電穿孔法在豐年蝦之基因轉殖研究

Abstract

甲殼類生物豐年蝦 (Artemia sp.) 的無節幼蟲，又稱為鹽水蝦，是最廣泛用於餵食養殖魚貝類幼苗的活體浮游餌料生物。藉由基因轉殖豐年蝦系統建立的優勢，其上游消費者的特性可被間接改良，例如：快速成長或抗病。此方法也許可以降低使用基因改良魚貝類的潛在風險。在此，我們發展出了一種基因轉殖方法，能有效地將外源性的去氧核醣核酸 (DNA) 導入目標物種 (Artemia sinica) 的基因體中。不同的構築質體經由電穿孔法，被導入經去殼處理後的豐年蝦休眠卵中，在2千伏特、20微秒的脈衝波長度、20次的脈衝數和在100微升的體積中DNA濃度為每微升20微克的設定條件下。在我們試驗的七個不同構築質體中，被命名為pCMV-MB-EGFPITR的DNA片段，含有被人類巨細胞病毒啟動子和稻田魚β型肌動蛋白啟動子所驅動的增強型綠螢光蛋白報導基因，能產生最強的綠螢光表現。雖然豐年蝦無節幼蟲於24小時後的孵化率 (30±1％) 顯著地低於未經處理的對照組 (61±7％)，但於孵化後72小時的綠螢光表現率為33±6％。轉殖豐年蝦親代、第一子代和第二子代的基因體DNA被萃取出來，一個含有719個鹼基對的產物，以聚合酶連鎖反應被擴增放大出來，這符合了被當作是正控制組的報導基因長度。這些證據都說明了轉殖基因確實有鑲嵌入轉殖豐年蝦的基因體中，而且可以遺傳至子代。這是第一篇報導證明了具有強螢光表現的豐年蝦轉殖品系，可以被創造出來。

The nauplii of crustacean Artemia, also named brine shrimp, are the most widely used live zooplankton bait for feeding larvae of aquaculture finfish and shellfish. By taking advantages of genetically transformed Artemia, some traits of the feeder can be improved indirectly such as fast-growing or disease-resistance. This approach might decrease the potential risk of using genetically modified finfish and shellfish. Here we developed a gene transfer method which enabled to introduce exogenous DNA into the genome of target species－Artemia sinica efficiently. Different DNA constructs were introduced into the decapsulated cysts of Artemia by electroporation under 2KV, 20 μsec pulse length, 20 number of pulses and a DNA concentration of 20μg/μl in a volume of 100μl. Among seven different constructions we examined, a DNA fragment, named pCMV-MB-EGFPITR, containing EGFP reporter gene driven by human cytomegalovirus (CMV) promoter and medaka β-actin promoter, produced the most intense GFP expression. Although the 24 hour-hatching rate of Artemia nauplii (30±1％) was significantly lower than untreated group (61±7％), the GFP expression rate was 33±6％ in 72 hours after hatching. Genomic DNA was extracted from the F0, F1 and F2 generation of transgenic Artemia, a 719-bp product was amplified which was correspondent with the reporter gene as a positive control, by polymerase chain reaction. These evidences suggest that transgene was integrated into the genome of transgenic Artemia and inherited to their offsprings. This is the first report which demonstrates that a transgenic line of Artemia with strong fluorescent expression can be generated.