EP1215959A1

EP1215959A1 - Methods for evaluating a compound for its effect on skin

Info

Publication number: EP1215959A1
Application number: EP00960031A
Authority: EP
Inventors: Robert Burgeson; Satoshi Amano; Jiro Kishimoto; Toshio Nishiyama; Ritsuko Ehama
Original assignee: General Hospital Corp
Current assignee: General Hospital Corp
Priority date: 1999-09-10
Filing date: 2000-09-08
Publication date: 2002-06-26
Also published as: WO2001017343A9; JP2003520946A; WO2001017343A1; EP1215959A4

Abstract

The invention provides methods of evaluating a treatment for its effect on skin. The invention also provides non-human transgenic animals, e.g., mice, having a reporter gene coupled to a skin-metabolism promoter.

Description

J METHODS FOR EVALUATING A COMPOUND FOR ITS EFFECT ON SKIN

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; TGFβ; of TGFα.

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 the presence of the second treatment, and optionally compared to the response seen in the absence of the second treatment.

In another aspect, 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, e.g., a mouse, having a reporter gene coupled to a, preferably human, versican promoter; administering a treatment, e.g., the removal of hair, e.g., by plucking or shaving, or the administration of a compound, to the transgenic animal, or to a tissue taken therefrom; and evaluating expression of the reporter gene, thereby evaluating the treatment for its effect on skin aging.

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 dead transgenic animal.

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, or a transgenic mutant mouse which exhibits a phenotype of accelerated aging. The most preferred animals are mice.

In particularly preferred embodiments, the versican promoter is a human versican promoter.

In a preferred embodiment the reporter is a luminescent or fluorescent product, which preferably, can lumenesce or fluoresce without the addition of exogenous substrates or cofactors, e.g., green fluorescent protein.

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, preferably 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 fransgenic animal.

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; TGFβ; of TGFα.

In preferred embodiments, the method further includes administering a second freatment ) 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 freatment can be determined in the presence of the second freatment, and optionally compared to the response seen in the absence of the second treatment.

In another aspect, 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, e.g., a mouse, having a reporter gene coupled to a, preferably human, matrix metalloproteinase promoter; administering the freatment to the fransgenic animal, or to a tissue taken therefrom; and evaluating expression of the reporter gene, thereby evaluating the treatment for its effect on skin aging.

The treatment, e.g., the administration of a compound, can be administered to a live animal. In other embodiments the freatment, 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 freatment, 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 dead transgenic animal.

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 fransgenic mini-pig, a transgenic guinea-pig, a fransgenic rat, or a transgenic mouse, e.g., a hairless mouse, a nude mouse, a senescence accelerated mouse, e.g., SAM mice, or a transgenic mutant mouse which exhibits a phenotype of accelerated aging. The most preferred animals are mice.

In particularly preferred embodiments, the matrix metalloproteinase promoter is a human matrix metalloproteinase 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 fransferase gene. In preferred embodiments, the freatment is administered repeatedly, preferably prior to evaluation of reporter gene evaluation.

In preferred embodiments, the method further includes administering a second freatment 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 the presence of the second freatment, and optionally compared to the response seen in the absence of the second treatment.

In another aspect, the invention features, a method of evaluating a treatment, e.g., the removal of hair, e.g., by plucking or shaving, or the adminisfration of a compound, for its effect on skin. The method includes: providing a transgenic animal, e.g. a mouse, having a reporter gene coupled to a vascular endothelial growth factor promoter; administering the freatment to the transgenic animal; and evaluating expression of the reporter gene, thereby evaluating the freatment for its effect on skin aging.

The freatment, 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 fransgenic animal.

The effect of the treatment, e.g., the adminisfration of a compound, can be evaluated in a living fransgenic animal, a dead transgenic animal, or tissue taken from either a living or dead transgenic animal. 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 fransgenic 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 fransgenic mouse, e.g., a hairless mouse, a nude mouse, a senescence accelerated mouse, e.g., SAM mice, or a transgenic mutant mouse which exhibits a phenotype of accelerated aging. The most preferred animals are mice. In particularly preferred embodiments, the vascular endothelial growth factor promoter is a human vascular endothelial growth factor promoter.

In a preferred embodiment the reporter is a luminescent or fluorescent product, which preferably, can lumenesce or fluoresce without the addition of exogenous substrates or cofactors, e.g., green fluorescent protein. 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 fransferase gene.

In preferred embodiments, the treatment is administered repeatedly, preferably 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 fransgenic 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 freatment.

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 method further includes administering a second freatment 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 freatment 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 the presence of the second treatment, and optionally compared to the response seen in the absence of the second treatment.

In another aspect, 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, e.g., a mouse, having a reporter gene coupled to a, preferably human, hyaluronan synthase promoter; administering the treatment to the fransgenic animal; and evaluating expression of the reporter gene, thereby evaluating the treatment for its effect on skin aging. The treatment, e.g., the administration of a compound, can be administered to a live animal. In other embodiments the treatment, e.g., the adminisfration 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 dead transgenic animal.

In preferred embodiments evaluating includes detection of a signal, e.g., a fluorescent signal, with a confocal microscope.

In preferred embodiments, the freatment includes the administration of a compound and the compound is administered by: applying the compound to the skin of the fransgenic 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 fransgenic animal is a non-human transgenic animal. For example, the fransgenic animal can be a transgenic mini-pig, a transgenic guinea-pig, a fransgenic rat, or a transgenic mouse, e.g., a hairless mouse, a nude mouse, a senescence accelerated mouse, e.g., SAM mice, or a transgenic mutant mouse which exhibits a phenotype of accelerated aging. The most preferred animals are mice.

In particularly preferred embodiments, the hyaluronan synthase promoter is a human hyaluronan synthase promoter. In a preferred embodiment the reporter is a luminescent or fluorescent product, which preferably, can lumenesce or fluoresce without the addition of exogenous substrates or cofactors, e.g., green fluorescent protein.

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 fransferase gene. In preferred embodiments, the treatment is administered repeatedly, preferably 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 fransgenic 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 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 the presence of the second treatment, and optionally compared to the response seen in the absence of the second treatment.

In another aspect, the invention features, a method of evaluating a freatment, e.g., the removal of hair, e.g., by plucking or shaving, or the adminisfration of a compound, for its effect on skin. The method includes: providing a transgenic animal, e.g., a mouse, having a reporter gene coupled to, a preferably human, MMP2 or MMP9, preferably a MMP9, promoter; administering the treatment to the fransgenic animal, or to a tissue taken therefrom; and evaluating expression of the reporter gene, thereby evaluating the treatment for its effect on skin aging. The freatment, 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 fransgenic animal, a dead transgenic animal, or tissue taken from either a living or dead transgenic animal.

In preferred embodiments, the treatment includes the adminisfration 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 particularly preferred embodiments, a MMP2 or MMP9, preferably a MMP9, promoter is a human MMP2 or MMP9, preferably a MMP9, 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 fransferase gene.

In preferred embodiments, the freatment includes the adminisfration 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 another aspect, 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, e.g., a mouse, having a reporter gene coupled to a, preferably human, neutrophil elastase promoter; administering the treatment to the transgenic animal, or to a tissue taken therefrom; and evaluating expression of the reporter gene, thereby evaluating the treatment for its effect on skin aging. The freatment, 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 dead transgenic animal.

In preferred embodiments, the transgenic animal is a non-human transgenic animal. For example, the transgenic animal can be a transgenic mini-pig, a fransgenic 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, or a transgenic mutant mouse which exhibits a phenotype of accelerated aging. The most preferred animals are mice.

In particularly preferred embodiments, the neutrophil elastase promoter is a human neutrophil elastase promoter. In a preferred embodiment the reporter is a luminescent or fluorescent product, which preferably, can lumenesce or fluoresce without the addition of exogenous substrates or cofactors, e.g., green fluorescent protein.

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 fransgenic 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 confrol value, e.g., the level of expression of the reported gene in an untreated transgenic animal.

In preferred embodiments, the method further includes administering a treatment (other than the compound) to the transgenic animal. The treatment can be one which injures or damages the skin, kills skin cells, or in general, induces an unwanted condition of the skin. The 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 compound can be determined in the presence of the treatment, and optionally compared to the response seen in the absence of the treatment. In another aspect, the invention features, a non-human transgenic animal described herein, e.g., a transgenic animal having a reporter gene coupled to a versican promoter.

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 fransgenic 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, . Am. Geriatr. 39:911-919, or a fransgenic mutant mouse which exhibits a phenotype of accelerated aging. The most preferred animals are mice. In particularly preferred embodiments, the versican promoter is a human versican promoter.

In another aspect, the invention features, a non-human fransgenic animal, e.g., a mouse, or a tissue taken therefrom, having a reporter gene coupled to a matrix metalloproteinase promoter.

In preferred embodiments, the transgenic animal is a non-human fransgenic animal. For example, the transgenic animal can be a transgenic mini-pig, a transgenic guinea-pig, a transgenic rat, or a fransgenic mouse, e.g., a hairless mouse, a nude mouse, a senescence accelerated mouse, e.g., SAM mice, or a transgenic mutant mouse which exhibits a phenotype of accelerated aging. The most preferred animals are mice.

In another aspect, the invention features, a non-human fransgenic animal, e.g., a mouse, or a tissue taken therefrom, having a reporter gene coupled to a vascular endothelial growth factor promoter.

In preferred embodiments, the fransgenic animal is a non-human transgenic animal. For example, the transgenic animal can be a transgenic mini-pig, a transgenic guinea-pig, a fransgenic rat, or a fransgenic mouse, e.g., a hairless mouse, a nude mouse, a senescence accelerated mouse, e.g., SAM mice, or a transgenic mutant mouse which exhibits a phenotype of accelerated aging. The most preferred animals are mice.

