| 研究開始時の研究の概要 |
This study will investigate how brain cholesterol levels are regulated by long non-coding RNAs (lncRNAs). Cellular cholesterol levels are controlled by a careful balance of synthesis, uptake and efflux. Notably, 40% of long ncRNAs are expressed in the brain. Therefore, this project will be the first to investigate lncRNA functionality on cholesterol metabolism in neurons.
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| 研究実績の概要 |
Through this collaboration we first established iPS cell lines conducible for CRISPR screening. Cell lines were engineered to express GFP in specific loci responsible for sodium channel. Using this newly established cell line, we generated single cell RNA gene expression profiles from over 5000 neurons and identified 1000 long non-coding RNAs involved in neuron differentiation. We next designed sgRNA libraries targeting these long non-coding RNAs compatible for large-scale CRISPR screening. The experimental design also led to the development of novel assays to evaluate the effectiveness of the screening, which involved optimization of cytometry analysis and cell sorting, immunohistochemistry, cell culture protocols, where we have successfully developed a method to selectively isolate population of neurons that can distinguish partial to full responsiveness to genetic perturbations using fluorescently tagged antibodies. Overall, the development of cell lines, single cell data, CRISPR screening assay have been successful and will result in discovering novel regulators of neuronal differentiation and sodium channel regulation.
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