The Annotation of Zebrafish Enhancer Trap Lines Generated with Transposon.

An enhancer trap (ET) mediated by a transposon is an effective method for functional gene research. Here, an ET system based on a transposon that carries a mini Krt4 promoter (the minimal promoter from zebrafish) and the gene () has been used to produce zebrafish ET lines. One enhancer trap line with eye-specific expression GFP named EYE was used to identify the trapped enhancers and genes. Firstly, GFP showed a temporal and spatial expression pattern with whole-embryo expression at 6, 12, and 24 hpf stages and eye-specific expression from 2 to 7 dpf. Then, the genome insertion sites were detected by splinkerette PCR (spPCR). The Krt4- was inserted into the fourth intron of the gene (, ) in chromosome 9 of the zebrafish genome, with the direction the same as that of the gene. By the alignment of homologous gene sequences in different species, three predicted endogenous enhancers were obtained. The trapped endogenous gene whose overexpression is related to hepatocellular carcinoma, showed a similar expression pattern as detected by in situ hybridization, which suggested that and were possibly regulated by the same enhancers. In short, the zebrafish enhancer trap lines generated by the transposon-mediated enhancer trap technology in this study were valuable resources as visual markers to study the regulators and genes. This work provides an efficient method to identify and isolate tissue-specific enhancer sequences.