In particularly preferred embodiments, the vascular endothelial growth factor promoter is a human vascular endothelial growth factor promoter.

In another aspect, the invention features, a non-human transgenic animal, e.g., a mouse, or a tissue taken therefrom, having a reporter gene coupled to a hyaluronan synthase promoter. In preferred embodiments, the transgenic animal is a non-human transgenic animal. For example, the transgenic animal can be a fransgenic 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, or a fransgenic mutant mouse which exhibits a phenotype of accelerated aging. The most preferred animals are mice. In particularly preferred embodiments, the hyaluronan synthase promoter is a human hyaluronan synthase promoter.

In a preferred embodiment the reporter is a luminescent or fluorescent product, which preferably, can lumenesce or fluoresce without the addition of exogenous substrates or cofactors, e.g., green fluorescent protein. 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 another aspect, the invention features, a non-human transgenic animal, e.g., a mouse, or a tissue taken therefrom, having a reporter gene coupled to a Type IV collagenase promoter. In preferred embodiments, the transgenic animal is a non-human transgenic animal. For example, the fransgenic 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, or a transgenic mutant mouse which exhibits a phenotype of accelerated aging. The most preferred animals are mice. In particularly preferred embodiments, a MMP2 or MMP9, preferably a MMP9, promoter is a human a MMP2 or MMP9, preferably a MMP9, promoter.

In another aspect, the invention features, a non-human fransgenic animal, e.g., a mouse, or a tissue taken therefrom, having a reporter gene coupled to a neutrophil elastase promoter.

In preferred embodiments, the fransgenic animal is a non-human transgenic animal. For example, the fransgenic animal can be a transgenic mini-pig, a fransgenic 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, or a transgenic mutant mouse which exhibits a phenotype of accelerated aging. The most preferred animals are mice. In particularly preferred embodiments, the neutrophil elastase promoter is a human neutrophil elastase promoter.

In another aspect, the invention features a promoter-reporter gene construct described herein.

In another aspect, the invention features a method of analyzing GFP presence or distribution in a tissue. The method includes: providing a tissue sample, e.g., a tissue section, which includes GFP; evaluating or detecting fluorescent emission, or the lack of fluorescent emission, wherein said detecting step is performed prior to washing or fixing with an aqueous solution, thereby analyzing GFP in a tissue.

In preferred embodiments the tissue is frozen prior to the detection step. The sample is not contacted with a fixing agent prior to detection.

In preferred embodiments: the tissue is from a transgenic animal, e.g., a transgenic mini-pig, guinea pig, rat or mouse, e.g., a hairless or 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 GFP is expressed from a transgenic sequence encoding GFP or under the control of a fransgenic control element, e.g., a fransgenic promoter or enhancer.

In preferred embodiments the promoter is a human promoter. In preferred embodiments detection includes examination of the sample with a microscope, e.g., a fluorescent or epi-fluorescent microscope.

Animals described herein can be used in this method. In another aspect, the invention features, a method of determining the stage of the hair cycle in an animal which expresses a reporter molecule in hair follicle, e.g., the outer root sheath of the hair follicle. The method includes evaluating or detecting the presence or absence of reporter expression, presence being associated with a growing hair cycle (anagen) and absence with a resting hair cycle (telegen and catogen).

In preferred embodiments the reporter molecule is under the confrol of a promoter expressed in the hair follicle, e.g., the outer root sheath.

In preferred embodiments the promoter is a human promoter. In a preferred embodiment the reporter is a luminescent or fluorescent product, which preferably, can lumenesce or fluoresce without the addition of exogenous substrates or cofactors, e.g., green fluorescent protein.

In preferred embodiments the reporter molecule is GFP. In preferred embodiments the animal is a transgenic animal, e.g., a transgenic mini-pig, guinea pig, rat or mouse, e.g., a hairless or nude mouse, a senescence accelerated mouse, e.g., SAM mice, or a fransgenic mutant mouse which exhibits a phenotype of accelerated aging;

Animals described herein can be used in this method.

In preferred embodiments the promoter is a VEGF promoter, a versican promoter, or other promoter described herein.

In another aspect, the invention features, a method of analyzing wound healing. The method includes: providing an animal which expresses a reporter molecule under the control of a VEGF promoter, detecting the presence or absence of reporter molecule in a wound, thereby analyzing wound healing.

In preferred embodiments the detection step is reported.

In preferred embodiments, the animal, tissue from the animal, is subjected to a treatment, e.g., the administration of a compound. In such embodiments the method can be used to evaluate the effect of the treatment on wound healing. It may be desirable to compare results from a treated subject or tissue with an untreated subject or tissue.

In preferred embodiments the reporter molecule is GFP. In preferred embodiments the animal is a transgenic animal, e.g., a transgenic mini-pig, guinea pig, rat or mouse, e.g., a hairless or nude mouse, a senescence accelerated mouse, e.g., SAM mice, or a transgenic mutant mouse which exhibits a phenotype of accelerated aging. Animals described herein can be used in this method.

In preferred embodiments the promoter is a human promoter.

In another aspect, the invention features, a method of analyzing GFP expression in a fransgenic animal having a GFP fransgene. The method includes: a first step of evaluating or detecting the presence or absence of GFP in the animal or in a tissue from the animal; and (optionally) a second step of evaluating or detecting the presence or absence of GFP in the animal or in a tissue from the animal; and thereby analyzing GFP expression. In preferred embodiments (in this method and in other methods disclosed herein) the GFP is red shifted GFP.

In preferred embodiments the animal is a transgenic animal, e.g., a transgenic mini-pig, guinea pig, rat or mouse, e.g., a hairless or nude mouse, a senescence accelerated mouse, e.g., SAM mice, or a fransgenic mutant mouse which exhibits a phenotype of accelerated aging. An animal described herein can be used in this method. In preferred embodiments at least 1, 5, 10, 20, 30, or 60, 180, 365 days elapse between first and second step.

In preferred embodiments the tissue is skin tissue.

In preferred embodiments the detection steps are performed on a live animal. In preferred embodiments the promoter is a human promoter.

In another aspect, the invention features, a method of analyzing the expression of a fransgene on a transgenic animal, e.g., a fransgenic mouse or pig. The method includes: providing a live transgenic animal; evaluating or detecting the presence or absence of a reporter gene, e.g., GFP, encoded by a fransgenic sequence or under the control of a fransgenic confrol element, e.g., a promoter or enhancer; thereby analyzing the expression of a fransgene.

In preferred embodiments the animal is a fransgenic animal, e.g., a transgenic mini-pig, guinea pig, rat or mouse, e.g., a hairless or nude mouse, a senescence accelerated mouse, e.g., SAM mice, or a transgenic mutant mouse which exhibits a phenotype of accelerated aging. Animals described herein can be used in the methods.

In preferred embodiments the reporter is under the control of a VEGF promoter or another promoter described herein.

In preferred embodiments the promoter is a human promoter.

In preferred embodiments the promoter is one which is expressed on the skin. In preferred embodiments evaluating includes detection of a signal, e.g., a fluorescent signal, with a confocal microscope.

In preferred embodiments the tissue is skin tissue.

In preferred embodiments the detection steps are performed on a live animal.

In preferred embodiments the method is repeated at least once during the life of the animal. The first and a subsequent repetition of the method can be separated by as much as 1, 10, 30, 60, 90, 180, 365, or 700 days.

In another aspect, the invention features, a method of evaluating gene expression in a live animal. The method includes: providing a live transgenic animal having a fransgene which includes a reporter gene, e.g., GFP, e.g., red shifted GFP; evaluating or detecting the presence or absence of reporter molecule, e.g., GFP, in the live fransgenic animal, thereby evaluating gene expression in the live animal.

In preferred embodiments, the sequence which encodes a reporter gene is under the control of a preselected promoter, e.g., a human promoter. The preselected promoter can be, a skin metabolism-related promoter, e.g., : a promoter from a gene which encodes a fransmembrane 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. The VEGF promoter is a preferred promoter. In preferred embodiments the promoter is one which is up or down regulated in inflammatory angiogenesis, or neoplastic growth.

In preferred embodiments, the animal is a non-human transgenic animal. For example, the transgenic animal can be a fransgenic mini-pig, a fransgenic 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, or a transgenic mutant mouse which exhibits a phenotype of accelerated aging. The most preferred animals are mice.

In preferred embodiments, a treatment is administered to the animal any of before, during, or after valuation of reporter gene expression. The freatment can be the adminisfration of a compound. The compound can be 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 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 a preferred embodiment the reporter is a luminescent or fluorescent product, which preferably, can lumenesce or fluoresce without the addition of exogenous substrates or cofactors, e.g., green fluorescent protein.

Methods of the invention can be performed in vivo, with whole animals, or in vitro, that is, with tissue, e.g., skin, or cells, which are derived from a fransgenic animal described herein or with cells, preferably skin cells or tissue, from cells transformed with a skin-metabolism promoter/reporter gene construct.

As used herein, a "fransgenic animal" is an animal, e.g., a non-human mammal, e.g., a mini-pig, a guinea-pig, or a rodent, e.g., a mouse or a rat, in which one or more, and preferably essentially all, of the cells of the animal include a fransgene. The fransgene can be introduced into the cell, directly or indirectly by introduction into a precursor of the cell, e.g., by microinjection, transfection or infection, e.g., by infection with a recombinant virus. The term genetic manipulation is directed to the introduction of a recombinant DNA molecule. This molecule may be integrated within a chromosome, or it may be extrachromosomally replicating DNA.

Transgenic animals can be, e.g., heterozygous or homozygous for a transgene. As used herein, the term "rodent" refers to all members of the phylogenetic order Rodentia.

