Towards the Development of Transgenic Reporter Lines for Assessing and Characterizing a LAC-Regulated UAS-GAL4 System
Nizami, Mohammed Husain
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I designed and prepared the components of an inducible gene expression system based on the mouse lac system to address limitations in conventional Drosophila melanogaster inducible gene expression systems that preclude their use from some aging studies. Based on both the D. melanogaster UAS/GAL4 system (adapted from yeast) and the mouse lac system (adapted from Escherichia coli), this system would ostensibly allow for precise control of the location, timing, and amount of a transgene’s expression. Previously, flies capable of expressing the lac repressor (lacI) in a GAL4-dependent manner were reported. Here, I report the remaining components needed to characterize the utility of these tools Two promoters incorporating two or three lac operator sequences into the conventional UAS promoter were also produced. Here, I generated promoter-reporter constructs to test the viability of this system. I prepared true-breeding transgenic flies and mapped the chromosome of transgene insertion in flies carrying the genes for pro-apoptotic reporter grim or the enzyme ß-galactosidase (lacZ) under the control of each chimeric UAS-lacO promoter. I prepared a total of 28 lines (eight 2-operator grim lines, five 3- operator grim lines, twelve 2-operator lacZ lines, and three 3-operator lacZ lines). Preliminary experiments to assess the baseline (unrepressed) expression of these reporters indicated weak expression relative to the conventional GAL4-UAS promoter, however as no appropriate benchmark for characterization of these lines existed at the time, these results were inconclusive. To assess the feasibility of this system in aging studies, preliminary longevity analyses in the presence of the inducers IPTG and lactose were performed. The presence of lactose was not shown to affect survival in a statistically significant manner. Survival was inconsistent when IPTG was present in the food. All the components of this system have been prepared, however further characterization is needed to confirm its utility in aging studies.