HSP-FLP重组质粒的构建及表达分析

The Construction of HSP-FLP Recombinant Plasmid and the Analysis of its Expression

为了制备用于在斑马鱼中热休克全身表达目的基因的转基因载体,通过分子克隆的方法从P{hsFLP}86E/TM6B转基因果蝇的基因组中克隆出FLP.并同时对HSP-Wnt5A-EGFP载体进行改造,Wnt5A由FLP代替,并在FLP和EGFP编码区之间插入带有多克隆位点的IRES序列,获得pTol2-HSP-FLP-IRES-EGFP转基因表达载体.利用得到的转基因载体显微注射到斑马鱼单细胞期胚胎中进行表达分析,结果表明,经热激外源目的基因FLP和报告基因EGFP均能在斑马鱼中表达.pTol2-HSP-FLP-IRES-EGFP转基因表达载体的成功构建对于建立FLP/FRT重组系统的斑马鱼转基因实验模型具有重要意义.

In an effort to generate a desired expression construct for making widely expression transgenic zebrafish . FLP was cloned from the P {hsFLP} 86E/TM6B transgenic drosophila genome through using subcloning technology . Meanwhile , the HSP-Wnt5A-EGFP plasmid was modified using subcloning technology . The Wnt5A was replaced by FLP . An IRES fragment was inserted between the FLP and EGFP , we got the pTol2 - HSP - FLP - IRES - EGFP recombinant plasmid . To test the effectiveness of this new expression plasmid , we injected this transgenic recombinant plasmid into one -cell stage embryos of zebrafish . Under fluorescence microscope , the green fluorescence produced by pTol2-HSP-FLP-IRES-EGFP was detected in zebrafish . This novel expression construct will become an important tool for our research on using FLP/FRT site - specific recombination system applying zebrafish as a model .