As used herein, the term "reporter gene" refers to a nucleic acid sequence which is fused downstream of a skin metabolism-related promoter, such that its expression is under the confrol of the promoter. Reporter genes usually encode a protein whose activity can be easily measured. For example, the reporter gene can be a gene encoding an assayable protein, e.g., an enzyme, not found in the cell in nature, e.g., a beta- galactosidase gene, a luciferase gene, a green fluorescent protein gene, an alkaline phosphatase gene, a horseradish peroxidase gene, a chloramphenicol acetyl transferase gene, luciferase, and the like. In a preferred embodiment the reporter product is capable of providing a signal which indicates the activity of the promoter to which it is linked. Preferred reporters are those which luminesce or fluoresce. Preferred reporters can luminesce or fluoresce, in vivo, without the addition of an exogenous substrate. A particularly suitable reporter is green fluorescent protein. Modified variants of green fluorescent protein, e.g., EGFP, EBFP, EYFP, d2EGFP, ECFP, GFPuv are included within the term green fluorescent protein. EGFP is particularly preferred. These variants of GFP are commercially available by CIONTECH, Laboratories, Inc. Palo Alto, CA. Furthermore, GFP and variants thereof, are provided in the following references, all of which are incorporated by referenced: Chalfie, M. et al. (1994) Science 263:802-805; Prasher, D.C., et al. (1992) Gene 111 :229-233; Inouye, S. & Tsuji, F.I. (1994) FEBS Letters 341 :277-280; Wang, S. & Hazelrigg, T. (1994) Nature 369:400-403; Cody, C. W., et al. (1993) Biochemistry 32:1212-1218; Inouye, S. & Tsuji, F. I. (1994) FEBS Letters 351 :211-214; Heim, R., et al. (1994) Proc. Natl. Acad. Sci., USA 91:12501-12504; Yang, T.T., et al. (1996) Nucleic Acids Res. 24(22): 4592-4593; Cormack, B.P., et al. (1996) Gene 173:33-38; Crameri, A., et al. (1996) Nature Biotechnol. 12:315-319; Haas, J. et al, (1996) Curr. Biol. 6:315-324; Galbraith, D.W., et al. (1995) Methods Cell Biol. 50: 1-12; Living Colors Destabilized EGFP Vectors (April 1998) CLONTECHniques XIII(2):16- 17, Living Colors pEBFP Vector (April 1997) CLONTECHniques XII(2): 16-17; Heim, R. & Tsien, R.Y. (1996) Curr. Biol. 6: 178-182; Ormδ, et al. (1996) Science 273:1392- 1395; Mitra, R.D. et al. (1996) Gene 173:13-17.

As used herein, the term "skin metabolism-related promoter" refers to a promoter which is franscriptionally active in the skin. It need not be skin-specific. The gene in which the promoter is naturally found can be a gene involved in the maintenance, or proper functioning of the skin. For example, the gene can be a gene encoding a protein which is part of the extracellular matrix, a protein involved in the maintenance or degradation of the extracellular matrix, or a protein involved in supplying nutrition to the skin. Active fragments or analogs of the promoters mentioned herein can be used in methods, compositions, and animals of the invention, e.g. an active fragment of the veriscan promoter. The 839 bp fragment of the functional human veriscan promoter (- 559 to +280) (Naso, M.F. , Zimmermann, D.R. & Iozzo, R.V. (1994) J. Biol. Chem., 269, 32999-33008) human genomic DNA can be used in methods which use a veriscan promoter. Embodiments of the inventions allow for evaluation of transgene expression, in situ, on live animals. The luminescent or fluorescent reporters, e.g., GFP reporter molecule, are particularly advantageous in such embodiments.

As used herein, "administering a compound to an animal" refers to dispensing, delivering or applying a treatment to an animal or cell. Administration can be by topical administration, by parenteral or oral route, intramuscular injection, subcutaneous/intradermal injection, intravenous injection, buccal adminisfration, transdermal delivery or adminisfration by the infranasal or respiratory tract route. The most preferred administrations are topical application or subcutaneous or intradermal injection. The methods of the invention allow rapid and efficient evaluation of compounds for their effect on skin.

Other features and advantages of the invention will be apparent from the following detailed description, and from the claims. Detailed Description

The drawings are first briefly described.

Brief Description of the Drawings

Figure 1 is a schematic representation of the human versican-Lac Z fransgene construction.

Figure 2 is a schematic representation of the vascular endothelial growth factor- green fluorescent protein [VEGF-GFP] transgenic construct.

Figure 3 is a depiction of the nucleotide sequence of the 5069 pb MMP9 promoter-GFP construct.

Figure 4 is a depiction of the nucleotide sequence of the 7383 pb MMP9 promoter-beta-Gal construct.

Promoters Methods of the invention allow evaluating a compound for its effect on the skin.

Methods of the invention can be used to evaluate a compound for its effect on the health or appearance of the skin, e.g., for use as a cosmetic. The effect on skin is usually determined as an effect on the expression of a gene under the control of a skin- metabolism-related promoter. Such promoters include those which confrol the expression of: a product which is a component of the skin, e.g., the dermis or epidermis; a product which affects hydration or nutrition of the skin; a product which promotes the synthesis, or degradation, of components of the skin; a product which affects the vasculature of the skin; a product which affects hair follicle metabolism; a product which affects skin glandular structures; a product which affects subcutaneous musculature; a product which affects adipose tissue; or a product which affects cutaneous nerves.

Methods of the invention are useful for evaluating a compound for an effect on a parameter related to the appearance or health of the skin, for example, the elasticity of the skin, the propensity of the skin to wrinkle, the ability of the skin to retain fluids, e.g., water or an oil, the ability of the skin to resist or repair damage, e.g., light or UV induced damage, the metabolism of hair follicles including growth cycling or pigment deposition, subcutaneous muscle tone and function, or neurotransmission by cutaneous nerves. Generally, effects on these parameters will be evaluated indirectly, e.g., by the effect on the expression of a reporter gene under the control of a promoter which is normally coupled to a gene which encodes a product which affects any of the these parameters.

Examples of such skin-metabolism-related promoters include a versican promoter; a matrix metalloproteinase promoter; a vascular endothelial growth factor promoter; a hyaluronan synthase promoter; a MMP2 or MMP9, preferably a MMP9, promoter; and a neutrophil elastase promoter. The versican promoter regulates the expression of the versican gene, described in

Naso M. F. et al. (1994) J. Biol. Chem. 269(52): 32999-33008, the contents of which are incorporated herein by reference. Versican is a large modular chondroitin sulfate proteoglycan expressed in the dermis and the epidermis of the skin. Versican can bind large amounts of water while remaining attached to the extracellular matrix and can, therefore, hydrate and fill the skin. Accordingly, compounds which result in upregulation of this gene are preferred.

A matrix metalloproteinase promoter regulates the expression of a matrix metalloproteinase gene. The matrix metalloproteinases (MMPs) belong to a family of extracellular matrix proteases, described in Mauch C. et al. (1994) Arch. Dermatol. Res. 287: 107-114. As the name implies, these matrix metalloproteinases are involved in the degradation of the extracellular matrix in, for example, the dermis and the epidermis of the skin. Accordingly, compounds which result in downregulation of this gene are preferred.

The vascular endothelial growth factor promoter regulates the expression of the vascular endothelial growth factor gene described in, for example, Tischer E. (1991) J. Biol. Chem. 266(18): 11947-11954, the contents of which are incorporated herein by reference. The vascular endothelial growth factor is a mitogen for vascular endothelial cells and, as a result, it can lead to proliferation of the microvasculature beneath the skin and increased vascular permeability. Increased microvasculature and vascular permeability allow for better nutrition (e.g., better nutrient delivery to the dermis and the epidermis) and hydration of the skin. Accordingly, compounds which result in upregulation of this gene are preferred.

The hyaluronan synthase (HAS 1-3) promoters regulate the expression of the hyaluronan synthase (HAS1-3) genes, described in Itano N. et al. (1996) BBRC 222: 816-820; Watanabe K. (1996) J. Biol. Chem. 271(38): 22945-22948; and Spicer A. P. (1997) J. Biol. Chem. 272(14): 8957-8961, respectively, the contents of which are incorporated herein by reference. Hyaluronan synthases, as the name implies, are enzymes involved in the synthesis of hyaluronan, a linear unbranched glycosaminoglycan. Hyaluronan binds versican and generally acts as an anchor for other proteoglycans, leading to the stabilization of the proteoglycan "network." As a result, hyaluronan (like versican) can assist in the hydration and filling of the skin. Accordingly, compounds which result in upregulation of this gene are preferred.

The type IV collagenase promoter regulates the expression of the type IV collagenase gene, described in Huhtala P. (1991) J. Biol. Chem. 266(25): 16485-16490, the contents of which are incorporated herein by reference. Type IV collagenase is another member of the extracellular matrix protease family and it is involved in the degradation of various components of the extracellular matrix in, for example, the dermis and the epidermis of the skin. Accordingly, compounds which result in downgulation of this gene are preferred.

The neutrophil elastase promoter regulates the expression of the neutrophil elastase gene, described in Takahashi H. (1988) J. Biol. Chem. 263(29): 14739-14747, the contents of which are incorporated herein by reference. Neutrophil elastase is a powerful serine protease capable of cleaving most protein components of the extracellular matrix (including elastin) in, for example, the dermis and the epidermis of the skin. Accordingly, compounds which result in downgulation of this gene are preferred.

