SB转座子介导的斑马鱼增强子捕获注解分析

The Annotation of Enhancer-trapping Mediated by the SB Transposon in Zebrafish

为了建立斑马鱼增强子捕获突变体库及研究功能基因的表达调控模式,制备了SB转座子介导的增强子捕获转基因斑马鱼(F0),通过繁育建立了组织或器官特异表达报告基因绿色荧光蛋白(Green fluorescent protein,GFP)的品系(F1)。选择F1代的SK-3系(脑部特异表达GFP)与野生型TU系斑马鱼交配,收集受精卵(F2),于24 hpf(Hour post fertilization)、2 dpf(Day post fertilization)、3 dpf、4 dpf、5 dpf、7 dpf 6个发育阶段检测报告基因绿色荧光蛋白的表达模式;然后通过Splinkerette PCR(SP-PCR)方法检测SB转座子在斑马鱼基因组中的插入位置,从而确定捕获的增强子和功能基因;最后通过胚胎原位杂交验证内源基因表达模式。结果表明,在F2代胚胎不同发育阶段,GFP表达具有明显的时空特性,前期在前脑,中脑,后脑三个部位均高水平表达,后期表达部位呈后移趋势,各个发育期表达强度基本无变化,结果提示该捕获增强子具有脑部表达特性。sp-PCR结果分析表明增强子位于基因组1号染色体35、914、498-35、914、621位置,在内源性基因ednraa位点附近。原位杂交结果表明该基因在胚胎24 hpf阶段具有转录活性。本研究结果提示SB转座子介导的增强子捕获技术可高效获得插入突变斑马鱼,对研究基因功能和获得具有自主知识产权的新基因具有重要作用。

In order to establish zebrafish enhancer-trapping mutant library and study the expression and regulation of functional genes, we prepared SB （ Sleeping Beauty ） transposon-mediated enhancer-trapping zebrafish F0, and built the line F1 with tissue- or organ-specific GFP （ Green Fluorescent Protein ） expression patterns through breeding. We selected SK-3 line of F1 （ head-specific expressing GFP ） to breed with wild type TU zebrafish, and collected fertilized eggs. The GFP expression patterns of early embryos at 24 hpf （ Hour post fertilization ）, 2 dpf （ Day post fertilization ）, 3 dpf, 4 dpf, 5 dpf and 7 dpf were analyzed. SP-PCR （ Splinkerette PCR ） was used to detect the inserted position of SB transposon in the zebrafish genome to determine the captured enhancer and functional gene. The e$：pression patterns of the functional gene were verified by in situ hybridization. The results showed that the expression of GFP had obvious temporal and spatial characteristics in SK-3 embryos at different developmental stages and highly expressed in the forebrain, midbrain and hindbrain at earlier stages, and was in a trend of backward at later stages. There was no variation in the expression level of each developmental stage, indicating that the captured enhancer was specifically expressed in the brain. SP-PCR results showed that the enhancer was located in the chromosome 1 ： 35, 914, 498- 35, 914, and 621 near the ednraa. The results of in situ hybridization showed that the gene had transcription activity at the 24 hpf. Above data suggested that the SB transposon-mediated enhancer-trapping technique can be efficiently used to acquire the inserted mutant zebrafish, which has a great significance on the study of gene function and the acquisition of new genes with independent intellectual property rights.