Functionality of the GAL4/UAS system in Tribolium requires the use of endogenous core promoters.
Functionality of the GAL4/UAS system in Tribolium requires the use of endogenous core promoters.
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DOI：
10.1186/1471-213x-10-53
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发表时间：
2010-05-19
影响因子：
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通讯作者：
Bucher G
中科院分区：
文献类型：
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作者：
Schinko JB;Weber M;Viktorinova I;Kiupakis A;Averof M;Klingler M;Wimmer EA;Bucher G
赤拟谷盗已发展成为仅次于果蝇的昆虫模式系统。此外，作为鞘翅目，它代表了物种最丰富的后生动物分类单元，其中还包括许多害虫物种。在过去的几年里，赤拟谷盗研究的遗传工具箱已经扩大，但到目前为止，时空控制的基因错误表达还不可能。本文报道了赤拟谷盗GAL 4/UAS双元表达系统的建立。由赤拟谷盗hsp 68基因的内源热激诱导型启动子驱动的GAL 4 Δ和GAL 4VP 16两者在上游激活序列（UAS）的控制下有效激活报告基因表达。在激活后4小时内，在胚胎中观察到UAS驱动的普遍存在的tGFP荧光，而针对tGFP的原位杂交显示在2小时后已经表达。与赤拟谷盗胚胎发育的持续时间相关的反应是快速的- 72小时，24小时后完成分割-这使得使用该系统研究早期胚胎过程成为可能。通过分别比较基于赤拟谷盗属、果蝇属和人工核心启动子的构建体的效率，我们发现内源核心启动子的使用对于转基因构建体的高水平表达是必不可少的。随着GAL 4/UAS二元表达系统的建立，异位表达的方法在拟谷盗属中是可行的。我们的研究结果支持的论点，即高水平的转基因表达通常需要内源性调控序列，包括内源性核心启动子在赤拟谷盗和可能也其他模式系统。
The red flour beetle Tribolium castaneum has developed into an insect model system second only to Drosophila. Moreover, as a coleopteran it represents the most species-rich metazoan taxon which also includes many pest species. The genetic toolbox for Tribolium research has expanded in the past years but spatio-temporally controlled misexpression of genes has not been possible so far. Here we report the establishment of the GAL4/UAS binary expression system in Tribolium castaneum. Both GAL4Δ and GAL4VP16 driven by the endogenous heat shock inducible promoter of the Tribolium hsp68 gene are efficient in activating reporter gene expression under the control of the Upstream Activating Sequence (UAS). UAS driven ubiquitous tGFP fluorescence was observed in embryos within four hours after activation while in-situ hybridization against tGFP revealed expression already after two hours. The response is quick in relation to the duration of embryonic development in Tribolium - 72 hours with segmentation being completed after 24 hours - which makes the study of early embryonic processes possible using this system. By comparing the efficiency of constructs based on Tribolium, Drosophila, and artificial core promoters, respectively, we find that the use of endogenous core promoters is essential for high-level expression of transgenic constructs. With the established GAL4/UAS binary expression system, ectopic misexpression approaches are now feasible in Tribolium. Our results support the contention that high-level transgene expression usually requires endogenous regulatory sequences, including endogenous core promoters in Tribolium and probably also other model systems.