蔡懷楨Tsai, Huaiu-Jen臺灣大學:分子與細胞生物學研究所張世弘Chang, Shih-HungShih-HungChang2010-06-022018-07-062010-06-022018-07-062008U0001-0907200817410100http://ntur.lib.ntu.edu.tw//handle/246246/184679甲殼類豐年蝦的無節幼蟲，是最廣泛被利用於餵食養殖魚蝦貝類的活體餌料生物。我們利用電穿孔轉殖於豐年蝦休眠卵中。觀察孵化後七天具有綠螢光之F0豐年蝦個體，其綠螢光表現率為13.3% (3219/24054)，進而選取200隻有強綠螢光表現的豐年蝦個體，再與野生型 (wild-type)進行配對後得到F1，選取F1豐年蝦中有綠螢光表現，進行近親自交得到F2，選取F2豐年蝦中有綠螢光表現，進行近親自交得到F3，並針對每一子代 (G0~F3)染色體DNA進行萃取，利用PCR偵測是否含有698 bp綠螢光基因PCR放大的片段及564 bp黃鰭鯛生長激素 PCR放大的片段，最後成功篩選出2株遺傳穩定的轉殖品系A3及A8。經SDS-PAGE分析後，發現在相同位置與野生型比對有明顯多出一條band，將該band切割下來進行LC-MS蛋白質定序，經過序列比對後，證實為重組黃鰭鯛生長激素rGH。並利用西方轉漬法偵測重組生長激素能在25 kDa 被偵測到。經ELISA 測定50隻來自轉殖豐年蝦A3及A8 homozygotic品系的無節幼蟲，其所含重組黃鰭鯛生長激素分別為0.089μg 及0.032μg。進一步地，斑馬魚(Danio rerio)於受精後第25天(dpf)的幼魚，分別施予每日餵食10隻野生型或轉殖豐年蝦，連續餵食十天後，結果顯示餵食野生型的組平均淨增長率為34.74% ;而餵食轉殖組的豐年蝦的平均淨增長率為53.24%，餵食轉殖組其平均體長比野生型組高出15.5%，具有顯著差異(p<0.01)，因此，我們成功地建立一種可以穩定表達外來基因的轉殖豐年蝦品系，且能經口服的方式促進魚苗生長。Brine shrimp nauplii (Artemia sp.) are mostly used as the livefeed for many cultured marine shellfish and finfish larvae, and it has also been used in the biological study as a model organism. Yet, the transgenesis of artemia has not well documented. Here, we applied electroporation on artemia cysts to generate the transgenic artemia. After the artemia cysts were decapsulated, the cysts were then electroporated with two plasmids, in which one contained GFP reporter gene and the other contained yellowfin porgy growth hormone (ypGH) cDNA. The transient GFP signal shown in the artemia larvae was observed at 7-d after hatching and the expression rate was 13.3% (3219 out of 24054 examined). We chose 200 G0 individuals harboring a high GFP fluorescence to mate with wild-type. Among F1 offsprings, we sampled high GFP- positive individuals to generate F2, and thereof F3 generations of Artemia. Genomic DNAs were extracted from the G0, F1, F2 and F3 and were checked with PCR detection. Two stable lines of transgenic Artemia were screened, named A3 and A8, because a 698-bp and a 564-bp PCR product were amplified, which were correspondent with PCR from GFP gene and ypGH cDNA, respectively. We found that one extra protein band with 25-kDa was shown in the transgenic Artemia, when it was compared with the total protein profile of wild-type. This extra band was cut out and digested in-gel for LC-MS peptide mapping, resulting that the amino acid sequence was confirmed as the recombinant ypGH. In addition, this 25-kDa protein was positive for antiserum against ypGH .The concentrations of ypGH produced by 50 homozygotic nauplii from two transgenic Artemia lines A3 and A8 were calculated as 0.089μg and 0.032μg by indirect ELISA. We respectively fed zebrafish (Danio rerio) on 10 wild-type or transgenic Artemia nauplii once a day from 25 dpf (days of postfertilization) to 35 dpf. The wild-type group average body length gain rate was 34.74%. The transgenic group average body length gain rate was 53.24%. Transgenic Artemia was statistically 15.5% larger than feeding wild-type at 35 dpf (p<0.01). Therefore, we conclude that we successfully establish two transgenic lines of Artemia expressing exogenous genes and enhance fish growth by oral administration.中文摘要 (Abstract in Chinese)…………...…..…...…...… 1文摘要 (Abstract in English)…………...…..…..………. 2言 (Introduction)………………………..………………. 4料與方法 (Materials and methods) …….……………… 10果 (Results)…………………………….………………..21論 (Discussion)……………………….………………... 26考資料 (References)……………………..…………….. 32與表 (Figures and tables) …………................................ 39錄(Appendix)……………………………………………. 49application/pdf1403175 bytesapplication/pdfen-US豐年蝦基因轉殖魚類生長激素Artemiatransgenefish growth hormone[SDGs]SDG14http://ntur.lib.ntu.edu.tw/bitstream/246246/184679/1/ntu-97-R95b43029-1.pdf