Novel Tools for Detecting FGF8 for Developmental Biology Research

描述（由申请人提供）：成纤维细胞生长因子8 （FGF8）是一种信号蛋白，在正常胚胎发生和组织稳态中具有关键功能；异常的FGF信号导致人类出生缺陷和FGF8分泌失调，这有助于许多癌症的发病机制。尽管该蛋白在脊椎动物原肠胚形成、左右不对称、颅面、肢体、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，关于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，也产生了定制抗体；尽管许多在免疫组织化学方面具有重要专业知识的实验室做出了努力，但这些抗体不够敏感，并且在发育研究中具有过高的背景。遗传标记策略是目前最好的选择，然而，她和其他人发现，Fgf8位点产生报告等位基因所需的改变会破坏正常的转录调控，也可能干扰翻译调控机制。此外，这种策略不允许评估内源性蛋白质的位置和功能。该应用程序的目标是开发高亲和力，FGF8特异性，标记适体，这将随后允许主要研究者和其他人解决关于定位，信号能力和范围，受体相互作用，以及组织特异性生产和加工不同的FGF8蛋白异构体在小鼠胚胎发育过程中的重要悬而未决的问题。在未来，这些适体可以用于研究癌变机制，并可能用于治疗特性的测试。

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.