Establishment of a fluorescence-based RNA interference screening assay and post-transcriptional down-regulation of the kainate receptor subunit GRIK2
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Neurogenesis occurs in the dentate gyrus of the hippocampus throughout adulthood in a broad range of mammalian species. Several behavioural studies have suggested a role for adult neurogenesis in learning and memory. During maturation the newborn granule cells extend their axons, the mossy fibres, towards the CA3 area in hippocampus, their dendrites towards the molecular layer in dentate gyrus, and are incorporated into existing neural circuits. This process is known to be regulated by neuronal activity and thought to make an important contribution to the function of new neurons in learning and memory. Kainite receptors are located presynaptically on the mossy fibre terminals, and have been shown to be important for the mossy fibre excitatory transmission, synapse formation and synaptic plasticity, and may be involved in maturation of new neurons in the adult dentate gyrus. In this project I aim to understand the mechanism of neuronal maturation mediated by the kainate receptor, and more specifically the kainate receptor subunit GRIK2, which is highly expressed in mossy fibres and has been shown to be necessary for the mossy fibre excitatory currents. The methodological approach in this study is to use retrovirus and RNA interference to block gene expression of GRIK2. As a first step to achieve this, I have established a fluorescence-based screening assay in cell culture, which has been used to test the efficacy of short hairpin RNA (shRNA) sequences for gene silencing. The down-regulation effect of four miRNA-GRIK2 shRNA plasmids has been tested by co-transfecting each of the plasmids with a plasmid containing a mCherry-GRIK2 fusion protein gene. The down regulation of the mCherry-GRIK2 fusion protein was investigated by measuring the fluorescence intensity of the fluorescent protein mCherry. By the use of the screening assay we have identified two shRNA sequences that successfully down-regulated the GRIK2 expression by 90%. The sequences will be further used to establish a role of the kainate receptor in the maturation of adult born dentate granule cells.