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  High-efficiency transfection of individual neurons using modified electrophysiology techniques

Rathenberg, J., Nevian, T., & Witzemann, V. (2003). High-efficiency transfection of individual neurons using modified electrophysiology techniques. Journal of Neuroscience Methods, 126(1), 91-98. doi:10.1016/S0165-0270(03)00069-4.

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Item Permalink: http://hdl.handle.net/11858/00-001M-0000-002A-1061-4 Version Permalink: http://hdl.handle.net/11858/00-001M-0000-002A-1062-2
Genre: Journal Article
Alternative Title : High-efficiency transfection of individual neurons using modified electrophysiology techniques

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JNeurosciMeth_126_2003_91.pdf (Any fulltext), 470KB
 
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 Creators:
Rathenberg, Jan1, Author              
Nevian, Thomas1, Author              
Witzemann, Veit1, 2, Author              
Affiliations:
1Department of Cell Physiology, Max Planck Institute for Medical Research, Max Planck Society, ou_1497701              
2Department of Molecular Neurobiology, Max Planck Institute for Medical Research, Max Planck Society, ou_1497704              

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Free keywords: Single-cell electroporation; Transfection; Neuron; Organotypic brain slice culture; Green fluorescent protein; Electrophysiology
 Abstract: Transfection of cells by electroporation is a widely used and efficient method. Recently, it has been shown that single neurons in brain slice cultures can be transfected using micropipettes loaded with plasmid DNA expression constructs. However, the transfection efficiencies were very low. Routine employment of single-cell electroporation (SCE) for transfection of neurons requires high and reliable efficiency together with good cell survival. Here, we describe the modification of electrophysiology techniques for SCE leading to very simple and efficient (up to 80%) transfection of neurons in organotypic rat hippocampus and mouse cortex slice cultures. Electroporation-mediated transfection was visualized in real-time by two-photon microscopy at the cellular level using fluorescently labeled oligonucleotides and plasmid DNA. Small oligonucleotides enter the cell immediately during pulse application while large plasmids remain localized for more than 10 min at the cell membrane before they enter the cell by an, as yet, unknown process. SCE does not affect the electrophysiology of transgene-expressing cells. Expression of several neuronal green fluorescent protein-tagged proteins demonstrates that the method can be employed to analyze subcellular trafficking and targeting in single living neurons.

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Language(s): eng - English
 Dates: 2003-02-282003-01-132003-03-072003-06-15
 Publication Status: Published in print
 Pages: 8
 Publishing info: -
 Table of Contents: -
 Rev. Type: Peer
 Degree: -

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Title: Journal of Neuroscience Methods
  Other : J. Neurosci. Meth.
Source Genre: Journal
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Publ. Info: Amsterdam : Elsevier
Pages: - Volume / Issue: 126 (1) Sequence Number: - Start / End Page: 91 - 98 Identifier: ISSN: 0165-0270
CoNE: https://pure.mpg.de/cone/journals/resource/954925480594