Reliable Identification of Living Dopaminergic Neurons in Midbrain Cultures Using RNA Sequencing and TH-promoter-driven eGFP Expression.
J Vis Exp
; (120)2017 02 10.
Article
em En
| MEDLINE
| ID: mdl-28287593
In Parkinson's Disease (PD) there is widespread neuronal loss throughout the brain with pronounced degeneration of dopaminergic neurons in the SNc, leading to bradykinesia, rigidity, and tremor. The identification of living dopaminergic neurons in primary Ventral Mesencephalic (VM) cultures using a fluorescent marker provides an alternative way to study the selective vulnerability of these neurons without relying on the immunostaining of fixed cells. Here, we isolate, dissociate, and culture mouse VM neurons for 3 weeks. We then identify dopaminergic neurons in the cultures using eGFP fluorescence (driven by a Tyrosine Hydroxylase (TH) promoter). Individual neurons are harvested into microcentrifuge tubes using glass micropipettes. Next, we lyse the harvested cells, and conduct cDNA synthesis and transposon-mediated "tagmentation" to produce single cell RNA-Seq libraries1,2,3,4,5. After passing a quality-control check, single-cell libraries are sequenced and subsequent analysis is carried out to measure gene expression. We report transcriptome results for individual dopaminergic and GABAergic neurons isolated from midbrain cultures. We report that 100% of the live TH-eGFP cells that were harvested and sequenced were dopaminergic neurons. These techniques will have widespread applications in neuroscience and molecular biology.
Texto completo:
1
Base de dados:
MEDLINE
Assunto principal:
Tirosina 3-Mono-Oxigenase
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Mesencéfalo
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Análise de Sequência de RNA
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Proteínas de Fluorescência Verde
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Neurônios Dopaminérgicos
Tipo de estudo:
Diagnostic_studies
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Prognostic_studies
Limite:
Animals
Idioma:
En
Revista:
J Vis Exp
Ano de publicação:
2017
Tipo de documento:
Article