請用此 Handle URI 來引用此文件:
http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/75999
標題: | 顯微注射lacZ基因在稻田魚早期胚胎發育中的暫時性表現 The transition expression of lacZ gene microinjected into medaka (Oryzias latipes) oocytes during early embryonic development |
作者: | Shu 一 Huei wang 王琡惠 |
出版年 : | 1993 |
學位: | 碩士 |
摘要: | 將三種不同病毒的基因控制區,Rous sarcoma virus ( pmiwZ )、Cytomegalovirus immediate-early gene ( pCAGGS-lacZ)、Moloney murine leukemia virus ( pMoZtk) ,接上reporter gene ( lacZ gene),分別利用顯微注射法,注入稻田魚的卵細胞中,再用X-gal的組織化學染色法,來測知此基因在稻田魚中的暫時性表現。結果顯示:( 1 ) .注射後的卵,人工受精率約為43~72%,而胚胎發育至第24hr的存活率為40~65%:與對照組(分別為76~ 85%與72~82%)相比,都有顯著性降低。(2) .以南方氏墨點法(Southern blot)來確認 reporter gene 的存在,結果在轉殖 pMoZtk DNA 片段的2hr、3hr、6hr 的胚胎出現模糊延伸的長條狀反應。(3 ) .此三種DNA均在胚胎發育至囊胚期 ( blastula stage)時開始被表現,其中pMoZtk較晚,約在囊胚後期才出現;且在2天後的胚胎就都不再出現。因此lacZ 基因表現的型式,與病毒基因的調控區域及胚胎發育的時期相關。而分佈的形式,則為鑲嵌型點狀出現於胚胎中。(4 ) .若注射沒lacZ encoding region的DNA 片段,則無法造成胚胎有藍色出現的結果;可知酵素呈色反應確是由轉殖基因所造成。(5 ) .競爭性的第二種DNA的加人,可造成基因提早6hr表現,故推測胚胎控制基因轉錄開始時間的方式,可能與Xenopus相同,屬於titration model。 Three different regulatory regions of viral genes , rouse sarcoma virus (pmiwZ), cytomegalovirus immediate-early gene (pCAGGS-lacZ), moloney murine leukemia virus (pMoZtk) microinjected into medaka oocytes. Histochemistry method were used to detect the transition expression of lacZ gene during early embryogenesis. Results indicated: (I).the rate of fertilization and survival (24hr after fertilization) of injected oocytes were 43-72% and 40-65%, respectively. Compared to control group (76-85% and 72-82% for fertilization and survival rate, respectively), microinjection cause a clearly decrease in fertilization and survival rate. (2). Southern blotting revealed that there was a diffused band shown at 2hr, 3hr and 6hr-old injected pMoZth embryos’ genome. (3). The initiation of lacZ gene expression was at mid-blastula stage of embryos injected either pmiwZ or pCAGGS-lacZ, but for embryos injected pMoZtk the expression initated at the late blastula stage. All the expression then disappeared in 2 days-old embryos. The timing of the gene expression was related to the regulatory region of viral gene and the stage of embryonic development. The pattern of gene expression was mosaic. (4). Injection of DNA fragment without containing lacZ gene encoding region failed in blue spots appearance. This result indicated the positive response was controlled by transferred lacZ gene. (5). Co-transferred the competitive DNA(pMT-X) and reporter gene (pmiwZ) showed that the expression of lacZ gene was 6hr earlier than injected lacZ gene alone. This suggested that the mechanism of controlling the gene transcription in medaka embryos maybe the same as the titration model reported in Xenopus embryos. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/75999 |
全文授權: | 未授權 |
顯示於系所單位: | 漁業科學研究所 |
文件中的檔案:
沒有與此文件相關的檔案。
系統中的文件,除了特別指名其著作權條款之外,均受到著作權保護,並且保留所有的權利。