Chemistry: molecular biology and microbiology – Apparatus – Mutation or genetic engineering apparatus
Reexamination Certificate
1999-11-16
2002-03-05
Redding, David A. (Department: 1744)
Chemistry: molecular biology and microbiology
Apparatus
Mutation or genetic engineering apparatus
C435S287200, C435S288400, C204S403060
Reexamination Certificate
active
06352853
ABSTRACT:
FIELD OF THE INVENTION
This invention relates to electroporation methods and apparatus for introducing exogenous molecules into lipid vesicles such as cells in vitro.
BACKGROUND OF THE INVENTION
Electroporation is a well established technique for introducting exogenous molecules such as plasmid DNA into cells. It involves the application of a large voltage across a liquid sample containing the cells and the molecules of interest. Typically, electroporation is conducted by positioning a single-channel apparatus that includes a pair of electrodes, i.e., a cathode and an anode, in a sample-containing chamber such as a disposable cuvette. See, e.g., Neumann et al.,
Biophysical Journal,
71, pp. 868-77 (1996).
Multi-channel electrode systems are used for high throughput introduction of exogenous molecules into cells, or to avoid the need for transferring cells from culture containers to electroporation cuvettes. A multi-channel electroporation apparatus includes a plurality of pairs of electrodes positioned in respective ones of a plurality of chambers that hold the exogenous materials and the cells. Currently available multi-channel electroporation devices contain 8 or 96 pairs of coaxial electrodes (Genetronics, Inc., San Diego, Calif.). These devices are used for electroporation in standard 96-well plates, which consist of 8 rows and 12 columns of wells and have a standard size of about 8.5 (W) cm×12.7 cm (L), with a standard center-to-center spacing of 9.0 mm between wells.
SUMMARY OF THE INVENTION
This invention features an electroporation apparatus for introducing exogenous molecules such as nucleic acids, proteins and chemical compounds into vesicles. The apparatus comprises a multi-channel electrode array which contains a plurality of electrode pairs. In each of these electrode pairs, the electrodes are flat and parallel to each other.
Vesicles that can be electroporated in accordance with this invention include, but are not limited to, microscopic or submicroscopic vesicles that contain lipid or fatty acid membrane, e.g., prokaryotic and eukaryotic cells, microsomes and micelles. The vesicles can be unilamellar, bilamellar or multilamellar and range in size, e.g., from 1 nm to 100 &mgr;m.
In one embodiment of the invention, the electrode array contains electrode pairs that are positioned to form a matrix consisting of a plurality of rows and columns of electrode pairs such that each electrode pair can fit into a different well of a multi-well plate.
In one of the preferred embodiments, the number of the electrode pairs in the array is a multiple of 96 (e.g., 192, 288, 384, 576, 672, 768 or 1536) and each electrode pair can fit into a different well of a multi-well plate having the same number of wells and the standard size of about 8.5 cm (W)×12.7 cm (L). The reason for this preference is that many currently available microplate instruments were designed specifically for the standard 96-well format and can be readily converted for use with multiples of the 96-well format. Thus, this preference does not in any way limit the scope of the invention.
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Exemplary methods and materials are described below, although methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present invention. All publications and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. The materials, methods, and examples are illustrative only and not intended to be limiting.
Other features and advantages of the invention will be apparent from the following drawings, detailed description, and the claims.
REFERENCES:
patent: 4882281 (1989-11-01), Hilliard et al.
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patent: 5137817 (1992-08-01), Busta et al.
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Beckman Coulter, Multimek 96, (1998).
Fiedler, et al., “Diffusional Electrotitration: Generation of pH Gradients over Arrays of Ultramicroelectrodes Detected by Fluorescence”,Anal. Chem.67:820-828 (1995).
Neumann, et al., “Calcium-Mediated DNA Adsorption to Yeast Cells and Kinetics of Cell Transformation by Electroporation”,Biophysical Journal71:868-877 (1996).
M. Peterfy et al., “Electroporation of COS-7 Cells With Transient Expression Vectors in 96 Well Microplates by Single, 8, and 96 Well Coaxial Electrodes,”Methods In Molecular And Cellular Biology,5:353-362, 1995.
Flak Tod A.
King Jeffrey S.
Redding David A.
Rosetta Inpharmatics, Inc.
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