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J.L. Rae; Single Cell Electroporation in the Eye . Invest. Ophthalmol. Vis. Sci. 2003;44(13):3456.
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Purpose: To characterize an electroporation method to put genes and Alexa molecules into single cells Methods: A modified patch clamp technique for electroporating tiny membrane patches of single cells. Confocal microscopy. Fluorescence microscopy. Results: Using a modifed current-voltage converter-patch clamp circuit, it has proven possible to electroporate membrane patches in single cells of the lens, corneal epithelium, corneal endothelium, ciliary body, and most cells of the retina as well as a variety of tissue culture cells. The technique uses a 100kOhm feedback resistor in a current-voltage converter to apply positive or negative voltages of up to 12V to the interior of a patch clamp electrode. When the electrode is placed close enough to a cell for the measured resistance to increment by 20% or so, the voltage in the pipette creates a sufficient voltage at the membrane surface to result in electroporation. This allows substances that will traverse electroporation "pores" to enter the cells. To date, we have used a wide variety of cDNA's coding for proteins that localize in particular organelles in single cells. In addition, we have used the sodium salts of two Alexa fluorescent probes which easily traverse gap junctions in a variey of cells and easily diffuse to terminals in neurons. The technique works on isolated cells or single cells in intact globes so long as there is a pathway allowing the electrode tip to contact the cells . The technique is a useful alternative to intracellular injection of genes or tracers. It has many potential uses including the creation of single cell knockouts through insertion of siRNA's into single identified cells. The technique does not kill the cells into which the injection occurs. In suitable cell systems, the affected cells can be followed through many cell divisions. Conclusions: Single cell electroporation is a useful tool for the insertion or DNA, RNA, tracers, carbohydrates and a wide variety of small molecules into single cells of a number of ocular preparations.
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