中文摘要：

采用两种类型的lox突变体和双色荧光报告基因,构建了双交换载体pCMVlox66-CreintEGFPlox2272和plox71RFPlox2272。将Cre酶基因插入载体pCS2＋,获得了体外转录模板质粒pCSCre。为检测Cre重组酶对斑马鱼胚胎发育的影响,将体外转录的Cre RNA稀释至不同浓度并分别注射入斑马鱼受精卵。结果显示,当Cre RNA浓度高于200ng/μL时会导致高死亡率和高畸形率;而浓度低于100ng/μL的Cre RNA对胚胎发育不造成影响。进一步将50ng/μL的Cre RNA同双交换载体共注射到斑马鱼受精卵中,筛选到发红色荧光的胚胎。PCR检测和测序鉴定证实,在Cre重组酶作用下,两个载体中lox位点间的荧光基因发生了置换反应。实验结果表明,Cre/lox系统介导的盒式交换反应是实现转基因鱼外源基因定点整合的有效策略。

英文摘要：

In this study, pCMVlox66 Greint EGFPlox2272 and Plox2272 RFPlox2272, two vectors containing fluorescence genes were constructed by using two different types of mutant lox sites. Another construct was obtained by inserting the Cre gene into the plasmid pCS2 ＋ for synthesizing Cre RNA in vitro. To test the impact of Cre recombinase on the development of zebrafish, Cre RNA was diluted into its solutions with different concentrations and then they were injected into zebrafish embryos respectively. It was shown that the concentrations of Cre RNA above 200ng/μL resulted in death and malforma- tion while the concentration below 100ng/μL had no adverse effect. Then the synthesized Cre RNA in vitro with two gene targeting vectors was co-injected into the zebrafish embryos and the appearance of red fluorescence was found. Genomic PCR analysis and DNA sequencing comfirmed that the gene replacement occurred at the precise site as predicted. The experiments show that Cre recombinase-mediated cassete exchange is a promising strategy to produce transgenic fish with site-directed integrated transgene.