Transgenic animals

Transgenic animals which can be used in the methods of the invention include non-human mammals, such as pigs, e.g., mini-pigs, or guinea-pigs; or rodents, e.g., mice or rats, e.g., hairless mice (described in, for example, Begona M. et al. (1994) Proc. Natl. Acad. Sci. 91 :7717-7721), nude mice, senescence accelerated mice (described in, for example, Takeda et al. (1991) L. Am. Geriatr. Soc. 39:911-19), or transgenic mutant mice which exhibit a phenotype of accelerated aging; in which one or more, and preferably essentially all, of the cells of the animal include a transgene. The transgenic animals can be homozygous or heterozygous for the fransgene. Mice are a preferred subject animal.

Construction of Transgenic Animals Methods of making transgenic animals, e.g., mice, are known in the art. One approach is described below.

Injection/Implantation of Embryos Procedures for embryo manipulation and microinjection are described in, for example, Manipulating the Mouse Embryo (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY., 1986, the contents of which are incorporated herein by reference). Mouse zygotes can be collected from six week old females that have been super ovulated with pregnant mares serum (PMS) followed 48 hours later with human chorionic gonadofropin. Primed females are placed with males and checked for vaginal plugs on the following morning. Pseudo pregnant females are selected for estrus, placed with proved sterile vasectomized males and used as recipients. Zygotes are collected and cumulus cells removed. Furthermore, blastocytes can be harvested. Pronuclear embryos are recovered from female mice mated to males. Females are treated with pregnant mare serum, PMS, to induce follicular growth and human chorionic gonadofropin, hCG, to induce ovulation. Embryos are recovered in a Dulbecco's modified phosphate buffered saline (DPBS) and maintained in Dulbecco's modified essential medium (DMEM) supplemented with 10% fetal bovine serum.

Microinjection of a fransgenic construct can be performed using standard micro manipulators attached to a microscope. For instance, embryos are typically held in 100 microliter drops of DPBS under oil while being microinjected. DNA solution is microinjected into the male pronucleus. Successful injection is monitored by swelling of the pronucleus. Recombinant ES cells can be injected into blastocytes, using similar techniques. Immediately after injection embryos are transferred to recipient females, e.g. mature mice mated to vasectomized male mice. In a general protocol, recipient females are anesthetized, paralumbar incisions are made to expose the oviducts, and the embryos are transformed into the ampullary region of the oviducts. The body wall is sutured and the skin closed with wound clips.

Screening for the Presence of the Targeting Construct

Transgenic animals can be identified after birth by standard protocols. DNA from tail tissue can be screened for the presence of the targeting construct using southern blots and/or PCR. Offspring that appear to be mosaics are then crossed to each other if they are believed to carry the targeting construct in their germ line to generate homozygous fransgenic animals. If it is unclear whether the offspring will have germ line transmission, they can be crossed with a parental or other strain and the offspring screened for heterozygosity. The heterozygotes are identified by southern blots and/or PCR amplification of the DNA. The heterozygotes can then be crossed with each other to generate homozygous fransgenic offspring. Homozygotes may be identified by southern blotting of equivalent amounts of genomic DNA from mice that are the product of this cross, as well as mice that are known heterozygotes and wild type mice. Probes to screen the southern blots can be designed as set forth above. Other means of identifying and characterizing the transgenic offspring are known in the art. For example, northern blots can be used to probe the mRNA for the presence or absence of transcripts encoding the reporter gene. In addition, western blots can be used to assess the level of expression of the fransgene in various tissues of these offspring by probing the western blot with an antibody against the protein encoded by the fransgene, or an antibody against the marker gene product, where this gene is expressed. Finally, in situ analysis (such as fixing the cells and labeling with antibody) and/or FACS (fluorescence activated cell sorting) analysis of various cells from the offspring can be performed using suitable antibodies to look for the presence or absence of the transgene product.

Other Transgenic Animals

Other transgenic animals can be used in methods of the invention. Methods for the preparation of a variety of animals are known in the art. A protocol for the production of a transgenic pig can be found in White and Yannoutsos, Current Topics in Complement Research: 64th Forum in Immunology, pp. 88-94; US Patent No. 5,523,226; US Patent No. 5,573,933; PCT Application WO93/25071; and PCT Application WO95/04744. A protocol for the production of a fransgenic rat can be found in Bader and Ganten, Clinical and Experimental Pharmacology and Physiology, Supp. 3:S81-S87, 1996. A protocol for the production of a transgenic cow can be found in Transgenic Animal Technology, A Handbook, 1994, ed., Carl A. Pmkert, Academic Press, Inc. A protocol for the production of a transgenic sheep can be found in Transgenic Animal Technology, A Handbook, 1994, ed., Carl A. Pinkert, Academic Press, Inc. All patents and references are incorporated herein by reference. Reporter Genes

The methods of the invention are based, at least in part, on coupling reporter genes to promoters of genes involved in skin metabolism. The reporter gene can be any gene which encodes a detectable product, preferably one which can be detected with relative ease, e.g., a gene product which is fluorescent, or which catalyzes a reaction which can be determined by formation of a colored product. For example, the reporter gene can encode an enzyme, e.g., an enzyme which produces a detectable product, e.g., a colored or a luminescent product. Reporter genes are known in the art and include a beta- galactosidase gene, a luciferase gene, a green fluorescent protein gene, an alkaline phosphatase gene, a horseradish peroxidase gene, or a chloramphenicol acetyl fransferase gene. Reporter genes are described in, for example, Sambrook, J., Fritsh, E. F., and Maniatis, T. Molecular Cloning: A Laboratory Manual. 2nd, ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989.

Examples

Example 1: Construction of the Transgene

To construct the fransgene, an 839 bp fragment of the human versican promoter (nucleotides -559 to +280; the transcription start site being +1) was obtained from human genomic DNA using art known PCR-based techniques. The versican promoter fragment was inserted into the pNASS2β vector (Clontech) which was linearized by restriction with Xho\-EcoR\ as shown in Figure 1. This vector contains the β-galactosidase reporter gene (lacZ) as well as a polyadenylation signal. This vector also includes an RNA splice donor and acceptor sequence to optimize the chance of high level of fransgene expression. To obtain the linearized transgene DNA fragment for pronuclear injection, the vector was cleaved with an EcoRλ and an Xbal enzyme. The linearized fransgene DNA fragment has a length of 4732 bp.

Example 2: Generation of Transgenic Mice The linearized fransgene DNA fragment was injected into fertilized oocytes of

DBA2 x C57BL6 (DBF1) mice (Charles River, Boston), and the eggs were implanted into pseudopregnant foster mothers. The offsprings (F0) were tested for chromosome integration of the human versican promoter fragment by southern blotting. Briefly, genomic DNA was isolated from the tails of 3-week old mice and digested with either the BamHl or the Psil restriction endonuclease. The DNA fragments were separated on a gel and then transferred on a nylon membrane. The membranes were hybridized with a 1.3 BP BamHl-EcoRV fragment of the human versican transgene which was used to generate the transgenic mice. Seven independent lines showed transgene insertion by Southern analysis. The copy number of the fransgene varied from one to more than ten.

Example 3: β-galactosidase Histochemistry

Collected mouse embryos of varying stages (from El 1.5d to E17.5d) of development were fixed with 0.5% glutaraldehyde in PBS for 30 minutes to 12 hours, depending on the embryo stage, transferred to 30% sucrose, and then frozen in OCT (Tissue Tek, CA) compound. 15μm-thick sections were prepared and loaded on slides. The sections were re-fixed in same fixative for 5 minutes and washed once with PBS, followed by a detergent wash solution for 10 minutes. Staining was performed in the reaction buffer containing 1 mg/ml X-gal (Sigma), 10 mM ferrocyanide, and 10 mM ferricyanide. Incubation was curried out for 3-6 hours at 37øC. After washing with PBS, sections were either mounted or counterstained with eosin. For adult tissue, mice were perfused with the same fixative and the desired organs were dissected and post-fixed, sucrose submerged, and then frozen with OCT compound. For skin tissue, 5 mm strips of back skin tissue block were fixed for 15 minutes to an hour and stained with X-gal, as described above for 3-6 hours. After washing, the skin tissue strips were embedded in paraffin and cut in 8 μm sections using a microtome.

Example 4: In situ hybridization

Radioactive in situ hybridization with 3' end labeled oligonucleotide probes was performed using methods known in the art. The specificity of the in situ signal was tested by hybridizing some sections with labeled oligonucleotides in the presence of excess unlabelled oligonucleotide.

The expression pattern of the transgene was examined by in situ hybridization of transgenic embryonic sections (E13.5) using lacZ and endogenous mouse versican probes. Using either probe, expression was observed in the developing limb bud, kidney, brain, and cartilage. LacZ expression was also examined by β-galactosidase histochemistry in E13.5, E15.5, E17.5 embryos, through 7-day, 40-day newborns, and 4 month (adult) transgenic mice sections. Strong mesenchymal expression was observed in the kidney, brain, cartilage, and limb bud, as early as E13.5d. The level of expression remained constant until birth. In 7-day old mice, the β-galactosidase staining was decreased compared to embryonic tissue and almost no expression was observed in adult tissue. In contrast to other tissue, dermal papilla (anagen hair cycle) from 30-day old mice exhibited intense β-galactosidase staining again and continued to express the same level of β-galactosidase during the second hair cycle.

In skin tissue, hair bud (future dermal papilla in hair follicle) expressed strong β- galactosidase activity at El 5.5 d and continued to do so until 7 days after birth (during the first hair cycle). Occasional β-galactosidase staining in dermal fibroblasts was also observed in E15.5d -newborn skin. However, β-galactosidase staining was decreased in 7 day-old skin tissue.

Example 5: UV irradiation

Irradiation with UVA was performed using a closely spaced array of five PUVA lamps. The energy output at 30 cm from array was measured with a UVA detector. 4-day old versican fransgenic mice were irradiated on back skin with 30 J/cm2

UVA. Back skin tissue biopsy samples were processed 24 hours later for β-galactosidase staining and compared to untreated confrol skin from fransgenic mice, β-galactosidase histochemistry showed increased β-galactosidase staining on the upper dermis of UVA irradiated skin compared to control.

