Application of CRISPR/Cas9 editing and digital droplet PCR in human iPSCs to generate novel knock-in reporter lines to visualize dopaminergic neurons

Uberbacher, Christa; Obergasteiger, Julia; Volta, Mattia; Venezia, Serena; Müller, Stefan; Pesce, Isabella; Pizzi, Sara; Lamonaca, Giulia; Picard, Anne; Cattelan, Giada; Malpeli, Giorgio; Zoli, Michele; Beccano-Kelly, Dayne; Flynn, Rowan; Wade-Martins, Richard; Pramstaller, Peter P.; Hicks, Andrew A.; Cowley, Sally A. und Corti, Corrado (2019): Application of CRISPR/Cas9 editing and digital droplet PCR in human iPSCs to generate novel knock-in reporter lines to visualize dopaminergic neurons. In: Stem Cell Research, Bd. 41, 101656

Abstract

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.

• Dokumententyp: Zeitschriftenartikel
• Fakultät: Medizin
• Themengebiete: 600 Technik, Medizin, angewandte Wissenschaften > 610 Medizin und Gesundheit
• ISSN: 1873-5061
• Sprache: Englisch