Multicellular living organisms and unmodified parts thereof and – Method of using a transgenic nonhuman animal in an in vivo...
Reexamination Certificate
1999-09-10
2004-02-10
Wilson, Michael (Department: 1632)
Multicellular living organisms and unmodified parts thereof and
Method of using a transgenic nonhuman animal in an in vivo...
C800S018000
Reexamination Certificate
active
06689936
ABSTRACT:
BACKGROUND OF THE INVENTION
The invention relates to transgenic animals which express reporter genes coupled to promoters of genes involved in the health, aging, or appearance of the skin, e.g., theversican, VEGF, or MMP promoters, and methods of using such animals in evaluating treatments, e.g., compounds, for their effect on skin.
SUMMARY OF THE INVENTION
The inventors have discovered that transgenic animals having one or more constructs which include a skin-metabolism promoter coupled to a reporter gene can be used to evaluate a treatment, e.g., the removal of hair, e.g., by plucking or shaving, or the administration of a compound, for use in enhancing the health or appearance of the skin.
Accordingly, the invention features, a method of evaluating a treatment, e.g., the removal of hair, e.g., by plucking or shaving, or the administration of a compound, for its effect on skin. The method includes:
providing a transgenic animal having a reporter gene coupled to a skin metabolism-related promoter, preferably a human promoter;
administering the treatment to the transgenic animal or a tissue therefrom; and
evaluating expression of the reporter gene, thereby evaluating the treatment for its effect on skin.
The treatment, e.g., the administration of a compound, can be administered to a live animal. In other embodiments, the treatment, e.g., the administration of a compound, is administered to a tissue, e.g., a cell, taken from a transgenic animal.
The effect of the treatment, e.g., the administration of a compound, can be evaluated in a living transgenic animal, a dead transgenic animal, or tissue taken from either a living or transgenic mammal.
In preferred embodiments evaluating includes detection of a signal, e.g., a fluorescent signal, with a confocal microscope. In preferred embodiments the evaluation of the expression of the reporter gene step is repeated at least once during the life of the animal. The first and a subsequent repetition of the step can be separated by as much as 1, 10, 30, 60, 90, 180, 365, or 700 days. Both the first and a subsequent repetition can be performed on a live animal, e.g., with the use of a confocal microscope.
In preferred embodiments, the treatment includes the administration of a compound and the compound is administered by: applying the compound to the skin of the transgenic animal; systemically administering the compound; orally administering the compound; or injecting the compound, preferably dermally or subcutaneously. In preferred embodiments, the compound is administered using a suitable delivery vehicle, for example, a surfactant or an agent which increases permeability in the skin, e.g., an SDS or DMSO containing formulation.
In preferred embodiments, the transgenic animal is a non-human transgenic animal. For example, the transgenic animal can be a transgenic mini-pig, a transgenic guinea-pig, a transgenic rat, or a transgenic mouse, e.g., a hairless mouse, a nude mouse, a senescence accelerated mouse, e.g., SAM mice, see Takeda et al., 1991
, L. Am. Geriatr
. 39:911-919, or a transgenic mutant mouse which exhibits a phenotype of accelerated aging. The most preferred animals are mice.
In preferred embodiments, the promoter is heterologous from the transgenic animal, i.e., the promoter is from another species. In other preferred embodiments the promoter is from the same species as the transgenic animal. In particularly preferred embodiments, the skin metabolism-related promoter is a human skin metabolism-related promoter. In particularly preferred embodiments, the skin metabolism-related promoter is: a promoter from a gene which encodes a transmembrane protein or a component of the extracellular matrix, such as a proteoglycan promoter, e.g., a versican promoter; a promoter from a protease expressed in the skin, e.g., a matrix metalloproteinase (MMP) promoter, e.g., an MMP1, MMP2, MMP3, MMP4, MMP5, MMP6, MMP7, MMP8, or MMP9 promoter; a promoter from a gene which affects vascular function, e.g., a vascular endothelial growth factor promoter; a hyaluronan synthase promoter, e.g., a hyaluronan synthase 1 promoter, a hyaluronan synthase 2 promoter, or a hyaluronan synthase 3 promoter; a promoter for a collagenase expressed in the skin, e.g., a MMP2 or MMP9, preferably a MMP9, promoter; or a neutrophil elastase promoter.
In preferred embodiments the reporter gene encodes a product which can be detected with relative ease, e.g., an enzyme, e.g., an enzyme which produces a colored or luminescent product. In particularly preferred embodiments, the reporter gene can be a beta-galactosidase gene, a luciferase gene, a green fluorescent protein gene, an alkaline phosphatase gene, a horseradish peroxidase gene, or a chloramphenicol acetyl transferase gene.
In preferred embodiments, the treatment is administered repeatedly, prior to evaluation of reporter gene evaluation.
In preferred embodiments, the treatment includes the administration of a compound and the method further includes one or more subsequent administrations of the compound to the transgenic animal. In preferred embodiments, the compound is administered to the transgenic animal for a period of at least one, two, three, or four weeks. The compound can be administered at a constant level or at a range of different levels. In preferred embodiments, the compound is administered to the transgenic animal before, during, or after UV irradiation or other skin damaging treatment.
In preferred embodiments, the method further includes comparing the expression of the reporter gene to a control value, e.g., the level of expression of the reported gene in an untreated transgenic animal.
In preferred embodiments, the transgenic animal further includes a second reporter gene coupled to a second skin metabolism-related promoter, wherein the second skin metabolism-related promoter is different from the first skin metabolism-related promoter. The reporter gene coupled to the first promoter can be the same or different from the reporter gene coupled to the second promoter. For example, the transgenic animal can include: a first reporter gene coupled to the versican promoter and a second reporter gene coupled to the vascular endothelial growth factor promoter; a first reporter gene coupled to the versican promoter and a second reporter gene coupled to a hyaluronan synthase promoter; a first reporter gene coupled to the hyaluronan synthase promoter and a second reporter gene coupled to the vascular endothelial growth factor promoter; a first reporter gene coupled to a matrix metalloproteinase (MMP) promoter and a second reporter gene coupled to a MMP2 or MMP9, preferably MMP9, promoter; a first reporter gene coupled to a matrix metalloproteinase (MMP) promoter and a second reporter gene coupled to the neutrophil elastase promoter; or a first reporter gene coupled to a MMP2 or MMP9, preferably a MMP9, promoter and a second reporter gene coupled to the neutrophil elastase promoter. In preferred embodiments, the transgenic animal can include two constructs both of which are upregulated. In preferred embodiments, the transgenic animal can include two constructs both of which are downregulated.
In preferred embodiments, the method further includes evaluating the expression of the reporter gene coupled to the second skin metabolism-related promoter.
In preferred embodiments, the compound is: a cosmetic; a non-toxic substance; a substance approved for human drug or cosmetic use in one or more jurisdictions; a retinoid or derivative thereof; TGF&bgr;; of TGF&agr;.
In preferred embodiments, the method further includes administering a second treatment) to the transgenic animal. The second treatment can be one which injures or damages the skin, kills skin cells, or can include the removal of hair, e.g., by plucking, shaving, or application of a depilatory, or in general, induces an unwanted condition of the skin. The second treatment can be the application of water, a drying agent, an irritant, an inflammatory agent, light or UV irradiation. Reporter gene expression in response to the treatment can be determined in
Amano Satoshi
Burgeson Robert
Ehama Ritsuko
Kishimoto Jiro
Nishiyama Toshio
Fish & Richardson PC
The General Hospital Corp.
Wilson Michael
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