CaltechAUTHORS
  A Caltech Library Service

Reliable Identification of Living Dopaminergic Neurons in Midbrain Cultures Using RNA Sequencing and TH-promoter-driven eGFP Expression

Henley, Beverley M. and Cohen, Bruce N. and Kim, Charlene H. and Gold, Heather D. and Srinivasan, Rahul and McKinney, Sheri and Deshpande, Purnima and Lester, Henry A. (2017) Reliable Identification of Living Dopaminergic Neurons in Midbrain Cultures Using RNA Sequencing and TH-promoter-driven eGFP Expression. Journal of Visualized Experiments (120). Art. No. 54981. ISSN 1940-087X. PMCID PMC5409190. doi:10.3791/54981. https://resolver.caltech.edu/CaltechAUTHORS:20170320-104204700

[img] PDF - Published Version
See Usage Policy.

328kB

Use this Persistent URL to link to this item: https://resolver.caltech.edu/CaltechAUTHORS:20170320-104204700

Abstract

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 libraries. 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.


Item Type:Article
Related URLs:
URLURL TypeDescription
http://dx.doi.org/10.3791/54981DOIArticle
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5409190/PubMed CentralArticle
ORCID:
AuthorORCID
Lester, Henry A.0000-0002-5470-5255
Additional Information:© JoVE 2017. Date Published: 2/10/2017. This work was supported by grants from the U.S. National Institutes of Health (DA017279, AG033954, DA037743), the Michael J. Fox Foundation, and the Caltech Innovation Initiative funding the Millard and Muriel Jacobs Genetics and Genomics Laboratory at the California Institute of Technology. We thank Brian Williams for optimising the RNA-Seq library protocol and for providing assistance. We thank Henry Amrhein for computational training. We thank Barbara J. Wold for the use of her equipment and laboratory space. We thank Igor Antoshechkin for library sequencing, and for facility management. The authors have nothing to disclose.
Funders:
Funding AgencyGrant Number
NIHDA017279
NIHAG033954
NIHDA037743
Michael J. Fox FoundationUNSPECIFIED
Caltech Innovation Initiative (CI2)UNSPECIFIED
Subject Keywords:Neuroscience, Issue 120, single-cell, RNA-sequencing, cell harvesting, midbrain cultures, dopaminergic neurons, Parkinson's disease
Issue or Number:120
PubMed Central ID:PMC5409190
DOI:10.3791/54981
Record Number:CaltechAUTHORS:20170320-104204700
Persistent URL:https://resolver.caltech.edu/CaltechAUTHORS:20170320-104204700
Official Citation:Henley, B. M., Cohen, B. N., Kim, C. H., Gold, H. D., Srinivasan, R., McKinney, S., et al. Reliable Identification of Living Dopaminergic Neurons in Midbrain Cultures Using RNA Sequencing and TH-promoter-driven eGFP Expression. J. Vis. Exp. (120), e54981, doi:10.3791/54981 (2017).
Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:75239
Collection:CaltechAUTHORS
Deposited By: George Porter
Deposited On:20 Mar 2017 19:40
Last Modified:15 Nov 2021 16:32

Repository Staff Only: item control page