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JL Rae; Electroporation of Single Cells in Natural Lens and Cornea Preparations . Invest. Ophthalmol. Vis. Sci. 2002;43(13):458.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: To describe procedures and results from single cell electroporation of genes into natural lens and corneal preparations. Methods: A patch clamp electrode is pressed against an identified cell. A simple voltage clamp circuit is used to electroporate the cell membrane adjacent to the tip thus allowing genes and other compounds to enter the cell.Sucess in gene expression is demonstrated by confocal microscopy and/or patch voltage clamping. Results: Genes as large as 14kB can be placed in single cells using pipette voltages in the 2-10V range. The insertion occurs through a membrane patch which is .25-.50 microns in diameter and so the gene insertions can be done at many locations on the surface of a single cell. Rectangular pulses as short as 10 microseconds or as long as 300 milliseconds are effective. Short pulses must be repeated so the total time of current flow is 300 millseconds.The procedure is effective for inserting genes for GFP-organellar fusion proteins and a wide variety of channel proteins. Other compounds including small peptides can also be inserted.Transfection rates can be ≷80%.Conclusion: A patch clamp based electroporation procedure is effective for placing genes and other compounds into single identified cells in natural lens and corneal preparations.
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