关键词: 增强子捕获, 注解, SB, 斑马鱼

Abstract: 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 expression 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.

Key words: enhancer-trapping, annotation, SB transposon, zebrafish