不同外來基因的組成對基因轉殖魚的嵌入性及其表現的影響

Abstract

魚類有卵數多且透明、卵徑大、可用光控制排卵、行體外受精及胚胎發育較快速等特點，再加上魚類也是脊椎動物，使得魚類成為基因轉殖研究的好材料。然而基因轉殖魚往往有外來基因表現率偏低且有差異性(variegated)及鑲嵌性(mosaic)表現的現象，以及不易插入到轉殖魚基因組(genome)致使外來基因遺傳到下一代的機率較低等缺點。 為瞭解決這些問題，本實驗以水母(Aequorea Victoria)綠螢光蛋白(green fluorescent protein, GFP)當報導基因，前面調控區則構築不同基因的啟動子如cytomeglovirus immediate early gene (CMVIE)及稻田魚本身的β-actin基因啟動子，並將線聯病毒(adeno-associated virus, AAV)的inverted terminal repeats (ITR)接到各質體的兩端後，以細胞質顯微注射(cytoplasmic microinjection)的方式，分別將含ITR序列以及不含ITR序列但帶有相同構成的DNA片段導入稻田魚胚胎內，之後用螢光顯微鏡觀察，結果顯示含ITR序列能幫助轉殖的外來基因大量的表現在稻田魚的胚胎內，特別在含有ITR序列及β-actin基因啟動子的構築（p β-EGFPITR 質體），有15.5%的注射胚胎在親代就會產生全螢光表現胚胎，且表現可持續到孵化後兩個月，大大地減少差異性及鑲嵌性表現的現象；Southern blot analysis則顯示p β-EGFPITR的片段有插入到胚胎基因組內，而其中三個全螢光表現胚胎的外來基因甚至會插入到相同的位置上，因此ITR序列確能促使轉殖基因插入到稻田魚胚胎基因組內。

There are some disadvantages in transgenic fish study: the expression rate of transgene is low, the expression pattern of transgene is so rare that it is hard to become germ-line transmission. In this study, we designed several gene constructs to improve the integration rate and to enhance the expression pattern. Promoter of cytomegalovirus immediate early gene (CMV IE) and medaka β-actin gene were respectively fused with green fluorescent protein cDNA (GFP) with or without being flanked with the inverted terminal repeats (ITR) sequence of adeno-associated virus. Results showed that the integration rates of embryos microinjected the DNA fragments which GFP were driven by β-actin and CMV IF were 75% (3 out of 4 embryos examined) and 0% (0 out of 9 embryos examined), respectively. Meanwhile the integration rates of embryos microinjected with same constructs but were flanked with ITR were 100% (8 out of 8 embryos examined) and 60% (3 out of 5 embryos examined), respectively. These results strongly suggest that ITR sequence increases the integration of foreign DNA fragments into the medaka genome. We also observed that the GFP expression was enhanced by the DNA fragments containing ITR sequence. The variegated and mosaic expression pattern of transgenic medaka decreased substantially because embryos microinjected with DNA fragment consisting of ITR sequence appeared higher degree of green fluorescent compared to that of embryos microinjected with DNA fragment without containing ITR. In addition, 38 out of 251 embryos (15.5%) microinjected with fragment which was β-actin promoter fused with GFP and flanked with ITR (β-actin-GFP-ITR) demonstrated that green fluorescence occurred in whole body and last at least two-month-old. Southern blot analysis revealed that the integration site of β-actin-GFP-ITR transgenic fish but GFP expressed in the whole body might be identical.