Development of enhancer-trapping and -detection vectors mediated by the Tol2 transposon in zebrafish
Development of enhancer-trapping and -detection vectors mediated by the Tol2 transposon in zebrafish
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斑马鱼中 Tol2 转座子介导的增强子捕获和检测载体的开发
DOI：
10.7717/peerj.6862
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发表时间：
2019-04-30
期刊：
影响因子：
2.7
通讯作者：
Song, Chengyi
中科院分区：
文献类型：
--
作者：
Chan, Shuheng;Shen, Dan;Song, Chengyi
增强子是基因表达的关键转录驱动因子。识别基因组中的增强子是理解基因表达程序的核心。虽然转座子介导的增强子捕获(ET)是鉴定斑马鱼增强子的一种有效方法，但其效率差别很大。为了提高ET的效率，我们构建了Tol2介导的ET载体，其报告基因(MCherry)表达盒分别由4个最小启动子(GATA、Myc、Krt4和Oct4)驱动。通过斑马鱼胚胎单细胞期注射，比较了4种启动子的ET效率和表达背景。结果表明，GATA最小启动子的基础表达水平最低，捕获率次之(12hpf时为44.6%，72hpf时为23.1%，n=305和n=307)。Krt4启动子具有最高的捕获率(12hpF时%和72hpf时67.1%，n=302和n=301)和最强的基本表达。为了检测增强子活性，将鸡5‘HS4双绝缘子克隆到带有GATA或Krt4最小启动子的两个ET载体中，位于mCherry表达框的两侧。将得到的检测载体注射到斑马鱼胚胎中。GATA启动子驱动的mCherry表达(约5%，n=301)显著低于注射ET载体的胚胎(72hpf时的23%，n=308)。这些结果表明，绝缘子阻断了基因组位置效应，该载体适合于增强子活性评估。为了评价增强子和最小启动子之间的亲和性，在增强子活性检测载体的GATA或β-珠蛋白最小启动子的上游克隆了4个增强子(CNS1、Z48、Hand2和Hs769)。用斑马鱼胚胎注射法对重组载体进行鉴定。我们发现Z48和CNS1对GATA最小启动子有反应，而Hand2只对β-珠蛋白最小启动子有反应。相反，Hs769对GATA或贝塔珠蛋白最低启动子都没有反应。这些结果表明增强剂和最低限度启动子之间存在配伍性。这项研究代表了一种发现可选的ET和增强子检测载体的系统方法。我们渴望为了解功能基因组学提供一个更好的工具。
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 genome-position effects and that this vector is fit for enhancer-activity evaluation. To assess the compatibility between the enhancers and the minimal promoters, four enhancers (CNS1, Z48, Hand2 and Hs769) were cloned upstream of the Gata or Beta-globin minimal promoter in the enhancer-activity-detection vectors. The resulting recombinant vectors were assayed by zebrafish embryo injection. We found that Z48 and CNS1 responded to the Gata minimal promoter, and that Hand2 only responded to the Beta-globin minimal promoter. In contrast, Hs769 did not respond to either the Gata or Beta-globin minimal promoters. These results suggest the existence of compatibility between enhancers and minimal promoters. This study represents a systematic approach to the discovery of optional ET and enhancer-detection vectors. We are eager to provide a superior tool for understanding functional genomics.