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http://hdl.handle.net/2445/148117
Title: | CRISPR/Cas9-mediated generation of a tyrosine hydroxylase reporter iPSC line for live imaging and isolation of dopaminergic neurons |
Author: | Calatayud Aristoy, Carles Carola, Giulia Fernandez-Carasa, Irene Valtorta, Marco Jiménez-Delgado, Senda Díaz, Mònica Soriano i Fradera, Jordi Cappelletti, Graziella García-Sancho, Javier Raya Chamorro, Ángel Consiglio, Antonella |
Keywords: | Malaltia de Parkinson Ciències de la salut Enginyeria genètica Parkinson's disease Medical sciences Genetic engineering |
Issue Date: | 2-May-2019 |
Publisher: | Nature Publishing Group |
Abstract: | Patient-specific induced pluripotent stem cells (iPSCs) are a powerful tool to investigate the molecular mechanisms underlying Parkinson's disease (PD), and might provide novel platforms for systematic drug screening. Several strategies have been developed to generate iPSC-derived tyrosine hydroxylase (TH)-positive dopaminergic neurons (DAn), the clinically relevant cell type in PD; however, they often result in mixed neuronal cultures containing only a small proportion of TH-positive DAn. To overcome this limitation, we used CRISPR/Cas9-based editing to generate a human iPSC line expressing a fluorescent protein (mOrange) knocked-in at the last exon of the TH locus. After differentiation of the TH-mOrange reporter iPSC line, we confirmed that mOrange expression faithfully mimicked endogenous TH expression in iPSC-derived DAn. We also employed calcium imaging techniques to determine the intrinsic functional differences between dopaminergic and non-dopaminergic ventral midbrain neurons. Crucially, the brightness of mOrange allowed direct visualization of TH-expressing cells in heterogeneous cultures, and enabled us to isolate live mOrange-positive cells through fluorescence-activated cell sorting, for further differentiation. This technique, coupled to refined imaging and data processing tools, could advance the investigation of PD pathogenesis and might offer a platform to test potential new therapeutics for PD and other neurodegenerative diseases. |
Note: | Reproducció del document publicat a: https://doi.org/10.1038/s41598-019-43080-2 |
It is part of: | Scientific Reports, 2019, vol. 9, p. 6811 |
URI: | http://hdl.handle.net/2445/148117 |
Related resource: | https://doi.org/10.1038/s41598-019-43080-2 |
ISSN: | 2045-2322 |
Appears in Collections: | Articles publicats en revistes (Patologia i Terapèutica Experimental) Articles publicats en revistes (Física de la Matèria Condensada) Articles publicats en revistes (Institut d'lnvestigació Biomèdica de Bellvitge (IDIBELL)) |
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