Example 6: VEGF-GFP Transgenic Mice

To elucidate the regulatory mechanisms of human VEGF gene expression in vivo, transgenic mice have been prepared which contain a green fluorescent protein reporter construct driven by a 3kbp fragment of the promoter region of human VEGF (shown in Figure 2). This construct was confirmed to be functional in vitro by transfection assays in cultured human keratinocytes. Three independent transgenic lines for VEGF-GFP were obtained and confirmed by PCR analysis. To examine constitutive endothelial expression, and keratinocyte inducible VEGF expression in wound skin, 2 mm skin punches were made in the back skin and biopsy samples were collected after 48 hours. Tissues were immediately fixed with 4% paraformaldehyde and cryostat sections were examined by fluorescent and confocal microscopy. VEGF-GFP fransgenic lines exhibited bright GFP fluorescence in neomicrovascular networks beneath the epidermis and upper dermis, and occasionally in the deeper dermis. Hair follicles were also positive in some tissue. In wound tissue, continuous GFP expression was observed at the wound edge of the epidermis, but no keratinocyte expression was seen in non-wounded areas, confirming the inducibility of VEGF expression in healing wounds. Primary keratinocyte cultures prepared from the VEGF-GFP transgenic line also showed GFP fluorescence after 3 days in culture. Taken together these results indicate that GFP is a potentially useful tool to examine in vitro and in vivo VEGF expression. This work is described in more detail below.

Dynamic changes in the expression of green fluorescent protein driven by the human vascular endothelial growth factor promoter in fransgenic mouse skin was analysed as follows.. Gene expression studies using transgenic mice most often utilize one of three classical reporter genes— lacZ (b-galactosidase) luciferase, or CAT (Chloramphenicol acetyl transferase). Detection of reporter gene activity usually requires the tissue to be removed from the animal for histochemical staining in the case of lacZ, or for complicated assays for luciferase and CAT, eliminating or at the least complicating the possibility of seeing a change in gene regulation in the living animal.

Recently an alternative fluorescent reporter molecule, green fluorescent protein (GFP), (Chalfee M, Tu Y, Euskirchen G, Ward WW and Prasher DC: Green fluorescent protein as a marker for gene expression. Science 263: 802-5, 1994) or modified GFP (EGFP), (Zhang G. Gurtu V and Kain SR: An enhanced green fluorescent protein allows sensitive detection of gene fransfer in mammalian cells. Biochem Biophys Res Commun 227: 707-11, 1996) has been developed. Because GFP is intrinsically fluorescent, the signal is visible without treatment (Misteli T and Spector DL: Applications of the green fluorescent protein in cell biology and biotechnology. Nat Biotechnol 15: 961-4, 1997). When GFP is induced in the epidermis, it is possible to observe changes in gene expression without sacrificing the animals using confocal laser microscopy. The promoter of human vascular endothelial growth factor (VEGF), which is an angiogenic factor that induces in vivo angiogenesis and vascular permeability in malignant tumor tissue is used here to express GFP as a reporter gene in transgenic mouse skin. The expression of VEGF in epidermal keratinocytes was shown to be up-regulated at the edge of a healing epidermal wound, and also up-regulated topical application of phorbolesters, such as TPA.

The expression and responsiveness of GTP in signaling changes in gene activity of human VEGF-GFP in fransgenic mice is described below. The expression patterns are consistent with that of endogenous VEGF, and show that GFP-derived fluorescent can be localized and visualized using confocal microscopy on intact tissue without any treatment following excisions.

Transgene construct Because PCR-based amplification of the GC-rish VEGF promoter is often difficult, a 5.0 kb fragment of VEGF genomic clone, which included the 5'-flanking DNA of human VEGF, was isolated from a human genomic library (Clontech, CA) using 500 bp cDNA fragment obtained by RT-PCR as a probe. The identified close was analyzed by restriction digestion, then a 2453 bp EcoRI-Agel fragment (-2271 to +91) (Tischer E, Mitchell R, Harman T, Silva M, Gospodarowicz D, Fiddes JC and Abraham JA: The human gene for vascular endothelial growth factor. Multiple protein forms are encoded through alternative exon splicing. J Biol Chem 266: 11947-54, 1991) was excised from the agarose gell. This promoter fragment was inserted into a GFP vector (pEGFP-1 cut EcoRI and Xmal). To maximize the efficiency of expression, 16s/19s splice donor and acceptor signals were also inserted between the promoter and GFP gene.

In vitro expression of GFP vector.

The resultant transgene in the mammalian expression vector was first fransfected into cultured human keratinocytes to confirm that the selected gene region promoted VEGF expression. Primary human keratinocytes were fransfected with either the BEGF- GFP vector or a CMV promoter-driven GFP confrol vector (pEGFP-Nl; Clontech). A porybrene and DMSO method was used for all fransfections.

Generation of Transgenic Mice The linearized fragment for pronuclear injection was excised with EcoRI and

Aflll, which also included a polyadenylation signal sequence. This fransgene fragment was then injected into fertilized oocytes of DBA2xC57B16 (DBF1) mice (Charles River Laboratories, Boston, MA), and the eggs were implanted into pseudo pregnant foster mothers. The offspring (F0) were tested for chromosomal integration of the fransgene by genome PCR. All experiments were done with Fl or F2 offspring mice.

For confocal microscopic observation of intact skin tissue, VEGF-GFP transgenic mice were generated in the hairless genetic background by breeding fransgenic founder with SKH-1 hairless mice (Charles River Laboratories). Thus, male hemizygous fransgenic mice (v/-HH) were mated with female hairless mice (-/-hh) to obtain the F2 generation of VEGF-GFP hairless mice (v/-hh), which could be easily selected by PCR detection and their hairless phenotype.

Tissue preparation for GFP detection and epimicroscopic observation solutions washed out or diffused GFP-derived fluorescence from the site of GFP expression. Ultimately, a freshly dissected, unfixed tissue block, was directly cut into 12 μm thick frozen sections using a cryostat. Epifluorescent microscopic observation was performed immediately after sectioning, without mounting medium or extended slide storage, using FITC/TRITC double excitation and an emission filter Chroma, *VT) to distinguish the GFP-derived fluorescent signal from tissue autofluorescence.

Wounding and TPA treatment procedure

Skin wounds were produced with a 2 mm biopsy punch on the shaved back skin of young adult fransgenic mice aged 4-6 weeks old. After 48 hours, normal and wounded tissues were collected and immediately frozen on dry ice. For induction of VEGF mRNA by TPA, a single dose of 5 μg TPA in acetone was topically applied to back skin of transgenic mice and skin biopsy samples were collected after 12 hours.

All animal procedures had the approval of the MGH Animal Care and Use

Committee.

Anti-VEGF and anti-GFP immunohistochemistry

Cryostate sections of transgenic mice skin were fixed with 4% paraformaldehyde in PBS (pH 7.2) at room temperature for 5 min. Immunohistochemical staining was performed on slide mount sections using the ABC method as described by the manufacturer (Vector Labs. UK), and visualized by peroxidase reaction using DAB as a color substrate. Anti-GFP antiserum was purchased from Clontech.

Confocal microscopy

For confocal microscopy observations, a coverslip was placed directly on the tissue. Sections or intact skin samples were analyzed using Leica TCS NT4D confocal microscope system with band pass filter 530/30nm, detecting emission at wavelengths between 515-545 nm. To visualize nuclei, sections were counterstained with 5μg/ml of 7-aminoactinomycin D (SIGMA, USA) solution for 20 min at ambient temperature, washed with PBS, and mounted with fluoromount-G (Southern Biotechnology, AL). VEGF promoter driven GFP expression in human keratinocytes

As the region of functional promoter activity for endogenous VEGF was not well characterized, 2.5 kb of 5'-flanking DNA of human VEGF was first inserted in a GFP mammalian vector and monitored in fransfected primary human keratinocyte cultures. Control cultures were transfected with a CMV promoter-driven GFP vector, VEGF-GFP fluorescence was first visible at 48 hours after transfection, and only in a small percentage of the cells. CMV-GFP fluorescence was visible within 24 hours, and nearly all the cells were positive. The VEGF-GFP signal was similar in intensity to that of CMV-GFP confrol confirming that this construct is a functional in in vitro culture system.

(Fluorescence in cultured keratinocytes fransfected with human cytomegalovirus (CMV) immediate early promoter driven GFP as a positive confrol, and with VEGF-GFP, 48 hours after transfection were observed.)

Expression of GFP in transgenic mice

In 30 founder mice, four lines positive for transgene chromosomal insertion were detected by PCR analysis. Three of these lines showed detectable GFP-derived fluorescent signal in the epidermis as assessed by epifluorescence microscopy of tail tissue, compared with negative wild type mice. GFP expression in new born fransgenic mouse (Fluorescence was determined in fresh, unfixed tissue from the tail from a VEGF- GFP fransgenic mouse and a similar tissue from a.negative wild-type litter mate. Fluorescence was also determined in tissues taken from new born VEGF-GFP fransgenic mice: including lung (fluorescence seem in alveoli); lateral ventricle of the brain, (fluorescence was observed in lateral epithelium); and vertebral cartilage; (heart epithelium). The expression pattern of VEGF promoter was evaluated in Fl newborn pups of these three lines. The lung, kidney, and brain, previously been shown to express VEGF by in situ hybridization studies were chosen for examined for GFP fluorescence. As expected, GFP expression in lung alveoli and in the lateral ventricle wall in the brain was observed. It was difficult to detect a GFP signal in the flomerulus of kidney. GFP fluorescence was also detectable in the chondrocytes of developing cartilage tissue (e.g. vertebra cartilage).

