斑马鱼pax2a(-3.1 kb):eGFP转基因品系的构建

为了更好地了解脊椎动物肾脏的早期发育,本研究利用斑马鱼这一研究脊椎动物器官发育的理想模式生物,通过构建肾脏特异性pax2a荧光标记转基因品系以实现实时在体地观察斑马鱼前肾的发育过程。我们分析了斑马鱼pax2a基因的启动子,扩增出其翻译起始位点上游3.1 kb的基因组DNA,利用Tol2转座子系统,经胚胎显微注射及三代筛选,成功构建了一个稳定的pax2a(-3.1 kb):eGFP转基因鱼品系。此转基因品系的绿色荧光蛋白在3体节时期就可以标记出中间中胚层,并在后续体节时期的中间中胚层前端(第3体节至第5体节对应的部分),及24 hpf的肾管前端和中段表达,基本模拟了pax2a在斑马鱼早期前肾发育中的时空表达模式。本研究所获得的pax2a(-3.1 kb):eGFP弥补了已有pax2a转基因品系中报告基因无法准确标记出中间中胚层和前肾管前端的缺陷,是研究斑马鱼前肾早期发育的良好材料。

Construction of the pax2a(-3.1 kb):eGFP Transgenic Zebrafish

In order to understand the early kidney development in vertebrates, zebrafish, an ideal animal model to study organogenesis, was used to generate a kidney specific reporter transgene pax2a:eGFP for the real time observation of the developmental process of pronephros in living embryos. In this study, we analyzed the promoter of pax2a, amplified a 3.1 kb genomic DNA fragment upstream of the translation start point, and cloned it into the Tol2 transgenic vector. After micro-injection and screens in three generations, a stable pax2a(-3.1 kb):eGFP transgenic line had been obtained. As early as 3 somite stage, eGFP was found expressed in the intermediate mesoderm in this transgenic line, and it continued to express in the anterior intermediate mesoderm parallel to the 3rd to 5th somite during the following somitogenesis, and the anterior and middle parts of pronephric duct at 24 hpf, which almost recapitulates the temporal-spatial expression pattern of endogenous pax2 a in early pronephros. The pax2a(-3.1 kb):eGFP transgenic line obtained in this study overcome the drawbacks of missing reporter gene expression in the intermediate mesoderm and the anterior part of pronephric tube in established pax2a transgenic lines. Thus,it is a useful tool for the study of pronephros development in zebrafish.