| 研究実績の概要 |
For the major goal to utilize DNA adenine methyltransferase identification sequencing (DamID-seq) on a small number of specific neurons, I have optimized a protocol working for in vivo sample of as few as 4 fly brains (expressing DAM in a pan-neuronal pattern, equivalent to ~4000 neurons). Further modification might be needed depending on the sample’s properties for the future experiments. Although the original plan is to purify the cell population by FACS, to further minimize the loss of cell during the sample processing, magnetic beads based sorting is also under test currently. A protocol for linearly amplify the RNA from purified neurons will be incorporated as well, to allow the samples be used for RNA-seq.
For visualizing neuronal activity upon activations, I have set up in vivo imaging for GCamP. More trials will be needed to find the best condition to catch the fast signal upon activation. I am currently continuing with setting up CamPARI and possibly using other neuronal activity marker to label the activated neurons. I have acquired a GFP tagged Hr38 (a gene response to several types of neuronal activations) strain to test the possibility of utilization of it to label activated neurons.
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| 今後の研究の推進方策 |
1. I plan to test the current DamID protocol with purified neurons and generate high quality samples for sequencing. As an alternative for DamID, I am currently testing in vivo ChiP-seq protocols to gain better resolution for transcription factors' binding sites, which are important for da neuron development.
2. I will set up CamPARI and other neuronal activity-based marker by visualizing c4da neuronal activity changes upon stimulations. 3.I will use those systems to test how chromatin binding proteins related to Autism affect sensory neuron activities.
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