Novel genetic approaches to explore neural stem cell homeostasis

Abstract : Drosophila has been used as a prime model organism for functional studies of genes due to sophisticated genetic manipulations and continuous development of novel technologies and reagents. To engineer precise genome modifications to disrupt or alter gene function in Drosophila, we have developed a Minos transposon based genome manipulation tool, the Minos-Mediated Integration Cassettes (MiMIC). When the MiMIC cassette is inserted in a coding intron (an intron flanked by two coding exons) in the gene trap orientation, it severely disrupts gene function. These intronic insertions also allow the tagging of genes/proteins endogenously with a desired tag via Recombinase Mediated Cassette Exchange (RMCE). Using this method, we have generated a resource of >700 endogenously EGFP tagged genes that allows us to assess expression pattern and perform proteomic analyses. These tagged genes also permit conditional knock down of proteins using the deGradFP, a recently developed method for protein knockdown, in a tissue specific, temporal and reversible manner. I am interested in using these tools and technique to explore the mechanisms underlying maintenance of Drosophila neural stem cells, the neuroblasts, in developing and adult brain. The first step towards this is to identify genes involved in neuroblast self-renewal and quiescence. For this, I have performed an extensive gene expression based screen on developing fly brain and identified 30 lines with enriched expression of GFP tagged genes in neuroblasts. Currently, I am performing a deGradFP based protein knockdown screen in neuroblasts, through which, I have identified novel genes whose knockdown results in overgrowth of the larval brains. In addition, I am establishing a traumatic brain injury model and developing MiMIC and CRISPR based methods for conditional knock out of genes and conditional protein knockdown in quiescent adult neural stem cells.