High-efficiency transfection of individual neurons using modified electrophysiology techniques
Introduction
Electroporation is a method to insert DNA, RNA, dyes, peptides and proteins into cells (Neumann et al., 1999). Compared to other methods, such as viral gene transfer and ‘gene gun’ biolistics potential toxic or mechanical damage is avoided. Furthermore, using these methods it is nearly impossible to transfect selectively a single cell within a brain slice culture.
Electroporation occurs when the voltage applied across the membrane exceeds the dielectric breakdown voltage of the membrane (∼0.2–1.5 V), leading to the formation of minute pores (20–120 nm diameter) through which molecules can enter the cell. The opening of the pores occurs in the microsecond range whereas their closure requires tens to hundreds of milliseconds (Teruel et al., 1997). Traditionally short-duration dielectric field pulses are applied between two relatively large plate electrodes (Teruel et al., 1999). However, this approach does not allow the transfection of single cells. For single-cell electroporation (SCE) a micropipette is filled with a DNA solution and connected to the cathode of a voltage pulse generator through a thin silver wire, which is in contact with the DNA solution. The ground electrode connected to the anode is in contact with the medium/buffer reservoir. Under visual guidance the pipette can be positioned with a micromanipulator into neural tissue. This method can be utilized to transfect neurons in organotypic hippocampal slice cultures, and even in living animals (Haas et al., 2001). However, there were low transfection rates. High transfection rates (up to ∼84%) could be achieved with optimized electroporation parameters and high-power visualization of the cell and the pipette using the lens epithelial cell line α-NT4 (Rae and Levis, 2002).
Direct visualization of the electroporation process is a prerequisite to improve the transfection efficiency for electroporated neurons. Fluoresceine-conjugated oligonucleotides and fluorescent dyes attached to DNA are tools to directly observe the electroporation process before, during and after pulse application. The green fluorescent dye TOTO-1 was employed to investigate electrically mediated gene delivery into cultured cells (Golzio et al., 2002). We analyzed factors which significantly enhance the transfection efficiency of individual neurons using SCE by visualization of the transfer processes by real-time imaging at high magnification. Our results show that electroporation does not affect the electrophysiological properties of SCE-transfected cells. Several green fluorescent protein (GFP)-tagged neuronal proteins were successfully expressed by this method. In addition to the transfection of hippocampal neurons we also show very efficient transfection of cortical neurons.
Section snippets
Organotypic brain slice cultures
Organotypic brain slice cultures were prepared as previously described (Stoppini et al., 1991). Hippocampi were isolated from 7-day-old Wistar rats, cortex was isolated from 7-day-old C57Bl/6 mice. Three hundred and fifty micrometer slices were cut using a McIlwain Tissue Chopper. The slices were cultured on Millicell CM culture plate inserts with 5-mm low wall (Millipore, Billerica, USA). The culture medium consisted of 50% MEM (Gibco Invitrogen, Karlsruhe, Germany), 25% EBSS (Gibco), 25%
Real-time SCE visualization of a fluoresceine-conjugated oligonucleotide
To increase the efficiency of SCE it is essential to understand what happens during the pulse application, and whether pulse application is correlated with oligonucleotide or plasmid DNA transfer through the cell membrane. Electroporation was therefore visualized using an electrophysiology setup equipped with a two-photon laser-scanning microscope. The pipette was filled with a fluoresceine-conjugated oligonucleotide and electrical pulses were applied as described in Section 2. A series of
Discussion
SCE, i.e. electroporation with a DNA filled micropipette, has been used for the transfection of individual neurons of organotypic brain slice cultures (Haas et al., 2001). However, the reported transfection efficiencies were too low for routine experiments. To improve SCE, we visualized in real-time the electroporation process to define optimal electroporation conditions.
The most critical and important factor for successful molecule transfer into the cell is the direct contact between the
Acknowledgements
We thank Dr R. Sprengel for the GluRB-GFP expression construct and Dr T. Kuner for the synaptophysin-GFP expression construct. Furthermore, we thank Dr Aron Jaffe for critically reading the manuscript.
References (15)
Membrane-bound choline-O-acetyltransferase in rat hippocampal tissue is associated with synaptic vesicles
Brain Res.
(1994)- et al.
Single-cell electroporation for gene transfer in vivo
Neuron
(2001) - et al.
Fundamentals of electroporative delivery of drugs and genes
Bioelectrochem. Bioenerg.
(1999) - et al.
Subunit-specific rules governing AMPA receptor trafficking to synapses in hippocampal pyramidal neurons
Cell
(2001) - et al.
A simple method for organotypic cultures of nervous tissue
J. Neurosci. Methods
(1991) - et al.
Electroporation-induced formation of individual calcium entry sites in the cell body and processes of adherent cells
Biophys. J.
(1997) - et al.
A versatile microporation technique for the transfection of cultured CNS neurons
J. Neurosci. Methods
(1999)
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These authors contributed equally to this paper.