Schinko Johannes B, Weber Markus, Viktorinova Ivana, Kiupakis Alexandros, Averof Michalis, Klingler Martin, Wimmer Ernst A, Bucher Gregor
Ernst Caspari Haus, Georg-August-University Göttingen, Justus-von-Liebig-Weg11, 37077 Göttingen, Germany.
BMC Dev Biol. 2010 May 19;10:53. doi: 10.1186/1471-213X-10-53.
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 GAL4 Delta 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.
赤拟谷盗已发展成为仅次于果蝇的昆虫模型系统。此外，作为一种鞘翅目昆虫，它代表了物种最为丰富的后生动物类群，其中也包括许多害虫物种。在过去几年中，用于赤拟谷盗研究的遗传工具箱有所扩展，但迄今为止，还无法实现基因的时空可控性错误表达。
在此，我们报告了在赤拟谷盗中建立GAL4/UAS二元表达系统。由赤拟谷盗hsp68基因的内源性热休克诱导型启动子驱动的GAL4 Delta和GAL4VP16，在上下游激活序列（UAS）的控制下，均能有效地激活报告基因的表达。激活后4小时内，在胚胎中观察到UAS驱动的普遍tGFP荧光，而针对tGFP的原位杂交显示，2小时后就已出现表达。相对于赤拟谷盗胚胎发育的持续时间（72小时，24小时后完成分节）而言，这种反应很快，这使得利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、果蝇和人工核心启动子构建体的效率，我们发现使用内源性核心启动子对于转基因构建体的高水平表达至关重要。
随着GAL4/UAS二元表达系统的建立，异位错误表达方法现在在赤拟谷盗中可行。我们的结果支持这样一种观点，即高水平转基因表达通常需要内源性调控序列，包括赤拟谷盗以及可能其他模型系统中的内源性核心启动子。