GFP is expressed at the edge of healing wounds Since non-stimulated, normal epidermal keratinocytes showed only weak GFP fluorescence, we tested the inducibility of VEGF-driven GFP during wound healing. 48 hrs after removal of a skin biopsy, the healing wound epidermis beneath the scab showed a very strong and distinct fluorescent signal compared with adjacent unwounded epidermis. This fluorescent signal correlated with VEGF/GFP expression detected by anti-GFP and anti-VEGF immunohistochemistry in adjacent sections. GFP expression induced by skin wound healing. (VEGF-GFP transgenic mice were wounded by punch biopsy. Tissue from the wound was taken for evaluation by fluorescence microscopy, (a) In healing wound edge 48 hours after biopsy strong fluorescence was observed in the epithelium underlying the scab, (b) An adjacent section was reacted with anti-GFP antibodies, (c) An adjacent section was immunostained with antimouse VEGF antibodies. A confrol section was treated with normal goat serum. Non-specific peroxidase staining observed in the scabs as also seen in b and c.) Induction of GFP by TPA treatment Induction of epidermal GFP by phorbolester application was also examined.

Accumulation of fluorescent signal was observed 12 hours after TPA was applied to transgenic skin, whereas acetone alone showed only basal levels of GFP expression. This signal seemed to localize to the basoplateral surface of the epithelium rather than in either the keratinocyt cytosol or nucleus. Anti-GFP immunostaining produced a signal in the basal cell cytosol, indicating that GFP originated in basal cells, and at least part of the GFP protein remained within the cell. Direct detection of GFP by confocal microscopy

In order to localize the GFP fluorescence signal more precisely, fresh cryostat sections were fixed and incubated with 7-AAD for nuclear counter staining and examined under laser confocal microscope. As expected, most diffusable GFP was lost during this procedure, although wound healing-induced-epidermal expression remained unaffected. Occasional fluorescence was detectable in the unwounded epidermis, where the fluorescence appeared to localize outside basal cells as we observed. Even after fixation and intense washing, GFP-derived fluorescence in the outer root sheath of hair follicle remained unchanged. At higher magnification of the hair follicles, it can be seen that the fluorescent GFP signal completely co-localized with a nuclear maker, indicating that hair follicle GFP remains stably within the nucleus. This was also the case for the chonodrocytes in the vertebrae cartilage. Direct detection of GFP in the epidermis of intact skin by confocal microscopy

To test the possibility that GFP might be detected in the skin of a living GFP transgenic mouse, dissected hairless skin was directly examined by confocal imaging. Horizontal optical sections of the living epidermis revealed bright fluorescence. The equivalent cryostate section showed that this fluorescence is derived from the basal cells. Higher magnification revealed that GFP was localized in the extracellular space around the keratinocytes. X-Z confocal imaging confirmed that this signal was within the basal layer of the epidermis. Detection of GFP expression in intact skin by confocal microscopy. Fress unfixed transgenic mouse skin was biopsied, and immediately evaluated by scanning laser confocal microscopy. Horizontal optical sections of the epidermis show fluorescence surrounding individual epithelial cells. Cryostate sections of an equivalent skin region counterstained with 7-AAD to indicate nuclei, showed fluorescence at the dermal-epidermal junction. The confocal X-Z image indicates the fluorescence near the basal keratinocytes.

This work shows that the expression pattern of GFP driven by a human VEGF promotor is spatially and temporally equivalent to that seen with mRNA detection.

Pups from breedings of transgenic mice expressing GFP in the skin epidermis were easily selected by simple epifluorescent microscopy, without the more tedious procedures of southern blotting or genomic PCR.

There was initial difficult in detecting GFP-derived fluorescence in sections, even though fresh untreated tissue blocks emitted bright fluorescence. After testing a variety of procedures, it was found that aqueous solution freatment immediately washed out the GFP flourescence. This was partially overcome by fixing the tissue block before cryostate sectioning, though fluorescence from some sites, such as that initially present in epidermal keratinocytes, was still lost or its staining pattern was altered. Fixation induced fluorescence indistinguishable from that produced by GFP in the skin microvasculature. Fluorescence at this site was not seen in unfixed tissue, and VEGF expression has not been reported in the microvasculature in studies using in situ hybridization methods. Thus, the most efficient method to preserve the original GFP signal is to immediately cryostat section freshly dissected skin without OCT compound, and then promptly examine it by fluorescence microscopy. This unexpected difficulty may explain why, until now, there have been so few reports of successful GFP transgenic mice. Prominent VEGF expression has been reported in lung, and in the lateral ventricle of the brain by in situ hybridization. Evaluation of the VRGF-GFP mice tissues confirm these findings. Failure of the kidney glomeruli to fluoresce may be due to a lack of tissue specific response element to 2.2 kb of 5'-flanking region of VEGF DNA used here for generating these fransgenic mice, or to a signal below the detection threshold.

In skin from a 1 week old pup, normal epidermal keratinocyte showed basal levels of GFP expression, while bright fluorescence was observed in the outer root sheath of hair follicle. The skin of older mice did not show equivalent GFP expression in hair follicles, therefore, VEGF expression in the follicles may be hair cycle-dependent. Fluorescence in hair follicle and in chondrocytes, as seen in the vertebra and previously reported was particularly impressive. These signals were even stronger than those observed in lung or brain. Laser confocal observations revealed that GFP at these locations is confined within the nuclei. This nuclear localization may intensify the apparent signal since nuclear localization is likely to reduce the diffusion of the fluorescent product during tissue processing. The reason for localization to the nucleas by some tissues and not by others remains unexplained, but suggests that inclusion of a nuclear localization conseneus sequence with in the fransgene might be useful.

The inducibility of GFP protein during wound healing model and after TPA freatment is shown herein, confirming that the reporter GFP expression pattern is highly VEGF promoter-dependent. Also, these data eliminated concerns about the possible delay in fluorescent signal production due to slow chromophore formation as reported in Drosophila embryos. (Davis I, Girdham CH, O'Farrell PH "A nuclear GFP that marks nuclei in living Drosophila embryos; maternal supply overcomes a delay in the appearance of zygotic fluorescence" Dev. Biol. 170:726-9 (1995)). In anticipation of this difficulty the red-shifted variant of GFP was selected for these studies, and the correct folding of the chromophore appears to occur in skin as well as in more internal tissues. There was also concern that GFP might have a prolonged half life in skin interfering with the ability to use the reporter to monitor long-term gene expression. The disappearance of fluorescence in hair follicles of older fransgenic mouse skin indicates that GFP protein has a reasonable half life.

Laser confocal microscopic evaluation of intact skin for detection of the GFP signal indicates that GFP is readily detected within the epidermis. These results suggest the potential use of GFP transgenic mice for non-invasive monitoring of long-term gene expression in vivo. The technology for microscopic evaluation of living mouse skin is already in use. When combined with use of a transgenic GFP reporter gene, the system has a great advantage over conventional transgenic reporter gene animals methods because it reduces the number of animals used, utilizes a simple monitoring system, and allows long term monitoring of changes in gene expression on the same individual. Our successful in vivo expression of GFP in fransgenic mouse skin should facilitate the understanding of VEGF gene expression in skin, by providing a useful monitoring and screening tool. Its use would be particularly attractive when bred to a mouse with a phenotype thought to involve VEGF expression. This model would also be advantageous in research focused on skin homeostasis during the aging process.

Example 7: Developmental and Age-Related Changes of Human Versican Promoter Activity in Transgenic Mice Skin and Hair Dermal Papilla

To investigate further for temporal-spacious expression pattern for human versican, particularly in skin tissue, transgenic mice were generated containing a lacZ reporter construct driven by the functional promoter for the core protein of human versican and tissue sections from resultant transgenic mice ranged from embryos, newborn to adult stage were examined by b-galactosidase histochemistry.

Construction of transgene

The 839 bp fragment of functional human versican promoter (from -559 to +280 according to the transcription start site as +1) including exon 1 ; (Naso MF, Zimmermann DR and Iozzo RV, "Characterization of the complete genomic structure of the human versican gene and functional analysis of its promoter", J. Biol. Chem. 269:32999-3008 (1994)) was obtained from human genomic DNA by PCR based technique and correct sequences were confirmed by direct sequencing. pNASS2β vector (Clontech) was used as a source of β-galactosidase reporter gene (lacZ) with polyadenylation signal. This vector also included RNA splice donor and acceptor sequence to optimize the chance of high level of transgene expression. Versican promoter fragment was inserted in front of splice donor/acceptor sequence using Xhol - EcoRI restriction site. Linearised fransgene DNA for pronuclear injection was obtained as EcoRI-Xbal 4732bp fragment.

Generation of Transgenic Mice The linearized construct was injected into fertilized oocytes of DBA2xC57BL6(DBFl) mice (Charles River, Boston), and the eggs were implanted into pseudo pregnant foster mothers. The offspring(FO) were tested for the chromosome integration of the human versican promoter construct by Southern hybridization. Thus, genomic DNA was isolated from the tails of the mice at 3 weeks old and digested either BamHI or Pstl restriction endonuclease. The fractionated DNA fragment on the nylon membranes were hybridized with 1.3kbp BamHI-EcoRV fragment of human versican fransgene were used to generate fransgenic mice. Seven independent lines which show transgene insertion by Southern analysis with different copy number from single insertion to more than ten.

b-galactosidase histochemistry

Collected mouse embryos of early to mid stages (from El 1.5d to E15.5d) of development were fixed with 0.5% glutaraldehyde in PBS for 30 min to 12 hrs depends on the stage and fransferred to 30% sucrose, then frozen in OCT compound. Sections were cut 15um thickness and loaded on slides. Sections were re-fixed in same fixative for 5 min and was once with PBS, followed by detergent wash solution for 10 min. Staining reaction was done in the reaction buffer containing lmg/ml X-gal (Sigma), lOmM ferrocyanide, and lOmM ferricyanide. Incubation was curried out for 3-6 hrs at 37 °C. After washing with PBS, sections were either mounted or counterstained with eosin. For adult tissue, mice were perfused with same fixative and desired organs were dissected and post-fixed, sucrose submerged, then frozen with OCT compound. In order to preserve better morphology some early to mid embryo (from El 1.5d to E15.5d) were processed for paraffin section. For skin tissue, 5mm strip of back skin tissue block were fixed for 15 min-1 hr and proceed X-gal staining for 3-6 hrs. After wash, blocks were processed for paraffin embedding and cut 8 um section with microtome.

