Transgenic Artemia: establishment and applicationof stable-transmitted lines

• Main Authors: Shih-Hung Chang, 張世弘
• Other Authors: Huaiu-Jen Tsai
• Format: Others
• Language: zh-TW
• Published: 2008
• Online Access: http://ndltd.ncl.edu.tw/handle/69541803886285504972

碩士 === 國立臺灣大學 === 分子與細胞生物學研究所 === 96 === 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.