在斑馬魚心臟表現 Tet - on 調控系統

Abstract

Gossen (1992 ）利用大腸桿菌（ Escherichia coli ）對四環黴素 ( tetracycline ）抗藥性基因操作子之 tetracycline repressor ( TetR ) DNA 序列，與單純皰疹病毒（ Herpes simplex virus ) virion protein 16 (VP16）之C-terminal domain 相互連接所形成的 tetracycline- controlled transcativator ( tTA ) ，研發出 tetracycline-controlled transcription system 。並進一步利用突變型的 TetR 序列衍生出 reversoTc- controlled transactivactor ( rtTA ) ，在加入 doxycycline 時， rtTA 可與 tetracycline operator ( tetO ）結合而活化其後的報導基因，而稱為Tet - On system 。這套系統廣泛應用於各種細胞株與模式動物中，但迄今尚未在魚類中經過測試。為了在斑馬魚（ Danio rerio ）體內建立穩定表現的 Tet- On system ，利用可在斑馬魚心臟專一表現的zebrafish cardiac myosine light chain ( cmlc2 ) promoter 來啟動、在斑馬魚纖維母細胞良好表現的 rtTA ，經顯微注射後的斑馬魚胚胎雖帶有少許非專一的溢出性螢光，但再經 Doxycycline ( Dox ）處理後，能穩定表現心臟專一性綠螢光。針對帶有Tet - On system 的斑馬魚基因轉殖品系 F1 進行測試，發現不同濃度的 Dox ( 10 ug/ ml 、 1 ug／ml、 100 ng / ml 、 1 0 ng ／ml、 and 1ng / ml ）具有不同誘導效果，並認為1 ug / ml是最適切的誘導濃度。授精後 72 小時的 Fl 胚胎經1 ug / ml Dox 誘導後，於第六小時開始出現肉眼可見的心臟專－性綠螢光；綠螢光的強度隨誘導時間增加，並在誘導後第 36 - 48 小時到達最高值。 Fl 與野生型交配所產下的 F2 胚胎顯示也擁有類似的誘導趨勢，但 Fl 雄雌互配產下的 F2 、誘導後心臟專一性綠螢光的最高亮度稍有增加，達到最高亮度的時間也稍有延遲至誘導後第 48 - 60 小時；與 Fl 性狀相較之下，伴隨著更顯著的誘導效果， Fl 互配產生的 F2 胚胎亦呈現較強的非專一溢出性綠螢光。也期待在斑馬魚體內建立的 Tet - On system , 能應用未來在基因功的研究上。

The tetracycline—controlled transcription system is well developed in many transgenic organisms. Yet, this system has not been reported in zebrafish. In order to develop tet-on regulatory system to allow the transgenic GFP to be expressed specifically in the zebrafish heart, we use the zebrafish cmlc2 promoter to drive the reverse-transactivator(rtTA). We selected the constructed vectors which had good capability to present GFP expression after Dox induction in zebrafish fibroblast cell line. Then, we challenged these vectors in the transgenic zebrafish in vivo. Although fluorescence appeared slightly leaky, transgenesis showed that rtTA in the transient F0 embryos could drive transgenic GFP to be specifically expressed in the hearts. When we examined the germ-line transmitted Fl embryos, we found that the induction efficiency obtained from differentconcentrations of Dox (10 ug/ml， 1 ug/ml，100 ng/ml 10 ng/ml and 1 ng/ml)was various: dosage of 1ug/ml was turned out to be the optimal concentration for induction. After 6 hr induction under treating with 1g/ml Dox, the rtTA-derived heart-specific GFP expression appeared in the transgenic lines embryos at 72-hpf with a relative low leakiness. The higher GFP levels, the longer induction time: the highest level was reached within 36-48 hr after induction. The germ-line transmitted F2 from Fl crossing wild-type has the same tendency of induction, and the F2 from F1 crossing Fl shows stronger GFP intensity of highest level within 48-60 hr after induction These results were consistent with cell line induction. The leaky GFP is also stronger than those of F1 and F2 from Fl crossing wild-type. This is the first tet-on system developed in transgenic fish, which should be very useful to study gene function at any stage whenever gene is induced.