A Novel Split-Input Genetic System for Tissue Regeneration Research in Drosophila melanogaster

The model organism Drosophila melanogaster, commonly known as the fruit fly, has been a cornerstone of genetics research for over a century. Due to its extensively characterized genome, highly conserved genes and mechanisms, and relatively short life cycle, Drosophila have become critical to the advancement of research in the field of regenerative medicine. This study introduces a novel split input genetic system to address the drawbacks in two existing methods. By separating the activation of tissue ablation and gene manipulation into two distinct inputs, this third system offers spatial and temporal control over experimental conditions in the imaginal wing disc. The system combines components from binary activation systems such as the GAL4/UAS and LexA/LexAop pathways along with modifications to prevent cross-activation. Initial experiments identified challenges in its functionality. Issues with stop cassette stability, phenotypic markers, and environmental factors prevented a thorough characterization of the system. Despite these concerns, this system successfully induced apoptosis through the activation of pro-apoptotic gene reaper after a 40-hour heat shift at 30℃, as shown through caspase activity. However, phenotypic analysis of adult wings revealed inconsistencies with expected regeneration outcomes. Interestingly, PCR and gel electrophoresis suggested recombination events involving the phenotypic marker sternopleural. These findings highlight the need for further refinement, such as redesigning the system to incorporate a more stable balancer on the second chromosome and exploring alternative activation inputs such as optogenetics or drug-induced gene expression. This novel system provides a strong base for more precise studies of tissue regeneration in Drosophila, which can offer insights into conserved pathways relevant to human health.