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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.
Pubmed ID: 31733438 RIS Download
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Non-profit plasmid repository dedicated to helping scientists around the world share high-quality plasmids. Facilitates archiving and distributing DNA-based research reagents and associated data to scientists worldwide. Repository contains over 65,000 plasmids, including special collections on CRISPR, fluorescent proteins, and ready-to-use viral preparations. There is no cost for scientists to deposit plasmids, which saves time and money associated with shipping plasmids themselves. All plasmids are fully sequenced for validation and sequencing data is openly available. We handle the appropriate Material Transfer Agreements (MTA) with institutions, facilitating open exchange and offering intellectual property and liability protection for depositing scientists. Furthermore, we curate free educational resources for the scientific community including a blog, eBooks, video protocols, and detailed molecular biology resources.
View all literature mentionsCommercial organization which provides reagents and services for molecular biology research. Its services include clone collections, microRNA solutions, genome editing, qPCR products, and fluorescent labeling and detection.
View all literature mentionsConsortium to address the increasing demand by researchers for quality-controlled, disease-relevant research grade induced Pluripotent Stem Cell (iPSC) lines, data and cell services by demonstrating an operational banking and distribution service of iPSC lines after 3 years and establishing subsequently for Europe a centralized, not-for-profit bank providing all qualified users with access to scalable, cost-efficient and customized products. The main facility will be at the Babraham Research Campus (Cambridge, UK) and will undertake cell expansion, QC and characterization. The European Cell Culture Collection (ECACC) of Public Health England (Department of Health, UK) will coordinate cell line distribution. The Fraunhofer IBMT (Saarbr��cken, Germany) will provide comprehensive operational back up. In a phased business strategy EBiSC will hot-start distribution of lines contributed by iPSC Centres in 2014, lines collected based on specified user demand, will reach full scale operations in 2016, and with extended funding will become self-sustaining as a not for profit banking operation by 2019. EBiSC will spearhead Europe in the international standardization of iPSC banking by forging collaborative links with similar endeavors in the USA and Asia. It will also provide training to encourage adoption and use of the bank. The project has up to one year after completion to disseminate intellectual property or data created by the project.
View all literature mentionsSoftware for single-cell flow cytometry analysis. Its functions include management, display, manipulation, analysis and publication of the data stream produced by flow and mass cytometers.
View all literature mentionsAn Antibody supplier; Dako was purchased by Agilent in 2012 and several years later the websites began to reflect the Dako products as part of the Agilent catalog.
View all literature mentionsCell line HEK293T is a Transformed cell line with a species of origin Homo sapiens (Human)
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