In situ hybridization The embryo tissues for in situ hybridization were fixed freshly prepared 4% paraformaldehyde in DEPC-treated PBS, dehydrated, processed through a standard paraffin embedding protocol under RNase-free condition. Digoxigenin labeled non- radioactive in situ hybridization was performed on 8 um paraffin sections of El 3.5 d embryo as described previously (Kishimoto J, Cox H, Deverne EB, and Emson PC "Cellular Localization of Putative Odorant Receptor Messenger RNAs in Olfactory and Chemosensory Neurons - A Non Radioactive Insitu Hybridization Study" Molecular Brain Research 23:33-39 (1994)). CDNA for lacZ and mouse versican antisense probe were prepared by PCR and sub-cloned into pBluescriptll(sfratagene). Digoxigenin labeled RNA probes were prepared using a RNA labeling kit (Boehringer) according to the manufacturers' instruction. Paraffin sections on slides were quickly dewaxed in xylene, dehydrated through 100%, 90%, 70% ethanol, washed with 0.1M PBS (pH7.5) and then treated with 0.2N hydrochloric acid for 10 minutes. Sections were treated with 0.02% pepsin (in 0.2N HC1) for 30 minutes at 37°C, following which the enzyme was deactivated by 4% paraformaldehyde. After rinsing in 0.2% glycine solution (in PBS) three times, slides were acetylated in 0.25% acetic anhydride in 0.1 M triethanolamine / 0.9% NaCl for 10 minutes at room temperature and partially dehydrated through 70, 80, 90% ethanol and briefly dried. Approximately O.lμg/ml digoxigenin labeled RNA probes (denatured by boiling prior to use) were added in hybridization buffer (50% deionized formamide, 4XSSC, 10% dexfran sulphate, IX Denhardt's solution) and slides were incubated at 55°C under coverslips for 16-18 hours. The coverslips were carefully removed and the sections were washed in 2X SSC/0.1% SDS for 30 minutes at room temperature, sequentially washed twice in 0.1X SSC / 0.1% SDS at 60°C for 30 minutes. Immunohistochemical procedures to visualize the hybridized probes used alkaline phosphatase conjugated anti-manufactures' instructions. The colorimetric reaction was started by adding nitroblue tetrazolium (Boehringer) and 5-bromo-4-chloro-3-indolyl- phosphate (Boehringer) and incubated for 6-24 hours at room temperature. Slides were mounted with glycerin jelly and were kept at 4°C.

Radioactive in situ hybridization with 3' end labeled oligo nucleotide probes was carried out as described previously (Kishimoto J. Keverne EB, Hardwick J, Emson PC, "Localization of nitric oxide synthase in the mouse olfactory and vomeronasal system: a histochemical, immunological and in situ hybridization study" European J. of Neuroscience 5:1684-1694 (1993)). Sequences of mouse versican antisense oligonucleotide probes were 5'-CCGTTCTGGGCCACCAAGACAGTCGTCTCC-3', 5'-TGACCGCCCCGATATCCAAACAAGCCTGTT-3' (SEQ IDNO: ),

5'-TCCGACAGCCAGCCGTAATCGCATTGGTCA-3' (SEQ IDNO: ).

Some sections were hybridized with labeled oligonucleotides in the presence of excess unlabelled same oligonucleotides to check the specificity of the in situ signal. Cell culture

For primary fibroblast culture from transgenic mice skin, newborn fransgenic mice skin were pealed off and incubated in 0.25% trypsin 18 hrs. Discarded epidermal sheet and dermis were further digested by collagenase for 1 hr. Cells were spread out to the MEDM medium (GIBCO)+ 10% FCS. cells were passaged in every 3 days after trypsin treatment. For b-galactosidase histochemistry cultured fibroblast cells were fixed with 0;.2% glutaraldehyde in PBS for 2 min., followed by detergent wash solution for 5 min. and staining reaction was performed for 18 hrs. at 30°C. After reaction was stopped cells were proceeded for counter-nuclear staining with 5μg/ml of 7-aminoactinomycin D (SIGMA, USA) solution for 20 min. at R.T. then, washed PBS, mount with fluoromount- G (Southern Biotechnology, AL).

Generation of hairless versican-lacZ transgenic mice

To eliminate hair follicle derived lacZ expression in order to examine age-related change of versican promoter activity, hairless genetic background of versican-lacZ fransgenic mice were obtained by breeding with SKH-1 hairless mice resultant founder with SKH-1 (Charles River Laboratories, Boston). Thus, male hemizygous transgenic mice (v/-HH) were mated with female hairless mice(-/-hh), and selected transgene positive pups (v/-Hh) by genomic PCR were then mated back to a hairless again to obtain some F2 generation of versican-lacZ hairless mice(v/-hh) which can be easily selected by combination of PCR detection and hairless appearance.

Generation ofversican-lacZ transgenic mice

Of a total of 27 founder mice six positive lines were detected by both PCR and southern blotting analysis for the transgene insertion. The copy number of transgene was 1 to more than 20 copies per cell. These were further screened for lacZ staining by using X-gal histochemical reaction on tail skin. The intensity of lacZ staining in tail did not correlated with the copy number of transgene as both two lines. A4681(l-2 copies) and A4688(over 20 copies) exhibited similar strong lacZ staining in tail hairs with A4679 which had also over 20 copies, showed only light staining in tail. The fransgenic line A4681 was chosen for further study as the staining of tail skin was most intense and had a good breeding behavior. All further analysis was performed on F1-F3 offspring derived by mating with DBA/2 strain.

Distribution oflacZ expression in embryonic tissue

Intense lacZ staining was observed in developing fore and hind limb (El 3.5) within an area of mesenchymal condensation and also in ectoderm. This ectodermal expression was restricted in the tip of limb. In situ hybridization with radio-labeled antisense oligoes showed similar endogenous mouse versican mRNA expression in condenses mesenchyme in limb bud.

In El 5.5 embryo strong lacZ staining was also observed in kidney glomeruli mesenchyme in olfactory epithelium, mesenchyme and muscles in tongue, and submandibular gland. Perichondrocyte surrounding cartilage, blood vessel, developing edge of hind and fore brain, smooth muscle were also positive.

The expected expression pattern other than limb was confirmed by digoxigenin non-radioactive in situ hybridization with lacZ and endogenous mouse versican probes on transgenic embryonic section (El 3.5) as well as confrol sense probes. Both mRNA expression was observed in, kidney, olfactory epithelium, humerus cartilage, and in vertebrae cartilage, confirming reasonable expression pattern and timing of expression of this fransgenic mice.

Human versican activity in embryonic skin

LacZ expression was examined by X-gal histochemistry on developing skin from E13.5, E14.5, E15.5 and E17.5 embryo. At E13.5d basically no staining was observed in whole body of ectoderm except hind and fore limb and with a few occasion trace of lacZ staining was found in single mesenchymal cell which seems to be earliest stage of condensation. Hair germ of whisker has already exhibited lacZ staining at this stage. At E14.5 condensed mesenchymal cells attached beneath ectodermal placode (hair plug) were clearly lacZ positive and this was highly contrasted against complete negative surrounding scattered mesenchymal cells. Phase contrast photo showed individual b-gal positive condensed mesenchymal cell. The number of these positive site were 4 -5 per sagittal - mediaeval embryonic section of whole embryo tissue. At El 5.5 these positive condenses mesenchyme under hair plug were getting more intense and increased in volume (i.e. the number of cell). At E17.5 the number and intensity of lacZ positive dermal papilla are dramatically increased, yet still highly restricted within the future dermal papilla. A few epidermal placode (i.e. epithelial keratinocytes) itself exhibited lacZ staining although the majority were still restricted in mesenchymal cells.

Hair cycle dependent human versican activity in transgenic mice skin

LacZ staining was examined on the skin section of fransgenic mice from newborn, 7 days, 14 days, 28 days, 40 days, to 4 month old. Strong lacZ staining in hair dermal papilla cells in late embryonic skin were proceed into new born skin and continue during first anagen hair cycle. Strong staining was confined within the dermal papilla cells. Interestingly at age 7, mid to late anagen stage relatively strong staining was also observed in inner root sheath. However, in late anagen stage lacZ staining was again restricted only in dermal papilla cells. Also later in telogen hair no lacZ staining observes in club hair. In second hair cycle growing dermal papilla (second anagen hair cycle) exhibited again intense lacZ staining and after second anagen hair cycle lacZ activity in hair dermal papilla was decreased almost completely through long telogen hair cycle.

Human versican activity in cultures fibroblast

Primary dermal fibroblast culture derived from dermal fibroblast cells of new born fransgenic line exhibited lacZ staining in only certain population of cells and center of hair clamp, obviously dermal papilla. Since all strongly stained cells were round shape these should be derived hair dermal papilla. After first passage, however, shape of positive cells were diversed and not confined round shape, including typical fibroblast- like cells with dendritic process in passage 2. These population of positive cells maintained after first passage to at least until six passages we cultured while certain number of cells were completely unstained through passages determined with counter- nuclear staining which clearly indicated total number of cells. Approximate number of positive cells were not exceed over 50%. Intensity of staining was gradually faded with passages.

