Polyethylenimine-Mediated DNA Transfection of Peripheral and Central Neurons in Primary Culture: Probing Ca2+Channel Structure and Function with Antisense Oligonucleotides

doi:10.1006/mcne.1996.0018

Molecular and Cellular Neuroscience

Volume 7, Issue 3, March 1996, Pages 239-246

https://doi.org/10.1006/mcne.1996.0018 Get rights and content

Abstract

To study neuronal ion channel function with antisense oligonucleotides, a reliable method is needed which allows different neuronal cell types to be transfected without artifactual disruptive effects on their electrical properties. Here we report that use of the recently introduced transfecting agent, polyethylenimine, fulfills this requirement. Four days after transfection, in both central and peripheral neurons, an antisense designed to block the synthesis of the Ca²⁺channel β subunits induced a maximal decrease of the Ca²⁺current amplitude and modification of their kinetics and voltage-dependence. Controls with scrambled oligonucleotides, as well as Na⁺current recordings of antisense transfected neurons, confirm both that the transfecting agent does not modify the electrophysiological properties of the neurons and that the effect of the antisense is sequence specific.

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Expression of all constructs was driven by a cytomegalovirus promoter. For FRET two-hybrid experiments, HEK293 cells were cultured on glass-bottom dishes and transfected with polyethylenimine [22] (PEI) before epifluorescence imaging. Whole-cell patch clamp and FRET two-hybrid experiments were performed 1–2 days after transfection.
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While a PEI concentration representative of the concentrations used to transfect cells showed only small effects, an important lysosomal disruption was obtained at 5-fold higher concentrations. In this respect, the inferred high intraendoensomal PEI concentrations may be sufficient to destabilize the endosomes, while in line with previous results on neuronal cells [41], the extracellular PEI concentration may not be sufficient to destabilize the plasma membrane. The accumulation of PEI on the inner surface of endosome membranes together with the membrane-destabilizing properties of PEI may by themselves explain the release of PEI into the cytoplasm.
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Analysis of gene regulatory sequences in primary cultures of neurons has been hampered by inefficient transfection of post-mitotic neurons with reporter plasmids. We describe detailed conditions that allowed a significant improvement of transfection efficiency in primary cultures of serum-supplemented rat fetal hypothalamic cells. Transfected cells expressed the green fluorescent protein (GFP) under the control of the strong but non-cell-specific cytomegalo virus (CMV) promoter or under the thyrotropin-releasing hormone (TRH) promoter, to direct expression only in TRH neurons. Using the CMV promoter-GFP plasmid, we tested several commercially available transfection reagents; the best results were obtained with polyethylenimine (PEI) and Lipofectamine 2000. We optimized the transfection procedure with PEI because it rendered more reproducible results. Transfection with PEI was optimal when cells were transfected at a cellular density of 2.9×10⁶ cells in 35-mm dishes, with 10 μg of DNA, a PEI/DNA ratio of 8.8 and PEI pH of 6.9. Using these conditions, we were able to detect GFP positive neurons after transfecting the TRH promoter-GFP plasmid. GFP positive cells were successfully purified by FACS. This opens the possibility to use transfection of mammalian CNS post-mitotic neurons for new applications including the purification of specific neuronal subtypes.

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¹: To whom correspondence should be addressed at Laboratoire de Neurobiologie Cellulaire, CNRS, Centre de Neurochimie, 5 rue Blaise Pascal, 67084 Strasbourg, France. Fax: (+33) 88 60 16 64. E-mail: [email protected].

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MethodsPolyethylenimine-Mediated DNA Transfection of Peripheral and Central Neurons in Primary Culture: Probing Ca2+Channel Structure and Function with Antisense Oligonucleotides

Abstract

Methods
Polyethylenimine-Mediated DNA Transfection of Peripheral and Central Neurons in Primary Culture: Probing Ca²⁺Channel Structure and Function with Antisense Oligonucleotides