Functional analyses of the tissue-specific enhancer elements of the zebrafish six1,six6 and human DMP1 and in vivo high throughput gene-trapping by Tol2 transposon system

• Main Authors: Yu-Chen Lin, 林育珍
• Other Authors: Bei-En Chang
• Format: Others
• Language: zh-TW
• Published: 2010
• Online Access: http://ndltd.ncl.edu.tw/handle/69135607292760554281

碩士 === 臺灣大學 === 口腔生物科學研究所 === 98 === The Tol2 element was isolated from the genome of the Japanese medaka fish. The Tol2 transposable element belongs to the hAT family of transposons including hobo of Drosophila, Ac of maize, and Tam3 of snapdragon. Transposons have been used as powerful tools for genetic studies in Drosophila and other model organisms. Such a tool had, however, not been developed in zebrafish. This is because no active DNA-transposable element has been found from the zebrafish genome. In 2000, Kawakami has used Tol2 transposn system successfully in zebrafish. They established a highly efficient transgenesis method in which a plasmid DNA containing the Tol2 transposon vector and the transposase mRNA synthesized in vitro were coinjected into one-cell stage embryos. It was estimated that about 51% founder fish would transmit foreign DNA via their germline cell to the next generation. The Tol2 transposon system should thus be used to develop novel transgenesis and insertional mutagenesis methods in zebrafish and possibly in other animal models. In view of the high efficiency of Tol2 trangennesis, I cloned Six1, Six6.1, Six6.2 and DMPE2 DNA sequences in Tol2-containing vector and coinjected with transposase RNA into zebrafish embryos. I used different dosages of DNA and mRNA to evaluate the best efficiency of Tol2 transposn system. At first, I added DNA and mRNA increasely on the same ratio containing 1X (DNA 25ng/µl；mRNA 25ng/µl), 2X (DNA 50ng/µl；mRNA 50ng/µl) and 4X (DNA 100ng/µl；mRNA 100ng/µl), three different dosages. And with this increasing dosages, I found that high dosages could lead to strong GFP expression in the zebrafish somite (Six1gene is tested). But high dosages could also lead to high ratio of embryonic lethality and abnormality. Therefore, I fixed DNA dosages with increasing mRNA dosages containing 2X0R (DNA 50ng/µl；mRNA 0ng/µl), 2X2R (DNA 50ng/µl；mRNA 50ng/µl) and 2X4R (DNA 50ng/µl；mRNA 100ng/µl), three different dosages. I found that transgenic zebrafish with the doses of 2X2R and 2X4R express GFP at levels comparable to 4X transgenic zebrafish and the death rate was also decreased. Furthermore, I also develop a gene trap method in zebrafish using the Tol2 transposon system. If the Tol2 transposon is inserted into the genome, the insertion could interfere with expression of the trapped gene leading to phenotypes difference. We can use the mutant containing GFP to find the trapped gene. I develop the gene trap method, Troy, which contains two mirror-image exons harboring IRES-GFP. When Tol2 transpoase identifies Tol2 construct containing Tol2 left-right end sequences, the Tol2 construct is excised from the donor plasmid and integrated into the genome. It is no matter what the direction of insertion is, the inserted trapped gene could transcrible into protein. In addition, IRES-GFP is not a fusion protein. There is no problem of frame-reading shift, when ribosomes bind to kozak sequence. The efficiency of our gene trap with these two features is six times higher the gene trap developed by Kawakami’s laboratory. The results of the experiments suggest that, it’s impotent to find the balance of dosages between DNA and mRNA to achieve the Tol2 transposon system established by Kawakami’s laboratory. Another difference is that Tol2 transposon inserted into genome is by single copy, whereas non-Tol2 transposon transgenes are inserted by DNA concatemers. These could lead to different intensities of GFP expression between Tol2 transgenic zebrafish and non-Tol2 transgenic zebrafish (example of Six6.1 gene).