A method for isolating cortical interneurons sharing the same birthdays for gene expression studies
Autor: | Hui Xuan Ng, Brenton Cavanagh, Seong-Seng Tan, Joanne M. Britto, Ean Phing Lee |
---|---|
Rok vydání: | 2017 |
Předmět: |
0301 basic medicine
Cell Membrane Permeability genetic structures Interneuron Green Fluorescent Proteins Gene Expression Mice Transgenic Cell Separation Biology Green fluorescent protein Mice 03 medical and health sciences 0302 clinical medicine Developmental Neuroscience Interneurons Gene expression medicine Animals Humans Cerebral Cortex Regulation of gene expression Glutamate Decarboxylase Pyramidal Cells musculoskeletal neural and ocular physiology fungi Gene Expression Regulation Developmental RNA Cell sorting Flow Cytometry 030104 developmental biology Real-time polymerase chain reaction medicine.anatomical_structure Genetic Techniques nervous system Neurology RNA extraction Neuroscience 030217 neurology & neurosurgery |
Zdroj: | Experimental Neurology. 295:36-45 |
ISSN: | 0014-4886 |
Popis: | The two neuronal populations in the cortex, pyramidal neurons and interneurons, can be separated based on neurotransmitter identity, however, within this segregation a large degree of diversity exists. Investigations into the molecular diversity of neurons are impeded by the inability to isolate cell populations born at different times for gene expression analysis. Developing interneurons may be distinguished by the expression of Glutamic Acid Decarboxylase-67 (GAD67). Neuronal birthdating using nucleoside analogs is an effective means of identifying coetaneous interneurons. Using these two features, neurotransmitter identity and birthdating, we have developed a method to isolate migrating interneurons using fluorescent-activated cell sorting (FACS) for RNA extraction and gene expression analysis. We utilized 5-ethynyl-2'-deoxyuridine (EdU) to birthdate interneuron cohorts and the GAD67 knock-in GFP transgenic mice to identify interneurons. In combination, we achieved simultaneous detection of GFP and EdU signals during FACS sorting of coetaneous interneurons with minimum loss of RNA integrity. RNA quality was deemed to be satisfactory by quantitative polymerase chain reaction (qPCR) for the interneuron-specific transcript Gad67. |
Databáze: | OpenAIRE |
Externí odkaz: |