A bioinformatics framework for targeted gene expression assay design: Application to in vitro developmental...

Brain development involves a series of intricately choreographed neuronal differentiation and maturation steps that are acutely vulnerable to interferences from chemical exposures. Many genes involved in neurodevelopmental processes show evolutionarily conserved expression patterns in mammals and may constitute useful indicators/biomarkers for the evaluation of potential developmental neurotoxicity. Based on these premises, this study developed a bioinformatics framework to guide the design of a gene expression-based in vitro developmental neurotoxicity assay targeting evolutionary conserved genes associated with neuronal differentiation and maturation in rat cerebellar granule cells (CGCs). Rat, mouse and human genes involved in neurodevelopment and presenting one-to-one orthology were selected and orthologous exons within these genes were identified. PCR primer sets were designed within these orthologous exons and their specificity was evaluated in silico. The performance and specificity of rat, mouse and human PCR primer sets were then confirmed experimentally. Finally, RT-qPCR analyses in CGCs exposed in vitro to well-known neurotoxicants (Chlorpyrifos and Chlorpyrifos oxon) uncovered perturbations of expression levels for most of the selected genes. This bioinformatics framework for gene and target sequence selection may facilitate the identification of transcriptional biomarkers for developmental neurotoxicity assays and the comparison of gene expression data across experimental models from different mammalian species.