Application of CRISPR/Cas9 editing and digital droplet PCR in human iPSCs to generate novel knock-in reporter lines to visualize dopaminergic neurons.
CRISPR/Cas9
Dopaminergic neurons
Human induced pluripotent stem cells
Knock-in, Digital droplet PCR, Fluorescent reporter, FACS
Journal
Stem cell research
ISSN: 1876-7753
Titre abrégé: Stem Cell Res
Pays: England
ID NLM: 101316957
Informations de publication
Date de publication:
12 2019
12 2019
Historique:
received:
19
12
2018
revised:
30
10
2019
accepted:
08
11
2019
pubmed:
17
11
2019
medline:
3
6
2020
entrez:
17
11
2019
Statut:
ppublish
Résumé
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.
Identifiants
pubmed: 31733438
pii: S1873-5061(19)30286-7
doi: 10.1016/j.scr.2019.101656
pmc: PMC7322529
pii:
doi:
Substances chimiques
enhanced green fluorescent protein
0
Green Fluorescent Proteins
147336-22-9
Types de publication
Journal Article
Research Support, Non-U.S. Gov't
Langues
eng
Sous-ensembles de citation
IM
Pagination
101656Subventions
Organisme : Medical Research Council
ID : MR/L023784/1
Pays : United Kingdom
Organisme : Medical Research Council
ID : MC_EX_MR/N50192X/1
Pays : United Kingdom
Organisme : Medical Research Council
ID : MR/L023784/2
Pays : United Kingdom
Organisme : Medical Research Council
ID : MR/M024962/1
Pays : United Kingdom
Organisme : Wellcome Trust
ID : WTISSF121302
Pays : United Kingdom
Organisme : Parkinson's UK
ID : J-0901
Pays : United Kingdom
Informations de copyright
Copyright © 2019 The Authors. Published by Elsevier B.V. All rights reserved.
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