Application of CRISPR/Cas9 editing and digital droplet PCR in human iPSCs to generate novel knock-in reporter lines to visualize dopaminergic neurons
Summary
Human induced pluripotent stem cells (hiPSCs) have become indispensable for disease modelling. They are an important resource to access patient cells harbouring disease-causing mutations. Derivation of midbrain dopaminergic (DAergic) neurons from hiPSCs of PD patients represents the only option to model physiological processes in a cell type that is not otherwise accessible from human patients. However, differentiation does not produce a homogenous population of DA neurons and contaminant cell types may interfere with the readout of the in vitro system. Here, we use CRISPR/Cas9 to generate novel knock-in reporter lines for DA neurons, engineered with an endogenous fluorescent tyrosine hydroxylase - enhanced green fluorescent protein (TH-eGFP) reporter. We present a reproducible knock-in strategy combined with a highly specific homologous directed repair (HDR) screening approach using digital droplet PCR (ddPCR). The knock-in cell lines that we created show a functioning fluorescent reporter system for DA neurons that are identifiable by flow cytometry. Copyright © 2019 The Authors. Published by Elsevier B.V. All rights reserved.
| Authors | Überbacher C, Obergasteiger J, Volta M, Venezia S, Müller S, Pesce I, Pizzi S, Lamonaca G, Picard A, Cattelan G, Malpeli G, Zoli M, Beccano-Kelly D, Flynn R, Wade-Martins R, Pramstaller PP, Hicks AA, Cowley SA, Corti C |
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| Journal | Stem cell research |
| Publication Date | 2019 Dec;41:101656 |
| PubMed | 31733438 |
| PubMed Central | PMC7322529 |
| DOI | 10.1016/j.scr.2019.101656 |