Transgenerational analysis of transcriptional silencing in zebrafish
Transgenerational analysis of transcriptional silencing in zebrafish
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DOI：
10.1016/j.ydbio.2011.01.002
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发表时间：
2011-04-15
影响因子：
2.7
通讯作者：
Goll, Mary G.
中科院分区：
文献类型：
--
作者：
Akitake, Courtney M.;Macurak, Michelle;Goll, Mary G.
酵母Gal4/UAS转录激活系统是调控果蝇基因表达的有力工具，在斑马鱼发育研究中的应用日益广泛。这对研究De nova转录沉默的基础也是有用的。在转基因斑马鱼胚胎中，由上游激活序列(UAS)的多个串联拷贝控制的荧光报告基因经常显示出不同的表达和DNA甲基化。为了系统地描述这一点，我们监测了UAS调控的转基因的转录沉默的进展，这些转基因的整合位点和UAS的重复性质不同。对三代转基因幼虫进行了绿色荧光蛋白(GFP)报告的组织特异性表达和UAS的DNA甲基化检测。包含四个不同上游激活子序列的单个插入片段比包含14个相同UA拷贝的插入片段对甲基化的敏感性要低得多。此外，整合在转座子序列中或邻近转座子序列的转基因表现出沉默，而与转基因中包含的UAS位点的数量无关。将启动子驱动的Gal4上游的UAS调节的应答基因放置在单个双顺反子构建体中似乎也可以加速沉默和甲基化。这些结果证明了斑马鱼在有效跟踪几代人的基因沉默机制方面的有效性，并为DNA甲基化生物中Gal4调节的基因最佳表达提供了有用的指南。(C)2011 Elsevier Inc.保留所有权利。
The yeast Gal4/UAS transcriptional activation system is a powerful tool for regulating gene expression in Drosophila and has been increasing in popularity for developmental studies in zebrafish. It is also useful for studying the basis of de nova transcriptional silencing. Fluorescent reporter genes under the control of multiple tandem copies of the upstream activator sequence (UAS) often show evidence of variegated expression and DNA methylation in transgenic zebrafish embryos. To characterize this systematically, we monitored the progression of transcriptional silencing of UAS-regulated transgenes that differ in their integration sites and in the repetitive nature of the UAS. Transgenic larvae were examined in three generations for tissue-specific expression of a green fluorescent protein (GFP) reporter and DNA methylation at the UAS. Single insertions containing four distinct upstream activator sequences were far less susceptible to methylation than insertions containing fourteen copies of the same UAS. In addition, transgenes that integrated in or adjacent to transposon sequence exhibited silencing regardless of the number of UAS sites included in the transgene. Placement of promoter-driven Gal4 upstream of UAS-regulated responder genes in a single bicistronic construct also appeared to accelerate silencing and methylation. The results demonstrate the utility of the zebrafish for efficient tracking of gene silencing mechanisms across several generations, as well as provide useful guidelines for optimal Gal4-regulated gene expression in organisms subject to DNA methylation. (C) 2011 Elsevier Inc. All rights reserved.