Adaptation of GAL4 Activators for GAL4 Enhancer Trapping in Zebrafish
Adaptation of GAL4 Activators for GAL4 Enhancer Trapping in Zebrafish
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DOI：
10.1002/dvdy.21863
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发表时间：
2009-03-01
影响因子：
2.5
通讯作者：
Kamachi, Yusuke
中科院分区：
文献类型：
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作者：
Ogura, Eri;Okuda, Yuichi;Kamachi, Yusuke
斑马鱼中基于增强子陷阱的GAL4-UAS系统需要强大的GAL4激活剂，副作用最小。然而，酵母GAL4在斑马鱼中的活性太低，而GAL4 DNA结合域和VP16激活域的融合蛋白对胚胎发育是有毒的，即使在低水平表达也是如此。为了减轻这种毒性，我们开发了不同的GAL4激活剂，通过将VP16最小激活域或NF-kappa B激活域的多聚体形式融合到GAL4 DNA结合域。这些变异的GAL4激活剂在斑马鱼的发育中是足够无害和高效的反式激活剂。利用睡美人转座子系统将含有这些GAL4激活子的增强子陷阱载体随机插入斑马鱼基因组。通过结合这些遗传元件，我们成功地开发出增强子陷阱系，它们以组织特异性的方式激活UAS依赖的报告基因，反映被捕获的增强子活性。《发展动力学》238：641-655,2009。(C)2009年Wiley-Liss，Inc.
An enhancer trap-based GAL4-UAS system in zebrafish requires strong GAL4 activators with minimal adverse effects. However, the activity of yeast GAL4 is too low in zebrafish, while a fusion protein of the GAL4 DNA-binding domain and the VP16 activation domain is toxic to embryonic development, even when expressed at low levels. To alleviate this toxicity, we developed variant GAL4 activators by fusing either multimeric forms of the VP16 minimal activation domain or the NF-kappa B activation domain to the GAL4 DNA-binding domain. These variant GAL4 activators are sufficiently innocuous and yet highly effective transactivators in developing zebrafish. Enhancer-trap vectors containing these GAL4 activators downstream of an appropriate weak promoter were randomly inserted into the zebrafish genome using the Sleeping Beauty transposon system. By the combination of these genetic elements, we have successfully developed enhancer trap lines that activate UAS-dependent reporter genes in a tissue-specific fashion that reflects trapped enhancer activities. Developmental Dynamics 238:641-655, 2009. (C) 2009 Wiley-Liss, Inc.