- binary expression systems (1) (remove)
- Development of broadly applicable transgenic tools for the transposon mutagenesis of the red flour beetle, Tribolium castaneum (2005)
- The discovery of non-species-specific, broad-range transposable elements and the establishment of a universal 3xP3 promoter revolutionized insect transgenesis. It overcame the limitations of the germ-line transformation to be restricted to the model organism Drosophila melanogaster. In combination with discernable fluorescent markers, multi-component systems, such as transposon-based insertional mutagenesis, can now be introduced to various insect species. To drive the transposase gene for transposable element remobilisation, suitable promoters are needed. The broadly conserved thermotolerance factor, Hsp70, is well-characterised in D. melanogaster and its promoter, which is inducible by high temperatures, provides a genetic tool for transient gene activation. In this thesis, I could prove that the D. melanogaster hsp70 promoter is functional in Tribolium castaneum as well. Its observed basal level activity, however, must be considered and limits its use for experiments, where no strict transient gene expression is required. Nevertheless, the D. melanogaster hsp70 promoter will suffice to provide an efficient transposase source in transposon-based mutagenesis screens in T. castaneum. The remobilization of non-autonomous transposable elements in such screens results in novel mutations and tagging of potentially interesting cis-regulatory elements. To further investigate gene functions, misexpression studies are necessary. In D. melanogaster, this can be done by directed binary expression systems. Here I could show, that the combination of Gal4delta/UAST works best in D. melanogaster somatic tissue, whereas the LexA/(LL)4 and the tetracycline-controlled systems seem to function only poorly. All constructs are based on broad range transposons as well as universal markers and promoters, so that they can be used in other insect species to determine the best system. Preliminary tests in T. castaneum, however, showed that there are a number of additional problems that need to be addressed, before a suitable binary expression system can be established for this species. The full genome sequence of T. castaneum is now available. Therefore, interesting mutations, cis-regulatory elements and their biological functions can be directly linked to the sequence level. When target sites of site-specific recombination systems are included in insertional mutagenesis screens, their insertion sites can be precisely identified and designed chromosomal rearrangements (inversions, duplications and deletions) created. Here I could present a universal system, which can be introduced into non-drosophilid species and enables such chromosomal rearrangements, which I could successfully demonstrate in D. melanogaster. Defined inversions suppressed meiotic recombination between inverted and non-inverted regions on homologous chromosomes and can thus serve as defined balancer chromosomes. Also defined deletions/duplications were generated in D. melanogaster. Such aberrations will be crucial in other insect species, like T. castaneum, to safely keep mutation stocks and identify gene functions. Moreover, the separation of terminal inverted repeats by inverting the chromosomal region between two transposable elements resulted in immobilization. This is of a particular interest for applied transgenesis approaches in insect pest management, when transgenic insects will be released into the nature and transposable elements must be efficiently protected from potential cross mobilization in host species.