巨核酸酶I-SceI转基因元件的构建及其在斑马鱼中转基因效率的评估

Construction of I-SceI transgenic elements and assessment of its transgenic efficiency in zebrafish

摘要:

巨核酸酶I-SceI是由酿酒酵母线粒体中核糖体大亚基上的Ⅰ型内含子编码的一种核酸内切酶,近年已被应用于转基因研究中。I-SceI介导的转基因大多是将I-SceI蛋白和带有其识别位点的质粒共注射于动物的受精卵,但注射酶蛋白前期处理耗时长且工作量大,直接用I-SceI的mRNA代替蛋白进行注射更为简捷。本研究应用I-SceI mRNA介导荧光蛋白基因在斑马鱼中的转植,进一步了解其转基因效率。首先设计合成带有巨核酸酶I-SceI编码序列的辅助质粒(pCS2-I-SceI),并分别构建了带有巨核酸酶I-SceI识别位点、EF1α启动子和荧光蛋白基因(eGFP/RFP)编码序列的供体质粒pI-SceI-EF1α-eGFP/RFP。辅助质粒pCS2-I-SceI体外转录成mRNA, 以50 ng/μL供体质粒和100 ng/μL的I-SceI巨核酸酶mRNA共注射到斑马鱼(Danio rerio)受精卵中,注射后48 h的斑马鱼eGFP的平均荧光表达率可达75.86%。在斑马鱼的头部、躯干到尾部均可观察到荧光蛋白的表达,且随着胚胎发育,荧光信号逐渐增强。结果表明I-SceI系统在斑马鱼中可介导较高效率的转植,具有在经济鱼类转基因研究中应用的潜力。

Abstract:

Meganuclease I-SceI is an endonuclease, which is derived from the intron of mitochondrial 21S rRNA gene of Saccharomyces cerevisiae and has been applied to transgenic study in recent years. Usually, to use this system, target plasmid and I-SceI protein were microinjected together. However, preparation of the I-SceI protein for microinjection is laborious and time consuming. Replacing the protein by I-SceI mRNA would be more convenient. In this study, the transgene of fluorescence protein was mediated by I-SceI mRNA to assess its transgenic efficiency in zebrafish. First, helper plasmid with I-SceI coding sequence pCS2-I-SceI was constructed and I-SceI mRNA was transcripted in vitro. Donor plasmid, pI-SceI-EF1α-eGFP and pI-SceI-EF1α-RFP donor plasmid with I-SceI recognition sites, EF1α promoter and eGFP(RFP) gene were constructed, and co-injected with I-SceI mRNA into the 1-2 cell stage fertilized eggs of Danio rerio. The concentrations of donor plasmid and I-SceI mRNA were 50 ng/μL and 100 ng/μL respectively. The eGFP fluorescence expression rate was 75.86% in 48 h. The fluorescence can be observed in the head, body and tail of zebrafish and became stronger and stronger with the development of embryos. Our data suggest that I-SceI can efficiently mediate gene transfer and has the potential to apply in the transgene of economic fish.