以Tol2跳躍基因系統剖析斑馬魚six1,six6 及人類 DMP1之組織專一性促進子之功能，並以此系統在斑馬魚基因體進行高輸出量「基因捕獵」之研究

Abstract

Tol2元素是從日本青鱂魚(medaka fish)的基因組中分離出來。Tol2元素是屬於hAT轉位子家族成員之一，hAT家族包括，果蠅中的hobo、玉米中的Ac及金魚藻中的Tam3。轉位子被用來在研究果蠅基因體或是其它模式動物中，是非常具有效力的工具。但是，這樣的工具卻尚未在斑馬魚上建立。由於斑馬魚基因組上，沒有具有活性的DNA轉位子。Kawakami實驗室在2000年，成功的將Tol2轉位子系統運用斑馬魚上。Kawakami等人，建立了高效率的轉殖方法，包括含有Tol2轉位子載體的質體DNA和體外(in vitro)合成的轉位酶酵素mRNA以顯微注射的方式一注射到一個細胞期的斑馬魚受精卵中。結果發現有約50%的F0(Founder)會將其生殖線遺傳給下一代。此外利用Tol2轉位子系統，也可用來建立新基因的捕獵(gene trap)及插入性的突變(insertional mutagenesis)方法，在斑馬魚或其他模式動物上。 鑒於，Tol2轉位子高效率的轉殖系統，我將Six1、Six6.1、Six6.2及DMPE2 DNA片段含有Tol2轉位子載體的質體與轉位酶酵素mRNA，以顯微注射的方式送到斑馬魚胚胎中。利用DNA與mRNA之間不同的劑量(dosages)，來測試Tol2轉位子的最佳效果。首先，我將DNA與mRNA的濃度等比往上加，分別配了濃度為1X(DNA 25ng/µl；mRNA 25ng/µl)、2X(DNA 50ng/µl；mRNA 50ng/µl)及4X (DNA 100ng/µl；mRNA 100ng/µl)三種劑量，結果發現隨著的濃度上升，的確可以使斑馬魚體節的綠色螢光變強(以Six1基因來測試濃度)。但是高劑量的濃度4X會導致胚胎的死亡率提高及產生畸形。於是，我將濃度固定在2X，mRNA的濃度則等比例的往上加，分別配了2X0R(DNA 50ng/µl；mRNA 0ng/µl)、2X2R (DNA 50ng/µl；mRNA 50ng/µl)及2X4R(DNA 50ng/µl；mRNA 100ng/µl)三種劑量，結果發現濃度為2X2R 及2X4R的轉殖斑馬魚綠色螢光可以達到4X濃度的亮度，而且死亡率也降低了。 此外，我也利用Tol2轉位子系統在斑馬魚建立了基因捕獵(gene trap)方法，如果Tol2跳躍子能夠剛好插入一段基因中間，會使得此基因無法表現，造成外表性狀的差異，便可利用此含GFP mutant找出基因。我所建立的基因捕獵(Troy)當中含有兩個鏡像的exons 且內含IRES-GFP，當Tol2轉位酶酵素辨認到含有Tol2左右兩端的序列的質體後進行切割(excised)，嵌入基因組中，不論方向性都可以使基因進行轉錄及轉譯成蛋白質。此外，IRES-GFP並非fusion蛋白，可以使ribosome與kozak sequence結合時，不會有in frame reading的問題。這兩個特性可使基因捕獵的效率(6倍)高於Kawakami實驗室所建立的基因捕獵。 由以上的實驗結果發現，要達到Kawakami實驗室建立的Tol2系統，找到 DNA與mRNA濃度之間的平衡點是很重要的。另一個不同點是，Tol2轉位子嵌入基因組類是以single copy的方式，非Tol2系統則是以多連體DNA(concatemers)的方式；這可能是導致Tol2系統斑馬魚綠色螢光表現強度較弱與非Tol2系統所建立的螢光不太一樣的原因(以Six6.1基因為例子)。

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).