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Introduction
membrane in the cell. If, however, the pores are too large, they will not re-seal,
leading to lysis and death of the cell.
Electroporation is potentially applicable to all cell types regardless of their origin,
stage of maturation, or preparation in which they are found. Traditionally,
electroporation has been used for transfection of dissociated cells in solution. Cells
bathed in a solution of DNA are placed in a cuvette and exposed to high-voltage
pulses delivered between two large plate electrodes in the cuvette. Recently,
electroporation has been used for the bulk transfection of cells within intact tissues,
including neurons within the spinal cord (Sakamoto
et al
., 1998), eye (Koshiba
et
al
., 2000) and brain (Haas, 2002). These techniques typically involve injecting a
solution of DNA into an enclosed space, such as the lumen, or directly into the
tissue, followed by application of high-voltage pulses between two electrodes on
either side of the tissue. Controlled application of an electric field and restricted
exposure to the molecules to be delivered allow precise targeting of electroporation
to specific cells (Teruel, 1999; Atkins, 2000; Haas, 2001).
Single-cell electroporation allows targeted delivery of molecules. A number of
technical approaches have been devised to target individual cells, including
microelectrodes (Lindqvist, 1998; Olofsson, 2003), electrolyte-filled capillary tubes
(Nolkrantz, 2001) and electronic chips (Huang, 2000). A most efficient method for
single-cell electroporation employs a glass micropipette that restricts the electric
field to a single cell (Haas, 2001; Rae, 2002). This is accomplished by placing a
solution with the molecules to be delivered into the tip of the glass micropipette
whose diameter is less than the width of the target cell (approximately 0.5 µm).
After the micropipette tip is placed near, or in contact with the target cell
membrane, voltage pulses are delivered between a chlorided silver wire within the
pipette and an external chlorided ground electrode. The voltage pulses cause pores
to form in the membrane of the adjacent cell and electrophoretically drive charged
molecules from the pipette, through the pores into the cell. After pulse termination
the pipette is removed, leaving a single cell loaded with the desired molecules.
This technique leaves any surrounding cells in culture or in the intact tissue
unaltered. Single-cell electroporation can be used to deliver molecules to
individual cells within dissociated cultures, organotypic tissue cultures, acute tissue
Axoporator 800A Theory and Operation, Copyright 2005 Axon Instruments / Molecular Devices, Corp.
