Chemistry: molecular biology and microbiology – Treatment of micro-organisms or enzymes with electrical or... – Cell membrane or cell surface is target
Patent
1999-02-10
2000-09-12
Gorgos, Kathryn
Chemistry: molecular biology and microbiology
Treatment of micro-organisms or enzymes with electrical or...
Cell membrane or cell surface is target
4351734, 4351735, 205701, C12N 1300
Patent
active
061176605
DESCRIPTION:
BRIEF SUMMARY
TECHNICAL FIELD
The present invention relates to the field of applying a defined pattern of pulsed electrical fields to materials, especially living cells. More specifically, the present invention is especially concerned with the fields of electroporation, electrofusion, and electromanipulation.
BACKGROUND ART
Electroporation and electrofusion are related phenomena with a variety of uses in manipulation of prokaryotic and eukaryotic cells. Electroporation is the destabalization of cell membranes by application of a brief electric potential (pulse) across the cell membrane. Properly administered, the destabalization results in a temporary pore in the membrane through which macromolecules can pass while the pore exists. Therefore, in electroporation, membranes of membrane-containing material open to admit treating substances. Electrofusion is the fusion of two or more cells by application of a brief electric potential across a cell membrane. In electrofusion, membranes of membrane-containing material open to merge with membranes of other membrane-containing material. In this respect, one membrane-containing material may be regarded as a treating substance for another membrane-containing material. The physical and biological parameters of electrofusion are similar to those of electroporation.
The potential applied to cell membranes is applied using instruments delivering various pulse shapes. The two most common pulse shapes are exponential decay and rectangular wave. The exponential decay pulse is generated with capacitance discharge pulse generators. It is the least expensive pulse generator and gives the operator the least control over pulse parameters. The rectangular wave pulse generator is more expensive, gives more control over pulse parameters and generates a pulse that is less lethal to cells. With both pulse shapes, the energy needed to generate resealable pores in cells is related to cell size, shape, and composition.
With electrofusion, cells must be in contact at the time of membrane destabalization. This is accomplished by physical means such as centrifugation, biochemical means such as antibody bridging, or electrical means through dielectrophoresis. Dielectrophoresis is the creation of a dipole within a cell by application of a low voltage potential across a cell membrane in an uneven electrical field. The dipole can be created in DC or AC fields. Since DC fields tend to generate unacceptable heat, radio frequency AC is often used for dielectrophoresis.
The uses of electroporation and electrofusion are many. A partial list follows: (1) transient introduction of DNA or RNA into both eukaryotic and prokaryotic cells; (2) permanent transfection of DNA into both eukaryotic and prokaryotic cells; (3) permanent and temporary transfection of DNA into human and animal cells for gene therapy; (4) introduction of antibodies, other proteins, or drugs into cells; (5) production of antibody producing hybridomas; (6) pollen electrotransformation in plants; (7) electroinsertion; (8) manipulation of animal embryos; (9) electrofusion of adherent cells; (10) production of plant somatic hybrids; (11) DNA vaccination; and (12) cancer therapy.
One of the ways that electroporation or electrofusion works is to induce the formation of holes or pores in the cell membrane. There is some controversy about the exact nature of the cell pore induced by the application of an electrical pulse to a cell, but the practical effect is an induced cell permeability and a tendency to fuse with other similarly affected cells that are in close contact. There is a DC voltage threshold for the induction of pores in or for the fusion of cell membranes. Voltages below the threshold will not bring about substantial cell membrane disturbance. The threshold potential for many cells is approximately one volt across the cell membrane. The total DC voltage applied per centimeter between electrodes to achieve one volt potential across the cell membrane is therefore proportional to the diameter of a cell. Small cells such as bacteria, requir
REFERENCES:
patent: 5304486 (1994-04-01), Chang
patent: 6010613 (2000-01-01), Walters et al.
King Alan D.
Walters Derin C.
Walters Richard E.
CytoPulse Sciences, Inc.
Gorgos Kathryn
Tran Thao
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