TIP39KDA AND TOOTH FORMATION

TIP39KDA 和牙齿形成

• 批准号: 6922044
• 负责人: Michael Lansdell Paine
• 金额: $ 36.56万
• 依托单位: UNIVERSITY OF SOUTHERN CALIFORNIA
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-07-01 至 2008-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6922044
• 关键词: MDCK cell; ameloblasts; amelogenin; antibody; antisense nucleic acid; binding proteins; dental development; enamel organ; functional /structural genomics; genetically modified animals; green fluorescent proteins; laboratory mouse; messenger RNA; oligonucleotides; organ culture; peptide library; posttranscriptional RNA processing; protein isoforms; protein localization; protein structure function; proteomics; ribozymes; transfection /expression vector; yeast two hybrid system

DESCRIPTION (provided by applicant): Understanding the physiologic relationship between genomics and proteomics is a common theme and focus for investigation. The use of cell models and organ systems remains a fundamental approach to achieving such an understanding. Enamel is a complex bioceramic tissue that is a reflection of its unique molecular and cellular formation. From their ectodermal origins, ameloblasts exist for the sole purpose of producing a mineralized shell. The shell is almost entirely devoid of protein and is deposited over the "bone-like" dentine. We have recently discovered TIP39kDa, a protein that favors the secretory surface of ameloblasts (Tomes' processes). Antibodies to TIP39kDa react to secretory vesicles within Tomes' processes, and also the immature enamel matrix. TIP39kDa expression is not unique to ameloblasts, but preliminary data suggest that it may be unique to cells with a significant secretory function. This project will pursue an experimental strategy to identify a definitive function for TIP39kDa, and also explain the nature of the multiple messenger RNA isoforms observed for TIP39kDa. The first aim will test the hypothesis that TIP39kDa is involved with the cells secretory activity by down-regulating TIP39kDa expression within cell and organ cultures, and look at the subsequent levels of a secreted protein (amelogenin). For this I will employ both an antisense RNA and a ribozyme approach. The second aim will be to use the TIP39kDa gene promoter in an appropriate reporter system and produce transgenic animals to define organs for which TIP39kDa plays a role. The third aim will identify the subcellular localization of TIP39kDa. For this I will employ available TIP39kDa antibodies and green fluorescent protein tags. Regular database searches will be performed to assist in the identification of functional domains. The fourth aim will be to screen a yeast two-hybrid mouse embryonic library to look for TIP39kDa protein partners. This data will be used to help identify a physiological role for TIP39kDa. The fifth and final specific aim will seek to answer the question of how multiple messenger RNA isoforms of TIP39kDa are generated as evident from previously published data. These first four specific aims will assign a subcellular location and function to a TIP39kDa that, in the case of ameloblasts, has a preference for Tomes' processes, secretory vacuoles and the immature enamel matrix. The fifth specific aim will answer a very basic question related to how TIP39kDa is processed post-transcriptionally, and the potential significance of this.

描述（由申请人提供）：了解基因组学与蛋白质组学之间的生理关系是调查的共同主题和重点。细胞模型和器官系统的使用仍然是实现这种理解的一种基本方法。搪瓷是一种复杂的生物陶瓷组织，是其独特的分子和细胞形成的反映。从它们的外胚层起源中，存在成成成成成成成黑骨细胞，目的是产生矿化壳。壳几乎完全没有蛋白质，并沉积在“骨状”牙本质上。我们最近发现了TIP39KDA，这是一种有利于成成布的分泌表面（TOMES的过程）的蛋白质。 TIP39KDA的抗体对TOMES过程中的分泌囊泡以及未成熟的搪瓷基质反应。 TIP39KDA的表达不是成成成成成成成素的独特，但是初步数据表明，它可能是具有重要分泌功能的细胞所独有的。该项目将采用实验策略来确定TIP39KDA的确定功能，还可以解释针对TIP39KDA观察到的多个Messenger RNA同工型的性质。第一个目的将检验以下假设：TIP39KDA通过下调细胞和器官培养物中的TIP39KDA的表达与细胞分泌活性有关，并查看随后的分泌蛋白（Amelogenin）的水平。为此，我将同时采用反义RNA和核酶方法。第二个目的是在适当的记者系统中使用TIP39KDA基因启动子，并生产转基因动物来定义TIP39KDA起作用的器官。第三个目标将确定tip39kda的亚细胞定位。为此，我将使用可用的TIP39KDA抗体和绿色荧光蛋白标签。将执行常规数据库搜索以协助识别功能域。第四个目标是筛选酵母两杂交小鼠胚胎库，以寻找tip39kda蛋白质伙伴。该数据将用于帮助确定TIP39KDA的生理角色。第五个也是最终的特定目的将寻求回答以前发布的数据中如何生成TIP39KDA的多个Messenger RNA同工型的问题。这四个特定的目标将为TIP39KDA分配一个亚细胞的位置和功能，在Ameloblasts的情况下，它偏爱Tomes的过程，分泌液泡和未成熟的搪瓷矩阵。第五个特定目标将回答与TIP39KDA在转录后如何处理的非常基本的问题，以及此的潜在意义。