Silent Mutations at piRNA Binding Sites in Green Fluorescent Protein to Resist Silencing of Exogenous Reporters in Caenorhabditis elegans
Jackson, Sadie S.
MetadataShow full item record
Caenorhabditis elegans is a common model organism used to study germline stem cells (GSCs), a specific type of adult stem cell of the gonad. Studying GSCs has revealed a lot about how adult stem cells are regulated in vivo. Understanding regulatory mechanisms of adult stem cells is important, since dysregulation of these cells are a common cause of most cancers. The green fluorescent protein (GFP) has been used as a tool to track gene expression in Caenorhabditis elegans for over two decades. GFP reporters are a much cheaper and easier method than antibody staining. However, GFP has not been an effective reporter for studying gene expression in the nematode’s GSCs, since transgenes are often permanently silenced by the PIWI/piRNA silencing pathway. The PIWI/piRNA complex targets specific piRNA seeds in foreign DNA and is crucial for germline maintenance in many species of animals. A previous study done by Zhang et al. (2018) showed that germline silencing is prevented by introducing silent mutations at these piRNA seeds in GFP (GFPdpi) and another fluorescent protein, mCherry (mCherrydpi). In this study, we test whether functional reporters for RNA-regulating genes in the C. elegans germline can be constructed by using CRISPR/Cas9 to repair simple GFP reporters with GFPdpi. We also developed CRISPR/Cas9 reagents for mCherry repairs.