Development of enhancer-trapping and -detection vectors mediated by the Tol2 transposon in zebrafish

作者：Shuheng Chan, Dan Shen, Yatong Sang, Wang SaiSai, Yali Wang, Cai Chen, Bo Gao, Chengyi Song · 发表于：PeerJ · 年份：2019 · DOI：10.7717/peerj.6862 · 被引用次数：12 · 研究领域：Genomics and Chromatin Dynamics、RNA Interference and Gene Delivery、CRISPR and Genetic Engineering

Enhancers are key transcriptional drivers of gene expression. The identification of enhancers in the genome is central for understanding gene-expression programs. Although transposon-mediated enhancer trapping (ET) is a powerful approach to the identification of enhancers in zebrafish, its efficiency varies considerably. To improve the ET efficiency, we constructed Tol2 -mediated ET vectors with a reporter gene ( mCherry ) expression box driven by four minimal promoters (Gata, Myc, Krt4 and Oct4), respectively. The ET efficiency and expression background were compared among the four promoters by zebrafish embryo injection at the one-cell stage. The results showed that the Gata minimal promoter yielded the lowest basic expression and the second-highest trapping efficiency (44.6% at 12 hpf (hour post-fertilization) and 23.1% at 72 hpf, n = 305 and n = 307). The Krt4 promoter had the highest trapping efficiency (64% at 12 hpf and 67.1% at 72 hpf, n = 302 and n = 301) and the strongest basic expression. To detect enhancer activity, chicken 5′HS4 double insulators were cloned into the two ET vectors with the Gata or Krt4 minimal promoter, flanking the mCherry expression box. The resulting detection vectors were injected into zebrafish embryos. mCherry expression driven by the Gata promoter (about 5%, n = 301) was decreased significantly compared with that observed for embryos injected with the ET vectors (23% at 72 hpf, n = 308). These results suggest that the insulators block the...