Chemistry: molecular biology and microbiology – Treatment of micro-organisms or enzymes with electrical or... – Cell membrane or cell surface is target
Reexamination Certificate
1999-01-08
2001-07-17
Weber, Jon P. (Department: 1651)
Chemistry: molecular biology and microbiology
Treatment of micro-organisms or enzymes with electrical or...
Cell membrane or cell surface is target
C435S173500
Reexamination Certificate
active
06261815
ABSTRACT:
FIELD OF THE INVENTION
This invention relates to a method of introducing exogenous compounds into cells by electroporation.
BACKGROUND OF THE INVENTION
Single cell assays to measure signal transduction and other cellular processes are useful for the identification of drug targets, as well as for drug screening. The number of known signaling molecules involved in particular cell functions and disease states is rapidly increasing. At the same time, combinatorial chemistry has dramatically expanded the number of substances that must be tested for their effect on specific signaling molecules and transduction pathways. Commonly used in vitro measurements of drug-target binding interactions and other screening methods are often inadequate to assess the effectiveness of drugs within the cellular context, and therefore, the ability to assay intact cells for particular cell functions would be a powerful way to investigate all signaling steps that lead to the monitored event. Currently, the main limitations for this strategy are: (i) the lack of an efficient method to rapidly introduce membrane impermeant molecules into adherent cells using cost efficient amounts of samples and (ii) the limited number of available single cell assays to test for particular cell functions.
SUMMARY OF THE INVENTION
A first aspect of the present invention is an electroporation apparatus for introducing exogenous material into cells. The apparatus comprises a base member configured for holding a cell support, the cell support having a top surface portion, the top surface portion configured for carrying adherent cells; an electrode carrier operably associated with the base member, the electrode carrier having a bottom surface portion; a first electrode connected to the electrode carrier; and a second electrode connected to the electrode carrier. The electrode carrier has a channel formed therein, with the channel positioned between the first electrode and the second electrode, so that the exogenous material may be introduced through the channel and into contact with the cells.
A second aspect of the present invention is a method for introducing an exogenous compound into cells. The method comprises providing a cell support, with said cell support having a top surface portion with the cells adhered thereto, and with the cells being in contact with (i.e., immersed in) an electroporation solution; positioning a pair of electrodes in said electroporation solution; positioning a micropipette between the pair of electrodes, with the micropipette having an outlet opening that is in fluid contact with the electroporation solution; introducing the compound through the outlet opening and into the electroporation solution; and subjecting the cells to a current pulse across the pair of electrodes, the current pulse sufficient to cause the compound to pass through the cell membranes of the cells and into the cells by electroporation.
A third aspect of the present invention is a method for visually detecting the location of binding events within a cell. The method comprises introducing a fusion protein into the cell, the fusion protein comprising a binding domain and a detectable domain; and then visually detecting the location of increased levels of the detectable domain within the cell, the location of increased levels of the detectable domain indicating the location of a compound within the cell to which the binding domain specifically binds.
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Fromm et al., “Electroporation of DNA and RNA into Plant Protoplasts”, Meth. Enzymol., 153, pp. 351-366, 1987.*
McCormick et al.; A Low Cost Microprocessor-Controlled Electrofusion and Electroporation System,J. Chem. Tech. Biotechnol.,54(2):159-169 (1992).
Lewin;Genes(Third Edition), John Wiley & Sons, New York, p. 736 (1987).
Duke University
Myers Bigel & Sibley & Sajovec
Weber Jon P.
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