷，而FGF8的异常产生参与了许多癌症的发病机制。尽管该蛋白在脊椎动物原肠形成、左右不对称、颅面、四肢、肾脏、心血管以及中枢神经系统发育中具有重要作用，但由于缺乏足够敏感的抗FGF8抗体，该蛋白在体内的分子生物学基本问题仍未得到解答。这项应用的目标是开发高亲和力、FGF8特异的标记适配子，从而使研究人员和其他人能够解决重要的悬而未决的问题，如定位、信号容量和范围、受体相互作用、组织特异性生产以及在小鼠胚胎体内发育过程中不同FGF8蛋白亚型的加工。在未来，这些适配子可以用于研究致癌机制，并可能测试治疗特性。

DESCRIPTION (Provided by Applicant): Fibroblast growth factor 8 (FGF8) is a signaling protein with critical functions during normal embryogenesis and tissue homeostasis; abnormal FGF signaling causes human birth defects and dysregulated FGF8 production, which contributes to pathogenesis of numerous cancers. In spite of the importance of this protein in vertebrate gastrulation, left-right asymmetry, craniofacial, limb, renal, cardiovascular, as well as central nervous system development, fundamental questions about the molecular biology of this protein in vivo remain unanswered due to the lack of sufficiently sensitive anti-FGF8 antibodies. The principal investigator and many others in the field have tested and attempted to optimize commercially generated antibodies and also generated custom antibodies; in spite of the efforts of many laboratories with significant expertise in immunohistochemistry, the antibodies are insufficiently sensitive and have prohibitively high background for developmental studies. Genetic labeling strategies are the best available choices at present, however, she and others have found that the required alterations to the Fgf8 locus to generate reporter alleles can disrupt normal transcriptional regulation, and it may also interfere with translational regulatory mechanisms. Moreover, such strategies do not allow one to assess endogenous protein location and function. The goal of this application is to develop high affinity, FGF8-specific, labeled aptamers which will subsequently allow the principal investigator and others to address important outstanding questions about the localization, signaling capacity and range, receptor interactions, as well as tissue-specific production and processing of different FGF8 protein isoforms during mouse embryonic development in vivo. In the future, these aptamers can be employed in studies investigating mechanisms of carcinogenesis and may be tested for therapeutic properties. PROJECT NARRATIVE: Fibroblast growth factor 8 (FGF8) is a signaling protein with critical functions during normal embryogenesis and tissue homeostasis; abnormal FGF signaling causes human birth defects, and dysregulated FGF8 production contributes to pathogenesis of numerous cancers. In spite of the importance of this protein in vertebrate gastrulation, left-right asymmetry, craniofacial, limb, renal, cardiovascular, as well as central nervous system development, fundamental questions about the molecular biology of this protein in vivo remain unanswered due to the lack of sufficiently sensitive anti-FGF8 antibodies. The goal of this application is to develop high affinity, FGF8-specific, labeled aptamers which will subsequently allow the investigator and others to address important outstanding questions about the localization, signaling capacity and range, receptor interactions, and tissue-specific production as well as processing of different FGF8 protein isoforms during mouse embryonic development in vivo. In the future, these aptamers can be employed in studies investigating mechanisms of carcinogenesis and may be tested for therapeutic properties.