Particle-mediated gene transfer to rat neurons in primary culture

Y M Usachev; A Khammanivong; Colin Campbell; Stanley A Thayer

doi:10.1007/s004240050999

Back

Particle-mediated gene transfer to rat neurons in primary culture

Journal article

Peer reviewed

Particle-mediated gene transfer to rat neurons in primary culture

Y M Usachev, A Khammanivong, Colin Campbell and Stanley A Thayer

Pflügers Archiv, Vol.439(6), pp.730-738

04/2000

DOI: 10.1007/s004240050999

PMID: 10784347

View Online

Abstract

Gene transfer into neuronal cells provides an important approach to study their function. Particle-mediated gene delivery was used to transfect rat dorsal root ganglion (DRG) and hippocampal neurons in primary culture with the genes for the enhanced blue and green fluorescent proteins (EBFP and EGFP) under control of the cytomegalovirus promoter. Quantitative analysis of marker protein fluorescence detected expression at 3 h that continued to increase for 48 h. For DRG neurons the optimal expression efficiency of 8+/-2% was obtained 24 h following transfection. In contrast, approximately 2+/-1% of hippocampal neurons in culture expressed EGFP at 3 h which subsequently declined. Co-transfection of DRG cultures with two plasmids produced reliable expression of both genes. Transfected DRG neurons exhibited normal electrophysiological properties, and resting and stimulated intracellular Ca2+ concentrations were unchanged. After transfection, 44% of hippocampal neurons remained in functional synaptic networks as indicated by glutamatergic Ca2+ spiking activity. Particle-mediated gene delivery provided a straightforward, reproducible and efficient method for transfection of neurons in primary culture. Transfected cells were easily identified by EGFP fluorescence, enabling subsequent physiological analysis. Biolistic particle bombardment was well tolerated by peripheral neurons, although caution was required when this method was applied to CNS cultures.

Electrophysiology

Transfection

Particle Accelerators

Green Fluorescent Proteins

Gene Transfer Techniques - instrumentation

Cells, Cultured

Rats

Ganglia, Spinal - cytology

Hippocampus - cytology

Animals

Neurons - physiology

Indicators and Reagents

Luminescent Proteins - genetics

Details

Title: Subtitle: Particle-mediated gene transfer to rat neurons in primary culture
Creators: Y M Usachev - Department of Pharmacology, University of Minnesota Medical School, Minneapolis 55455, USA
A Khammanivong
Colin Campbell
Stanley A Thayer
Resource Type: Journal article
Publication Details: Pflügers Archiv, Vol.439(6), pp.730-738
Publisher: Germany
DOI: 10.1007/s004240050999
PMID: 10784347
ISSN: 0031-6768
eISSN: 1432-2013
Grant note: DA11806 / NIDA NIH HHS DA9293 / NIDA NIH HHS DA7304 / NIDA NIH HHS
Language: English
Date published: 04/2000
Academic Unit: Iowa Neuroscience Institute; Anesthesia; Neuroscience and Pharmacology
Record Identifier: 9984040362202771

Metrics

21 Record Views

13 Times Cited - Web of Science