Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
1999-04-01
2002-05-21
Prouty, Rebecca E. (Department: 1652)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S007910, C435S018000, C435S021000, C435S196000, C435S206000, C435S288500, C435S007200, C435S325000, C435S252300
Reexamination Certificate
active
06391549
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to a method, system and kit for use in monitoring of DNA expressions within cells.
PRIOR ART
The following is a list of references which are intended for a better understanding of the background of the present invention:
REFERENCES
Groskreutz, D. and Schenborn, E. T., Reporter systems. In: Methods in molecular biology, Vol. 63: Recombinant protein protocols; detection and isolation. R. S. Tuan (ed.), pp. 173-218, Humana Press Inc., Totowa, N.J.
Jain, V. K. and Magrath, I. T., A chemiluminescent assay for quantitation of &bgr;-galactosidase in the fentogram range: application to quantitation of &bgr;-galactosidase in lacZ-transfected cells,
Anal. Biochem.,
199:119-124 (1991).
Kulys, J., Razumas, V and Malinauskas, A., Electrochemical oxidation of catechol and p-aminophenol esters in the prsence of hydrolase,
J. Electroanal. Chem. [Bioelectrochem. Bioenerg.
7] 116.11-24 (1980).
Masson, M., Liu, Z., Haruyama, T., Kobatake, E., Ikariyama, Y and Aizawa, M., Immunosensing with amperometric detection, using galactosidase as label and p-aminophenyl-&bgr;-D-galactopyranoside as substrate.
Anal. Chim. Acta
304;353-359 (1995).
Miler, J. H., A short course in bacterial genetics, p. 72-74, Cold Spring Harbor Press., Cold Spring Harbor, N.Y. (1992).
Rosen, I. and Rishpon, J., Alkaline phosphatase as a label for heterogeneous immunoelectrochemical sensor,
J. Electroanal. Chem.,
258:27-39 (1989).
Silhavy, T. J. and Beckwith, J. R., Uses of lac fusions for the study of biological problems,
Microbiol. Rev.
49;398-418 (1985).
U.S. Pat. No. 5,149,629.
Weichart, D., Lang, R., Henneberg, N., & Hengge-Aronis, R., Identification and characterization of stationary phase-inducible genes in
Escherichia coli. Mol. Microbiol,
10:407-420 (1993).
Yin, H. H. and Villarejo, M., OsmY, a new hyperosmotically inducible gene, encodes a periplasmic protein in
Escherichia coli., J. Bacteriol.
174:3637-3644 (1992).
BACKGROUND OF THE INVENTION
Reporter gene systems are used in gene expression studies and for biosensor developmont, Various analytical methods are available for monitoring the protein expressed by a reporter gene. These methods include photometry, radiometry, fluorescence, colorimetry and immunoassays (Groskreutz, D. et al., 1997). A light emitting gene was also used as the reporter gene and several techniques have been developed to monitor the light emitting from the cells (Legocki, R. P. et al., 1993).
The predominantly used methods for the identification and quantification of the reporter gene products in cell cultures involve repeated samplings of the culture and an assay for the enzymatic activity, that often involves an additional step of lysis or permeabilization of the cell and must be performed under aerobic conditions. These procedures perturb the culture and are time consuming at times, providing results only after several hours.
The gene lacZ, coding for the
Escherichia coli
enzyme &bgr;-galactosidase is one of the most widely used reporter genes (Silhavy & Beckwith, 1985) and has been used in colorimetric assays (Miler, 1992), or using fluorometry or chemilluminometry (Jain & Magrath, 1991). These methods involve the permeabilization of the cells followed by a multi-step procedure.
The enzymatic activity of &bgr;-galactosidase can be determined electrochemically by using the substrate p-aminophenyl-&bgr;-D-galactopyranoside (PAPG). The product of the enzymatic reaction, p-aminophenol (PAP) is oxidized at an electrode.
Several electroanalytical methods for PAP detection have been reported (Kulys & Malinauskas, 1980; Masson et al, 1995). An electrochemical imunoassay, in which a constant potential is applied on the electrode and the current generated by the oxidation of PAP is measured was also described (U.S. Pat. No. 5,149,629).
GENERAL DESCRIPTION OF THE INVENTION
In accordance with the present invention novel means are provided for monitoring of gene expression within cells. As will be detailed further below, the invention may be used for determination of cell parameters in a cell culture, for cell biology research, for determining parameters or conditions of a sample, etc. as well as for biosensor development.
In accordance with one aspect, the present invention provides a method for detecting a parameter of a host cell, comprising:
(a) transfecting said cell with an expressible DNA sequence under expression control of an inducible promoter sequence, said promoter sequence being inducible in correlation wit said parameter, said expressible sequence encoding an enzymatically active product which can catalyze a reaction giving rise to an electrical signal which is detectable in an electrochemical measurement;
(b) placing the transfected cells in an electrochemical cell; and
(c) measuring the level of the electrical signal, a signal above a threshold level indicating the presence of said parameter.
The present invention also provides a method for determining a parameter of a medium, comprising:
(a) providing cells transfected with an expressible DNA sequence under expression control of an inducible promoter sequence, said promoter sequence being inducible in correlation with said parameter, said expressible sequence encoding an enzymatically active product which can catalyze a reaction giving rise to an electrical signal which is detectable in an electrochemical measurement;
(b) placing said cells in the medium or a sample thereof; and
(c) providing an electrochemical cell and measuring level of said electrical signal, the level of said signal being correlated to the level of said parameter.
Still further provided by the invention is a method for monitoring a growth status parameter, said parameter being a parameter characteristic of a defined growth or cell cycle phase or a defined culture phase of a cell culture, comprising:
(a) providing a culture of cells, wherein at least some of the cells are transfected with an expressible DNA sequence under expression control of an inducible promoter sequence, said promoter sequence being inducible in correlation with the growth status parameter, said expressible sequence encoding an enzymatically active product which can catalyze a reaction giving rise to an electrical signal which is detectable in an electrochemical measurement;
(b) in an electrochemical cell, continuously or periodically measuring said electrical signal, said signal being indicative of said growth status parameter.
By a further of its aspects the present invention provides a system for monitoring expression of a target promoter, comprising:
(a) an electrochemical cell;
(b) cells transfected with an expressible DNA sequence under expression control of said target promoter, said expressible sequence encoding an enzymatically active product which can catalyze a reaction giving rise to an electrical signal which is detectable in an electrochemical measurement; and
(c) apparatus for measurement of said electrical signal.
By a still further aspect the present invention further provides a kit for use in detecting a parameter of host cell, a medium parameter or a cell or cell culture growth stats indicator parameter, comprising:
(a) host cells transfected wit an expressible DNA sequence under expression control of an inducible promoter sequence, said promoter sequence being inducible in correlation with said parameter, said expressible sequence encoding an enzymatically active product which can catalyze a reaction giving rise to an electrical signal which is detectable in an electrochemical measurement;
(b) at least one component of an electrochemical cell, said electrochemical cell being adapted for receiving and holding the host cells and for performing said electrochemical measurement.
GLOSSARY
The following is an explanation of some terms used above and in the following description and claims:
Parameter—when applied to a cell, referring to a certain property of interest within the cell which may include a certain phase of the cell cycle, a response of the cell to stimulants existing in an extracellular med
Babai Reuven
Biran Israel
Rishpon Judith
Ron Eliora Z.
Juneau Todd L.
Nath Gary M.
Nath & Associates PLLC
Prouty Rebecca E.
Ramont University Authority for Applied Research & Industrial De
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