起動子捕捉法在吳郭魚卵巢細胞之研究

Abstract

?動子補捉法是利用導入缺乏?動子的報導基因，藉由鑲嵌到細胞活化的?動子或表現中之基因的下游位置，去一併表現報導基因。藉由篩選基因 Neomycin phosphotransferase ( Neo）的活化，細胞可在 G418 篩選下存活下來。關於?動子補捉法之研究集中在小鼠胚胎幹細胞，利用這種技術可以補捉到參與胚胎發育中之基因，對於小鼠基因功能分析幫助頗大。目前未曾有關魚類細胞?動子捕捉法之研究報告發表。本實驗之目的在探討魚類細胞之?動子補捉法之研究，以吳郭魚卵巢細胞（Tilapia ovary cell）為實驗材料，設計?動子補捉載體對吳郭魚卵巢細胞，進行補捉?動子的工作，以期完成?動子的補捉，提高補捉的成功率，並藉以探討此種技術應用到魚類胚胎幹細胞之可能性。 ?動子捕捉載體利用 EMC 病毒的5’端非轉譯區、 Neo 基因與水母綠色螢光蛋白（Green fluorescent protein）及β-galactosidase 構集而成。為了使補捉載體順利的鑲嵌到細胞染色體上，兩次氯化銫純化之載體，以Xho I限制?切成線性，再利用 lipofection 或電穿孔法送入吳郭魚的卵巢細胞，做?動子補捉的工作。穩定選殖之細胞以 G418 篩選並選殖出來，再以聚合?鏈反應（polymerase chain reaction ）或南方氏點墨法（Southern hybridization）做分析。

The promoter trapping method is to introduce a promoter-less reporter gene to integrate onto the down stream of a activated promoter or an expression gene as well as to express the reporter gene. Through the activation of the selecting gene, Neomycin phosphotransferase (Neo), cells with the integrated gene survived under the selection of G418. Most studies of the promoter trapping methods were focused on the mouse embryonic stem cells. By applying this technique, the genes involved in the embryonic development were trapped, which is pretty helpful for the function analysis of mouse genes. Up to now, no study of promoter trapping was reported on fish cells. The objective of our research is to study the promoter trapping method applied on fish cells. We used the tilapia ovary cell (TO-2) as material and designed. a promoter trapping vector to work on the TO-2 cells. The aims of our study are to trap promoters, to increase the success of trapping, and ultimately, to find out the possibility to apply this technique on fish embryonic stem cells. The promoter trapping vector was constructed by using the 5’ untranslated region of encephalomyocarditis virus, Neo gene, the gene of green fluorescent protein of gelly fish (Aequorea victoria) , and β-galactosidase. In order to integrate the promoter trapping vector into the cellular chromosome, we purified the vector by twice cesium chloride gradient, linearized the construct by Xho I restriction enzyme digestion, and introduced the vector into the TO-2 cells by lipofection or electroporation. The stable cloned cell colonies were selected by G418 and further analysis were done by polymerase chain reaction or Southern hybridization.