https://scholars.lib.ntu.edu.tw/handle/123456789/163749
標題: | 行政院國家科學委員會補助專題研究計畫成果報告:視網膜退化在犬模式的候選致病基因之分離、轉植、及表現 | 作者: | 林中天 | 關鍵字: | 視網膜退化;視網膜基因;犬基因;遺傳疾病;表現基因序列;基因表現;retinal degeneration;retinal genes;canine genes;inherited disease;expressed sequence tags;gene expression | 公開日期: | 2000 | 出版社: | 臺北市:國立臺灣大學獸醫學系暨研究所 | 摘要: | 視網膜退化,或稱為"犬漸進性視網膜萎縮 (PRA)",是犬隻最常見的遺傳性眼睛疾病之 一,致病基因十分複雜。由於大部分犬視網膜 基因仍是未知的,深入研究犬視網膜基因之特 性及功能是很重要的。 在本計劃中,採取非染色體定位式候選基 因鑑定法(position-independent candidate gene approach)及EST (expressed sequence tags)法來篩選犬的重要之視網膜重要基因及 PRA 候選疾病基因。利用分離、選殖、及表現視 網膜基因,以了解重要視網膜基因在分子層級 的特性。本計畫分成三個主要的部份:自犬視 網膜cDNA 庫鑑定和分離基因;選殖並定序基因; 以及視網膜基因的表現。 在第一階段,先建造犬視網膜cDNA 庫並嵌 入l噬菌載體中,作為犬視網膜基因之來源。此 cDNA 庫中含視網膜特異性表現之基因比率可經 由篩減雜交法(subtractive hybridization) 增高, 以產生犬的篩減視網膜cDNA 庫 (subtractive retinal library)。此兩個cDNA 庫的視網膜特異性表現之基因可利用差別表現 篩檢雜交法(differential plaque/colony hybri-dization)選出。在分離出視網膜特異性 表現之基因後,在第二階段,純化並定序基因 之DNA,以獲得之DNA序列,並利用BLAST 和 FASTA 程式作GenBank資料庫之搜尋及序列比較 分析。在比較資料庫後,在第三階段,選取6 個重要基因(主要為人視網膜退化致病基因)作 表現實驗,利用北氏雜交法(northern blotting hybridization)及RT-PCR 來分析候選基因在各 犬組織的表現情形。 犬視網膜cDNA 庫之噬菌體量(titer)為 1.5 x 109 p.f.u./ml,96%成功地含有犬視網膜 cDNA (inserts),平均cDNA 長度為1.6 kb。自犬 視網膜cDNA 庫,共分析並定序76 株視網膜特 異表現基因及100 隨機棌樣株。76 株中有54 株(71%)和資料庫之序列有高相似性,其中意外 地首次發現功能很重要之犬sFRP2 基因,為犬 視網膜最豐富存在之特異表現基因,超過過去 其他種別視網膜在文獻所述之opsin 基因。最 近發現,sFRP 基因在控制發肓、影響腫瘤發生、 及調控細胞凋亡具重要之功能。其餘22株(29%) 為未知之新基因。在100 隨機株中,53 株和資 料庫之序列有高相似性,其餘47 株為未知之新 基因。在基因表現實驗, 其中opsin, transducin 1A, cGMP-PDEA,p61 及HRG4 為視 網膜特異表現之基因,不表現於其他犬組織;而 一犬視網膜株p23 則無任何組織特異性之表 現。 Canine generalized progressive retinal atrophies (gPRA) are a group of degenerative retinal diseases that are a major cause of hereditary blindness in a number of dog breeds. Expressed sequence tag (EST) approach was used to identify and characterize potential candidate genes from canine retinal cDNA libraries. A conventional and subtractive canine retinal cDNA libraries were constructed. For the conventional cDNA library, the titer was 1.5 x 109 p.f.u./ml. The average insert size was 1.6 kb and 96% of the clones contained inserts. Differential hybridization was performed to identify abundantly retinal expressed cDNA clones. Random and differentially expressed clones were fluorescently labeled and sequenced. The sequences were analyzed using GCG software and searched against GenBank database. For genes of interest isolated from the libraries, northern blotting and RT-PCR were performed to determine mRNA expression of the genes. DNA sequences from 76 differentially expressed clones and 100 random cDNAs and analyzed. 54 out of 76 differentially expressed cDNA clones (71%) showed homology to known genes in the database. An unexpected and interesting finding is the isolation of a functionally important gene sFRP2, which is the most abundant gene in the canine retina. The remaining 22 cDNA clones (29%) showed no homology to database sequences, representing new sequences. In 100 random canine retinal ESTs, 53 clones (53%) showed homology to database sequences (including known genes and ESTs). The remaining 47 cDNAs (47%) showed no homology to database sequences. Four candidate genes and 2 anonymous retinal ESTs were selected to analyze mRNA expression. The four known genes, namely opsin, cGMP-PDEA, transducin 1A, and HRG4 showed retina-specific expression. In anonymous ESTs, clone p61 revealed retina-specific expression, while p23 showed no tissue specificity. The isolation of sFRP2 and HRG4 is the first finding in the canine retina. |
URI: | http://ntur.lib.ntu.edu.tw//handle/246246/28688 | 其他識別: | 892313B002138 | Rights: | 國立臺灣大學獸醫學系暨研究所 |
顯示於: | 獸醫學系 |
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