Functional characterization of uncharacterized genes often relies on strategies which ultimately aim to either increase (overexpression) or reduce (knockdown, knockout) the expression of these genes followed by phenotypic analysis. We investigate a newly discovered gene that is transcriptionally activated by p53 in response to radiation-induced DNA damage in mouse embryos and thus may be partly responsible for the observed long-term neurodevelopmental defects. P53-mediated activation of the gene depends on a p53 response element located in an alternative promoter and results in expression of several short transcript variants. Furthermore, this gene is highly upregulated during normal brain development as well as in maturing primary neurons, arguing for a possible role in these important processes.
In this study, we aim to further characterize the function of this gene in neuronal differentiation and maturation via CRISPR/Cas9-mediated gene knockout in mouse neural progenitor cells (NPCs). To highlight the role of this gene in the DNA damage response, we will aim to generate cells in which the p53 response element is mutated, resulting in a cell line in which the gene can no longer be activated by p53. The research project will involve different techniques such as cell culture, CRISPR/Cas9-mediated genome editing, functional assays (e.g. luciferase reporters, live cell imaging), and qRT-PCR.