利用Tol2转座子系统构建响应缺氧的转基因斑马鱼  

Generation of transgenic zebrafish responding to hypoxia using the Tol2 transposon system

作　　者：王苗苗 郑栋栋 崔晓楠 唐艳飞 刘云章[1] 李筠[1] 卢玲[1] WANG Miaomiao;ZHENG Dongdong;CUI Xiaonan;TANG Yanfei;LIU Yunzhang;LI Yun;LU Ling(Key Laboratory of Marine Drugs,Ministry of Education,School of Medicine and Pharmacy,Ocean University of China,Qingdao 266003,China)

机构地区：[1]中国海洋大学海洋药物教育部重点实验室,医药学院,山东青岛266003

出　　处：《中国海洋药物》2024年第4期60-66,共7页Chinese Journal of Marine Drugs

基　　金：国家自然科学基金面上项目(32171141)资助。

摘　　要：目的 构建可以响应缺氧的转基因斑马鱼并用于以缺氧诱导因子HIF-1α为作用靶点的药物筛选。方法 构建p T2(HRE-sv40mp-GFP)和p T2(HREM-sv40mp-GFP)2种转座质粒,在细胞中转染质粒并在常氧和缺氧的条件下观察细胞内绿色荧光蛋白(green fluorescent protein,GFP)表达情况;将2种转座质粒分别显微注射到斑马鱼胚胎中,将胚胎进行缺氧处理并观察胚胎绿色荧光强度以验证转基因斑马鱼的缺氧响应性。经过多代筛选得到稳定遗传的Tg(HRE-sv40mp:GFP)和Tg(HREM-sv40mp:GFP)转基因斑马鱼;用HIF-1α抑制剂LW6和白杨素分别对缺氧的Tg(HRE-sv40mp:GFP)斑马鱼胚胎进行处理,在胚胎发育至48 hpf(Hour post fertilization,受精后小时)时进行拍照并对不同荧光强度的斑马鱼数量进行统计。结果 通过细胞实验证明了2种转座质粒构建成功;缺氧能够诱导转基因斑马鱼Tg(HRE-sv40mp:GFP)体内GFP的表达,但对转基因斑马鱼Tg(HREM-sv40mp:GFP)体内GFP的表达没有影响;LW6和白杨素均能够抑制缺氧条件下Tg(HRE-sv40mp:GFP)斑马鱼体内GFP的表达。结论 成功构建可以响应缺氧并用于HIF-1α靶点药物筛选的转基因斑马鱼模型Tg(HRE-sv40mp:GFP)。Objective To generate transgenic zebrafish that can respond to hypoxia for drug screening with HIF-1αas the target of action.The validity of the zebrafish model was also initially verified.Methods Two transposable plasmids,pT2(HRE-sv40mp-GFP)and pT2(HREM-sv40mp-GFP),were generated,and the plasmids were transfected in cells and the intracellular green fluorescent protein(GFP)expression was observed under normoxic and hypoxic conditions;the two transposable plasmids were microinjected into zebrafish embryos,hypoxia treatment of embryos and observation of green fluorescence intensity of embryos to verify the hypoxia responsiveness of transgenic zebrafish,Tg(HRE-sv40mp:GFP)and Tg(HREM-sv40mp:GFP)transgenic zebrafish were obtained after multi-generation screening;the hypoxic Tg(HRE-sv40mp:GFP)zebrafish embryos were treated with the HIF-1αinhibitor LW6 or Chrysin,respectively,the embryos were photographed and the number of zebrafish with different fluorescence intensities was counted at 48 hpf(Hour post fertilization)of development..Results The successful generation of both transposable plasmids was demonstrated by cellular experiments;hypoxia induced the expression of GFP in Tg(HRE-sv40mp:GFP)but not in Tg(HREM-sv40mp:GFP);both LW6 and Chrysin were able to inhibit GFP expression in Tg(HRE-sv40mp:GFP)zebrafish under hypoxic conditions.Conclusion A transgenic zebrafish model Tg(HRE-sv40mp:GFP)that can respond to hypoxia and be used for HIF-1αtarget drug screening was successfully generated.

关 键 词：缺氧 HIF-1Α 斑马鱼 药物筛选 

分 类 号：R965[医药卫生—药理学]