Application of CRISPR/Cas9 editing and digital droplet PCR in human iPSCs to generate novel knock-in reporter lines to visualize dopaminergic neurons
| Autor: | 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, P, Hicks, A, Cowley, S, Corti, C |
|---|---|
| Jazyk: | angličtina |
| Rok vydání: | 2019 |
| Předmět: |
0301 basic medicine
Cell type Population Green Fluorescent Proteins Induced Pluripotent Stem Cells Biology Polymerase Chain Reaction Article Flow cytometry Cell Line 03 medical and health sciences 0302 clinical medicine Gene knockin medicine CRISPR Humans Gene Knock-In Techniques Transgenes Induced pluripotent stem cell education CRISPR/Cas9 lcsh:QH301-705.5 Gene Editing education.field_of_study Tyrosine hydroxylase medicine.diagnostic_test Cas9 Dopaminergic Neurons Human induced pluripotent stem cells Cell Biology General Medicine Knock-in Digital droplet PCR Fluorescent reporter FACS 3. Good health Cell biology 030104 developmental biology Microscopy Fluorescence lcsh:Biology (General) Dopaminergic neurons CRISPR-Cas Systems 030217 neurology & neurosurgery Developmental Biology |
| Zdroj: | Stem Cell Research, Vol 41, Iss, Pp-(2019) Stem Cell Research |
| DOI: | 10.1016/j.scr.2019.101656 |
| Popis: | Highlights • ddPCR is a highly sensitive and accurate screening tool for CRISPR/Cas9 engineering. • Dopaminergic differentiation results in faithful co-expression of TH and reporter. • Endogenous fluorescence detectable in dopaminergic neurons by flow cytometry. 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. |
| Databáze: | OpenAIRE |
| Externí odkaz: |
|