Are the Requirements for microRNA-Mediated Gene Silencing Uniform Across Different Cell Types and Developmental Time Points?

We aim to experimentally determine which genes are essential in microRNA mediated gene silencing using Drosophila melanogaster. MicroRNAs are small, noncoding RNA molecules that silence genes, but all of the genes necessary for miRNAs to function have not been identified. A forward genetics screen was performed using a reporter system with green fluorescent protein and an eye specific enhancer. The reporter is known to be regulated by miRNAs, so flies with defects in silencing can be identified by increased GFP fluorescence. Subsequent experiments have focused on identifying the mutations generated in the screen, and categorizing the role of the corresponding genes in silencing. However, this screen only identified mutations that cause defects in silencing in adult eye tissue. Our lab is frequently asked whether silencing has the same requirements in different tissues and at a different developmental time point. To address this question, we generated a new approach to view silencing. We utilized a reporter that expresses GFP ubiquitously, and we looked at larvae at an early developmental time point. Larvae that have two copies of the mutation are not viable, so we used a genetic trick called mitotic recombination to generate clones in the larval tissue. Heat shock was utilized to induce the mitotic recombination. One caveat with this new system is that wild type and mutant tissue could not be unambiguously identified. To clarify the cell type, a line of flies was created with red fluorescent protein to mark the wild type tissue. The pattern of fluorescence indicating that silencing was disrupted was visible, but it is likely that RFP fluorescence was bleeding through into the GFP channel on the microscope, confusing the results. To address this, a line of flies is being created that have no GFP. If fluorescence is present in the GFP channel, our hypothesis will be supported and the settings on the microscope will be adjusted to prevent this. Based on the data we collect, we will optimize the microscope settings or use a laser scanning confocal microscope to obtain clearer images without any interference