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頁籤選單縮合
題 名 | 基因轉殖小鼠基因嵌插點定位技術之建立=Establishing a Technique for Identifying the Transgene Integration Site in Mice |
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作 者 | 楊凱傑; 潘筱茜; 劉郁佳; 賴瑋婷; 吳希天; | 書刊名 | 台灣農學會報 |
卷 期 | 14:1 2013.02[民102.02] |
頁 次 | 頁66-85 |
分類號 | 368.4 |
關鍵詞 | 基因轉殖小鼠; 染色體步移法; 反向式PCR; Transgenic mouse; Chromosome walking; Inverse PCR; |
語 文 | 中文(Chinese) |
中文摘要 | 由 DNA 顯微注射法所產製的基因轉殖小鼠,其轉殖基因容易在染色體中嵌入多個重複的組合,而這些組合會影響轉殖基因表現的強弱。基因定位的方法傳統上常採用螢光原位雜交法 (fluorescence in situ hybridization, FISH),但其染色體圖像解析度受限於技術不易辨別,無法得知轉殖基因嵌插於宿主染色體之確切位置。本研究結合聚合酶連鎖反應 (PCR) 和序列分析等分子生物技術,與生物資訊資料庫比對等技術之染色體步移法 (chromosome walking),尋找 FVB/Ncrl-Tg(PGK1-EGFP)01Narl 基因轉殖小鼠轉殖基因片段嵌插於染色體確切區域。試驗首先利用 digoxigenin (DIG) 標誌之探針與定量的基因轉殖小鼠基因組 DNA 進行雜交,再經影像軟體計算,推算出轉殖基因在基因轉殖小鼠染色體之套數為 32.6±5.7。進一步將轉殖小鼠基因組 DNA 以多種限制酶處理,判斷嵌入之 PGK1-EGFP 轉殖基因的疊接模式,結果證實為頭接尾 (head-to-tail) 的疊接模式。應用反向式 PCR (inverse PCR) 方法擴增出含有轉殖基因與小鼠染色體連接的部分區域片段,再經由選殖至載體,基因定序,並與 NCBI (BLAST, NCBI bioinformatics) 小鼠基因組資料庫作序列比對分析,結果顯示轉殖基因的嵌入位於小鼠第 6 號染色體長臂 (q arm) 上,A 至 G 區域距離 30 cM 之 C區域,編號為第 1 之次區域 (Chrom. 6, 30 cM qC1) 之免疫球蛋白 κ 輕鏈 (immunoglobulin kappa chain complex, Igκ) 基因座內,位於 exon 84 基因座變異區之間。本試驗利用分生技術配合完整的小鼠生物資訊庫,精確的檢測出轉殖基因套數及排列方式與定位外源基因嵌入染色體之確切位點。 |
英文摘要 | Transgenes introduced into transgenic mice by microinjection are often integrated into the host genome in a form of repeated combinations. These combinations affect the expression of the transgene. Fluorescence in situ hybridization (FISH) is frequently used in the traditional analysis of gene mapping, but the resolution of the chromosome is inadequate due to its technical limitation and cannot provide information about the chromosomal location of the inserted transgene. In this study, we used DNA cloning, PCR and sequence analysis of chromosome walking, contrasted with the mouse bioinformatics database to determine transgene insertion in the chromosome of a FVB/Ncrl-Tg(PGK1-EGFP)01Narl transgenic mouse. By using a DIG probe to hybridize with quantitative transgenic mouse genomic DNA, the PGK1-EGFP transgene copy number was estimated to be 32.6 ± 5.7. The direction and integration pattern of the inserted transgene were analyzed by multiple restriction enzyme digestion followed by Southern blotting. The results showed that the transgene PGK1-EGFP was inserted into the chromosome in a head-to-tail pattern. The junction region between the transgene and mouse chromosome was then cloned by inverse PCR. The cloned fragment was sequenced and analyzed via BLAST (NCBI, Bioinformatics). DNA sequence revealed that the transgene was inserted into exon 84 of the mouse immunoglobulin kappa chain complex gene (Igκ), with a distance of 30cM qC1 at chromosome 6. In conclusion, by using a combination of molecular techniques and the mouse bioinformatics database, we now can accurately determine the site of the inserted gene, copy number and integration pattern in mice chromosomes. |
本系統中英文摘要資訊取自各篇刊載內容。