Comparative analyses between traditional model organisms, such as the fruit fly Drosophila melanogaster, and more recent model organisms, such as the red flour beetle Tribolium castaneum, have provided a wealth of insight into the evolution of gene regulatory networks. While the study of trans-regulatory components is relatively straightforward, the study of cis-regulatory elements (CREs, or enhancers) remains challenging outside of Drosophila. A central component of this challenge has been finding a core promoter suitable for enhancer-reporter assays in diverse insect species. Previously, we demonstrated that a Drosophila Synthetic Core Promoter (DSCP) functions in a cross-species manner in Drosophila and Tribolium. Given the over 300 million years of divergence between the Diptera and Coleoptera, we reasoned that the DSCP is a promising candidate for the establishment of enhancer-reporter assays in a variety of insect models across the holometabola and possibly beyond. Our vectors utilize the DSCP with broadly functional piggyBAC and PhiC31 integration and the universal 3xP3 eye marker. These plasmids have now been used to study enhancers in three holometabolous orders: the Diptera, Lepidoptera, and Coleoptera. Additionally, we have developed the piggyLANDR concept, a platform for the establishment of PhiC31 landing sites free from position effects in emerging model insects. The functionality of our new reporter constructs across holometabolous orders suggests a high potential compatibility with a variety of other insects. These tools will help advance the study of gene regulatory networks beyond traditional insect models.
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