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Title: | 研究以精子載體法進行淡水長臂大蝦的基因轉殖 Study on the Sperm-mediated gene transfer in fresh water giant prawn,Macrobrachium rosenbergii |
Authors: | 李思賢 |
Publication Year : | 1998 |
Degree: | 碩士 |
Abstract: | 精子載體法是一種方便且快速的基因轉殖法,能在短時間內處理大量的樣本,且能夠克服某些具有堅硬卵殼的生物在進行基因轉殖時所遭遇的困難。本論文研究利用精子載體法來進行淡水長臂大蝦(Macrobrachium rosenbergii) 基因轉殖。所用的 DNA 片段是由 CMV (cytomegalovirus) 早期表現基因的?動子,後接綠螢光蛋白(green fluorescen protein)的 cDNA 及 SV40 多腺核酐訊號(polyadenylation signal)所組成,以 GFP 當作 report gene 。研究結果顯示(?) 以 7.5 伏特的電壓可以取得雄蝦的精莢,且不會對雄蝦造成傷害。(?)以蒸餾水作為 DNA 的溶劑不會影響蝦卵的受精與發育。(?)當 DNA 溶液的濃度超過 50 ng/?l l時,會造成蝦卵無法自然孵化。若濃度降低到 28 ng/?l l時,則可以受精成功且能自然孵化,孵化率卻下降為 52.5%(控制組為82.5%)。(?)抽取20隻以 DNA 溶液(28 ng/?l l)進行基因轉殖處理所得幼蝦(zoea l)的體染色體 DNA,經由聚合脢戀反應和南方氏轉漬法檢測。發現在20個樣本之中有一個樣本(5%)和 positive control 有一樣有 positivc signal,其分子量大小均為 650 bp 。由此,可以確定淡水長臂大蝦可以基因轉殖成功。(?)若將 DNA 溶液的濃度提高至 50 ng/?l l時可以受精成功,其轉殖率高達100% (8/8) ,且可以觀察到 GFP 的表現(zoea l),其表現率為 13.3% (4/30),但無法以自然方式孵化。本研究證實精子載體法可以將外源性基因導入淡水長臂大蝦體內,並可利用 GFP 選出轉殖成功的幼蝦。 Sperm mediated gene transfer is a rapid and convenient method to transfer foreign genes into organisms. A large amount of samples could be treated in a short period of time. Also, the problems we would encounter when transferring genes into eggs with tough egg shells could be eliminated. In our study, we assessed the feasibility of transferring foreign genes into Macrobrachium rosenbergii with this method. The DNA fragment used in the experiments is the promoter of CMV (cytomegalovirus) immediate early gene followed by cDNA of GFP and SV40 polyadenylation signal in sequence. The GFP gene is used as a reporter gene here. In the results of our experiments. (1). The spermatophore could be obtained from male shrimp by using 7.5 volts of electrical shock. No permanent injuries are observed after this treatment. (2).Using ddH2O as the solvent in dissolving DNA samples would not affect the fertilization and subsequent development of shrimp eggs. (3). When the foreign DNA concentration exceeds 5Ong/?l, the eggs would not be nature hatched. The fertilization is successful as the concentration of DNA aqueous solution is down to 28ng/?1.But,the hatch rate is down to 42.5% (Hatch rate of the control group is 82.5%). (4).We extract genomic DNA from 20 shrimps (at zoea 1 stage) which were treated with 28ng/?l of DNA solution. After PCR amplification and southern blot analysis, we observed that a sample (5%) shows the same positive signal with positive control. Their molecular weight are both 650bp. Therefore, the feasibility of transferring foreign DNA into Macrobrachium rosenbergii is definite. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/74999 |
Fulltext Rights: | 未授權 |
Appears in Collections: | 漁業科學研究所 |
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