2013-01-012024-05-17https://scholars.lib.ntu.edu.tw/handle/123456789/678833摘要：生殖細胞對於所動物的繁衍至為關鍵。在發育時期的生殖細胞，後生遺傳機制必須重新設定，不僅促進生殖細胞特化基因的表現與抑制了其他種類基因的表現，且為精卵受精後全化潛能鋪路。當後生遺傳機制在發育時期的生殖細胞經歷全細胞再程序化過程時，原本受到多個層次的後生遺傳機制的調控而呈現重度靜默的反轉錄跳躍子，將會去掉其受抑制的現象，此一主要的邊際效應對於細胞程序重新設定(reprogramming) 的過程是無可或缺。為了避免或盡量減低因活化的反轉錄轉錄物所引起之惱人的反轉錄移位事件，生殖細胞發展出數種跨種族之緊急的後生遺傳調控的靜默機制，但這些機制都涉及到 small RNA 後轉錄基因的靜默活性。在本計畫中，我們打算利用雞與蚜蟲這兩種新發展的後基因模式物種中 PIWI-interacting RNAs (piRNAs) 如何生成，以及 piRNA 如何調控反轉錄子靜默之機制。結合現有由模式昆蟲黃果蠅(Drosophila melanogaster) 獲得之piRNA 生成路徑與piRNA 調控功能，雞與蚜蟲piRNA 分子網路的探究將對 piRNA 如合抑制跳躍子之機制提供重要之演化發育資訊，使我們明瞭piRNA 功能與參與 piRNA 分子網路的成員如何在無脊椎與脊椎動物中演化。目前在其他物種中piRNA 之研究尚屬起步階段，特別是Piwi 和 Ago3 蛋白是否涉入、或是如何涉入 piRNA 之產生與調控跳躍子之活性幾乎未知。本計畫利用奈米級微型核酸晶片針對雞與蚜蟲的微量生殖細胞的 pi-RNA、transcriptome 及 small RNA 進行偵測，所測得之核酸樣本將可以從探針上分離出來並進行 NGS 之研究。所選殖出的 NGS 小分子核酸可視為後轉錄時期基因靜默相關小分子核酸 (PTGS-related small RNA)，有助於在微量的生殖細胞中找尋新的 miRNA, siRNA 及 piRNA。此外，利用染色質免疫沈澱技術 (ChIP)，將其運用於奈米晶片上，亦可以進行 ChIP 及其 NGS 分析，探究生殖細胞中 DNA 及 Histon modification。計畫顧問鍾正明院士與博士生廖虹富、共同主持人張俊哲博士分別具備成熟之操作雞與蚜蟲胚胎發育經驗，計畫主持人林劭品博士在生殖系統及後生遺傳學多年的經驗，加上近年在小鼠及人類PiRNA pathway相關之研究進展，輔以共同主持人林詩舜博士與協同主持人陳逸聰教授共同開發之奈米級微小RNA之分析平台，我們必能掌握雞與蚜蟲這在基因體與發育生物學之研究特色與先機，在piRNA 之研究取得領先國際之利基。<br> Abstract: Germ cells are the critical cells that transfer genetic information from generation to generation in animals. During the germline development, the epigenome have to be reset as to facilitate the expression of germline-specific genes and to suppress genes irrelated to germline development. While the epigenome in developing germ cells are undergoing genome wide reprogramming, the retrotransposons that are heavily silenced by layers of epigenetic machineries, will be de-repressed as a major side effect of the necessary reprogramming process. In order to prevent or minimize the dreadful retrotransposition event caused by active retrotranscripts, the germ cells have evolved to silence the activity of transposons and the inhibition of transposition has been regarded a universal phenomenon in germ cells across invertebrates, yet the mechanisms remain almost unknown. Here we propose experiments for studying the biosynthesis of PIWI-interacting RNAs (piRNAs) and how piRNAs are involved in silencing transposition in chicken and aphids, two emerging genomic model organisms. In combination with the growing information of piRNA in the fruit fly Drosophila melanogaster, we expect to understand how the piRNA pathways are evolved in invertebrates and vertebrates through the studies in chicken and aphids. To date little is known about functions of piRNA in other species, in particular whether PIWI and Ago3 proteins are involved in the generation of piRNA and suppression of transposition, as that known in Drosophila. Hence the exploration of piRNA in the chicken and aphid will be of great advantage in terms of understanding the evolution and development of the piRNA pathway. With the exciting nano-Chip small RNA detection and analysis platform developed by our Co-PIs, we will analyze the piRNA including small RNA species from chicken and aphids that are soociated with post transcriptional gene silencing. In addition, combining the nano-Chip technology with chromatin immunoprecipitation, followed by high throughput sequencing, we will have the opportunity to perform DNA methylation and histone modification studies with a few germ cells from specific developmental stages of chicken and aphids. With the PI, Dr. Shau-Ping Lin’s extensive reproductive and epigenetic research background and the good progress in the PiRNA pathway related research in human and mouse, plus the exciting newly developed nano-chip small RNA analytical platform and the enormouse research experience in chicken and aphid embryos provided by our consultant, Prof. Cheng-Ming Chuong and a very capable PhD student, Mr. Hung-Fu Liao and CoPI Dr. Chun-Che Chang, we have the unique international competitive eage in solving the piRNA associated retrotransposon silencing mechanisms that are crucial for chicken and aphid germ cell development. These researches have not only evolutionary significance but also implications in regenerative medicine in general.