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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.

Original publication

DOI

10.1016/j.scr.2019.101656

Type

Journal article

Journal

Stem cell research

Publication Date

12/2019

Volume

41

Addresses

Institute for Biomedicine, Eurac Research, Affiliated Institute of the University of Lübeck, Bolzano, Italy; Department of Biomedical, Metabolic and Neural Sciences, Università di Modena e Reggio Emilia, Modena, Italy. Electronic address: christa.ueberbacher@i-med.ac.at.

Keywords

Cell Line, Humans, Green Fluorescent Proteins, Microscopy, Fluorescence, Polymerase Chain Reaction, Transgenes, Gene Knock-In Techniques, Induced Pluripotent Stem Cells, Dopaminergic Neurons, CRISPR-Cas Systems, Gene Editing