A versatile bimodal QF/QS gene expression system for insect and vertebrate models based on conditional protein self-splicing

About This Grant

SUMMARY. Precise tissue- and temporally- specific manipulation of gene expression has driven mechanistic studies across many different model organisms. Bimodal expression systems such as the Q-system from N. crassa and the GAL4-system from S. cerevisiae are key genetic tools for elucidating gene function and cellular properties. These systems were introduced and optimized in Drosophila melanogaster, contributing to groundbreaking discoveries in embryonic development, animal growth, physiology, metabolism, and neuroscience, and have had a significant impact in the understanding of human biology, in both health and disease. Using CRISPR/Cas-9 technology, the Q-system (the QF2 driver and QUAS reporters) has been successfully introduced to other insects with major impact on human health, such as Aedes and Anopheles mosquitoes. Additionally, it has been applied to vertebrate model systems including the zebrafish Danio rerio. In contrast, while the GAL4 system, has been used in zebrafish, its transfer to mosquitoes has been unsuccessful due to cell toxicity. However, even the Q-system poses a number of limitations, including toxicity and the lack of an effective suppressor that functions independently of chemical compounds. In his application, the Q-system will be re-engineered to remedy these drawbacks by incorporating temperature-sensitive self-splicing intein modules (INTts), tested for efficacy in D. melanogaster and transferred to the zebrafish Danio rerio. Inteins are self-splicing endopeptidases embedded within proteins found in bacteria and unicellular eucaryotes (e.g., yeast), with the enigmatic property of excising themselves from pre-proteins, leading to the generation of a functional protein. Remarkably, inteins can be inserted into foreign proteins, where they self-splice as in their normal host protein. The experimental strategy leverages temperature-sensitive self-splicing inteins that have been validated in S. cerevisiae to self-splice at temperatures up to 270 C in foreign proteins. Specifically, inteins with permissive and restrictive setpoints between 15 and 300 C, compatible with growth and development of insects and fish, will be integrated into the DNA binding domain of QF2 (QF2_ INTts) and the protein interaction domain of QS (QS_ INTts), to disrupt their function when retained at restrictive temperature (24 to 300 C). When kept at permissive set points (17 to 230 C), intein removal via self-splicing activates QF and QS. Transgenic Drosophila will be tested for functionality of QF2_ INTts and QS_ INTts using QUAS-GFP reporter genes at different temperature. Genes encoding validated QF2_ INTts will be conferred to plasmids for D. rerio transgenic fish, tested for functionality using QUAS-GFP reporters. These new tools provide a much-needed temporal control element for the Q-system, making it invaluable for the research community to analyze gene and cell function relevant to human health.