2011-08-012024-05-13https://scholars.lib.ntu.edu.tw/handle/123456789/642687摘要：α-internexin是一種神經元中間絲蛋白，先前的研究發現，在哺乳動物中樞神經系統發育的過程中，α-internexin表現時間早於神經元中間絲蛋白三合體(neurofilament triplet proteins)。這些研究結果顯示α-internexin此細胞骨架蛋白在早期神經發育細胞骨架之動態變化扮演重要的角色。雞是一種演化重要的模式動物，牠填補了哺乳類與其他脊椎動物在生物演化學研究上的空缺，同時也是第一個被全基因定序的非哺乳類羊膜動物。由於其易得性與操作方便，雞胚在神經發育生物學研究中是經常被使用的動物模式。目前已知有部分的雞神經中間絲蛋白已被選殖與定性。然而，卻沒有相關研究探討α-internexin在禽鳥類中樞神經系統發育過程中的的表現與生理機制。在本研究計畫中，第一年我們將分析雞的基因體，從中辨別並選殖雞的α-internexin同源基因，確認基因序列、結構與其在雞胚發育過程中之表現情形。同時，我們將利用該基因序列生產雞α-internexin的多株抗體，並確認其效力與專一性。在第二年的計畫中，我們將以原位雜交與免疫組織染色技術，確認α-internexin在雞胚神經系統中之表現與分布狀況。同時，我們將嘗試建立穩定表現此中間絲蛋白的雞細胞株，以利後續的細胞生物學研究。最後，在第三年的計畫裡，我們將在雞胚中建立α-internexin gene knockdown系統，以研究基因在動物體內之生理功能。此基因之功能確定後，可望應用於以雞為模式生物之神經發育學研究，並為基因體學、演化發育生物學以及生物資訊學研究提供有力的線索。<br> Abstract: α-Internexin is one of the neuronal intermediate filament (IF) proteins, which also includes neurofilament triplet proteins (NFTPs) designated as NF-L, NF-M and NF-H for low, middle and high molecular weight subunits. The expression of α-internexin precedes those of NFTPs in most neurons of the developing brain. α-Internexin is now recognized to be structurally and functionally associated with the NFTP in the developing mammalian CNS. The chicken is an important model organism that fills the evolutionary gap between mammals and other vertebrates. It also represents the first non-mammalian amniotes to have its genome sequenced. The chicken embryo has been a useful model system in developmental biology due to experimental advantages of in ovo embryogenesis. Some neuronal IFs genes of chicken were cloned in the previous studies. However, there is little known about the gene sequence and physiological function of neuronal IF α-internexin in avian. In this project, we are going to identify the mRNA sequence encoding chicken α-internexin and characterize the gene structures and expression levels during developmental process. Furthermore, we are going to generate specific polyclonal anti-chicken α-internexin antibodies. In the second year of this project, we’ll confirm the gene expression patterns and protein distribution by in situ hybridization and immunohistochemistry. Meanwhile, we’ll tend to establish stable chicken cell lines that constantly express tagged chicken α-internexin for extended cytological studies. In the third year of this project, we’ll investigate the function of chicken α-internexin genes by establishing the gene knockdown system in the chicken model. The annotation of chicken α-internexin may be applied for neuroscience researches. Moreover, this may fill the gaps of the molecular evolution of neural intermediate filaments, and provide hints in genomics, evolutionary developmental biology or bioinformatics.α-internexin雞神經發育基因體神經元中間絲蛋白α-internexinchickenneural developmentgenomeneurofilament