Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving virus or bacteriophage
Reexamination Certificate
2001-03-07
2002-05-28
Guzo, David (Department: 1636)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving virus or bacteriophage
C435S006120, C435S029000, C435S320100, C435S455000, C435S456000, C435S325000, C435S366000, C435S370000, C435S371000, C435S091400, C435S091410, C435S091420
Reexamination Certificate
active
06395473
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to an assay system in mammalian cells using a recombinant adenovirus vector.
BACKGROUND OF THE INVENTION
Some drugs and drug metabolites effect a transcriptional response in key genes within the cell. Promoter/reporter assay systems are one way to examine transcriptional regulation in vitro. In general, promoter/reporter assay system join a promoter to a reporter gene whose transcriptional or translational product is known. If the promoter actually directs transcription and/or translation of the reporter gene, its transcriptional and/or translational product can be easily determined.
To date, standard plasmid vectors have been used for delivery of promoter/reporter cassettes to cells. However, traditional methods, such as lipofection, calcium phosphate precipitation, and electroporation, are cumbersome and generally inefficient delivery systems when the cells are mammalian, and in particular primary hepatocytes.
The liver is the primary site for metabolism of most drugs. The levels of several drug metabolizing enzymes in the liver are altered in response to drug exposure and in some instances this alteration is a result of transcriptional regulation. Liver cell cultures would provide a good model of drug metabolism in vivo, but liver cells are difficult to transfect efficiently.
It would also be desirable to efficiently transfect mammalian cells with foreign DNA to study promoter regulation of a gene involved in drug metabolism. It would also be desirable to obtain an assay system to examine transcriptional regulation of genes coding for a drug metabolizing enzyme in mammalian cells.
DETAILED DESCRIPTION OF THE INVENTION
The present invention relates to a method of determining if a candidate promoter is responsive to an inducing agent in a mammalian cell culture comprising:
a) constructing an adenovirus vector wherein an E1 gene region is replaced by the candidate promoter operatively linked to a reporter gene construct;
b) introducing the vector into mammalian cells;
c) contacting the cells with an inducing agent; and
d) determining if the reporter gene is expressed.
The present invention further relates to a method of determining if a promoter is responsive to a candidate promoter inducing agent in a mammalian cell culture comprising:
a) constructing an adenovirus vector wherein an E1 gene region is replaced by the promoter operatively linked to a reporter gene construct;
b) introducing the vector into mammalian cells;
c) contacting the cells with a candidate inducing agent; and
d) determining if the reporter gene is expressed.
In both of the methods described, there may be an optional step of quantifying the expression of the reporter gene.
In a specific embodiment, the present invention relates to a rat glutathione S-transferase Ya subunit promoter sequence linked to a reporter construct, chloramphenicol acetyltransferase (CAT).
REFERENCES:
patent: 5654168 (1997-08-01), Bujard et al.
patent: 5733745 (1998-03-01), Kowalski et al.
patent: 6204060 (2001-03-01), Mehtali et al.
Bernard Massie et al, Inducible Overexpression of a Toxic Protein by an Adenovirus Vector with a Tetracycline-Regulatable Expression Cassette, Journal Of Virology, Mar. 1998, pp. 2289-2296.
Morsy Manal A.
Richards Karen
Rushmore Thomas H.
Giesser Joanne M.
Guzo David
Merck & Co. , Inc.
Tribble Jack L.
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