Age-related change of human versican activity in transgenic mouse skin Increasing reaction time for x-gal staining to 18 hrs revealed more staining in upper dermis other than hair dermal papilla in young to adult skin of the transgenic mouse. In newborn mouse skin under this long incubation condition, most of dermis including hair follicle (hairless mouse have normal hair only in first cycle) showed fairly detectable staining. Young mouse skin showed only isolated fibroblast derived diffused staining preferentially in upper dermis, and older mouse only exhibited faint staining, indicating age-related decrease of human versican promoter activity in transgenic mouse skin.

Example 8: Evaluation of Reporter gene expression in a live animal.

Method:

Confocal microscopy

To examine whole intact skin on living fransgenic mouse with confocal microscope, hairless VEGF-GFP fransgenic mice were anaesthetized with avertin by intraperitoneal injection. They were placed directly on the petri dish with dorsal position. TPA or acetone treatment was performed on the back skin same as described above marking treated area with an ink to ease the orientation of the skin. Sections or intact skin of the mice were analyzed using Leica DM IRBE inverted microscope and Leica TCS NT4D confocal microscope system with band pass filter 530/30 nm, detecting emission at wavelengths between 515-545 nm.

Direct detection of GFP in the epidermis of intact skin of living hairless transgenic mice by confocal microscopy

To test whether the induction of GFP is directly detected in the skin of a living VEGF-GFP fransgenic mouse, acetone or TPA applied skin of the fransgenic hairless mouse (same individual) was directly examined by confocal imaging under anaesthetized condition. Horizontal optical sections through the normal living epidermis revealed detectable fluorescence and the skin treated with TPA after 12hrs showed the increase of fluorescence positive area. Non-fransgenic wild type litter mate showed no detectable specific fluorescence. Higher magnification of the TPA treated skin revealed that GFP was localized in the extracellular space around the keratinocytes. X-Z confocal imaging confirmed that this signal was within the basal layer of the epidermis.

Laser confocal microscopic evaluation of an intact skin of living transgenic mouse for detection of the GFP signal indicates that GFP is readily detected within the epidermis. These results suggest the potential use of GFP transgenic mice for non- invasive monitoring of long-term gene expression in vivo. When the transgenic GFP reporter gene is combined with the technology for microscopic evaluation such as confocal microscope, the system has a great advantage over conventional transgenic reporter gene animals methods because it reduces the number of animals used, utilizes a simple monitoring system, and allows long term monitoring of changes in gene expression on the same individual.

Successful in vivo expression of GFP in transgenic mouse skin provides for the understanding of the regulation of VEGF gene expression, such as in inflammatory and neoplastic skin diseases, by providing a useful monitoring and screening tool. It is particularly attractive when bred to a mouse with a phenotype which involves VEGF expression. This novel experimentation model will allow in vivo studies on the regulation of VEGF gene. This GFP reporter system on transgenic animal would also be advantageous in research focused on skin homeostasis during the aging process.

Example 9: human MMP9 transgenic mice

MMP9 (gelatinase B, 92-kDa type IV collagenase) is one of the member of matrix metalloproteinases (MMPs) capable to degrade extracellular matrix (ECM) components. This enzyme (MMP9) is known to degrade type IV and V collagens, gelatin and elastin. It is also shown to be induced by UVB which causes photoaging. (Fisher, G.J., Nature 379, 335, 1996). 5'flanking region of this gene from -670 sequence position to +1 transcription start site is sufficient to drive expression of a reporter gene (CAT) in HT1080 cells. (Sato, H., Oncogene 8, 395, 1993).

Construct of the Transgene To construct the fransgene, an 733 bp fragment of the human MMP9 promoter (nucleotides -714 to +19; the transcription start site being +1) was obtained from human genomic DNA using art known PCR-based techniques. Two different reporter genes, beta-galactosidase reporter gene (lacZ) and green fluorescent protein (GFP) gene, were employed for construction.

For the construction with beta-galactosidase reporter gene (lacZ), the MMP9 promoter fragment was inserted into an modified pNASSbeta vector (Clontech), which is inserted neutrophil elastase promoter and has Bgl II site, linearized by restriction with Bgl II-Xho I. To obtain fransgene DNA fragment for pronuclear injection, the vector was cleaved with Bgl II and Hind III. The length of the linearized transgene DNA was 4630bp.

For the construction with green fluorescent protein (GFP) gene, the same MMP9 promoter fragment was inserted into the pEGFP-1-SV, which was modified vector to have splice donor/acceptor based on pEGFP-1 vector (Clontech), linearized by restriction with Hind III-Kpn I. To obtain fransgene DNA fragment for pronuclear injection, the vector was cleaved with Hind III and Afl II. The length of the linearized transgene DNA was 1928bp.

Generation of transgenic mice The lenearized fransgene DNA fragment was injected into fertilized oocytes of hairless mice (SKH1; Charles River, Boston). Those eggs were implanted into pseudo pregnant foster mothers. The offsprings (F0) were tested for chromosome integration of lacZ or GFP fragment by southern blotting. For the detection of lacZ fransgene, the genomic DNA extracted from the tail was digested with Nci I and separated on a gel, then transferred on a nylon membrane. The Nci I digested fransgene DNA fragment was used for the probe. Two independent lines showed a transgene insertion. For the detection of GFP transgene, the tail genomic DNA was digested with Hinc II Two independent lines showed a fransgene insertion.

beta-galactosidase histochemistry

F 1 mice obtained from founders (F0) were used for beta-galactosidase histochemistry. Bones of 2-wk-old mice hindlimb were fixed with 0.5% glutaraldehyde in PBS for 30 minutes. After three times washing with PBS and once with detergent wash, the tissue was incubated with reaction buffer containing 1 mg/ml X-gal (Sigma), 10 mM ferrocyanide and 10 mM ferricyanide at 37°C. After 2 hours incubation, blue staining was observed in a Fl mouse which has fransgene, while no staining was observed in the sibling without transgene. These are consistent with a report in which MMP9 was shown to express at bone of 2-wk-old mouse limb by in situ hybridization (Reponen, P., Journal of Cell Biology 124, 1091, 1994).

Example 10: human Neutrophil Elastase transgenic mice

Neutrophil elastase is one of the powerful serine proteinase capable attacking a broad range of proteins. Hairless mice deficient in this enzyme do not suffer from elastosis, which is one of signs of photoaging caused by UVB irradiation. (Starcher, B., Connective Tissue Research, 31, 133, 1995). Therefore, neutrophil elastase is believed to play an important roll in photoaging.

Construction of the Transgene

To construct the transgene, an 1299 bp fragment of the human neutrophil elastase promoter (nucleotides -1280 to +19; the transcription start site being +1) was obtained from human genomic DNA using art known PCR-based techniques. This neutrophil elastase promoter fragment was inserted into the pNASSbeta vector (Clontech) linearized by restriction with Eco RI - Xho I. To obtain transgene DNA fragment for pronuclear injection, the vector was cleaved with EcoR I and Xba I. The length of the linearized fransgene DNA was 5171 bp .

Equivalents

Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following claims.

Claims

CLAIMS:

1. A method of evaluating a treatment for its effect on skin, comprising: providing a transgenic animal having a reporter gene coupled to a skin metabolism-related promoter, wherein said reporter gene encodes a luminescent or fluorescent product; administering said treatment to said fransgenic animal; and evaluating expression of said reporter gene, thereby evaluating said treatment for its effect on skin.

2. The method of claim 1 , wherein said treatment includes a compound which d is administered by applying said compound to the skin of said fransgenic animal

3. The method of claim 1, wherein said transgenic animal is selected from the group consisting of a fransgenic mini-pig, a fransgenic guinea-pig, a fransgenic rat, and a transgenic mouse.

4. The method of claim 1 , wherein said skin metabolism-related promoter is a human skin metabolism-related promoter.

5. The method of claim 1 , wherein said skin metabolism-related promoter is selected from the group consisting of a promoter which controls the metabolism of an extracellular matrix protein, a versican promoter, a matrix metalloproteinase promoter, a vascular endothelial growth factor promoter, a hyaluronan synthase promoter, a MMP2 or MMP9, promoter, and a neutrophil elastase promoter.

6. The method of claim 1, wherein said reporter gene is selected from the group consisting of the genes which encode the beta-galactosidase, luciferase, green fluorescent protein, alkaline phosphatase, horseradish peroxidase, and chloramphenicol acetyl fransferase.

7. The method of claim 1, further comprising a subsequent administration of said treatment.

8. The method of claim 1, further comprising comparing the expression of said reporter gene to a control value.

9. The method of claim 1 , wherein said fransgenic animal includes a reporter gene coupled to a second skin metabolism-related promoter, wherein the first skin metabolism-related promoter is different from the second skin metabolism-related promoter.

10. The method of claim 1, wherein the evaluation of the expression of the reporter gene step is repeated at least once during the life of the animal.

11. The method of claim 11 , wherein 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.

12. The method of claim 11 , wherein the first and a subsequent repetition are performed on a live animal.

13. A non-human fransgenic animal having a reporter gene coupled to a skin- metabolism promoter, wherein said reporter gene encodes a luminescent or fluorescent product.

14. The non-human transgenic animal of claim 16, wherein said promoter is selected from the group consisting of a versican promoter, a matrix metalloproteinase promoter, a vascular endothelial growth factor promoter, a hyaluronan synthase promoter, a MMP2 or MMP9, promoter, and a neutrophil elastase promoter.

15. A method of analyzing the expression of a transgene in a fransgenic animal, comprising: providing a live fransgenic animal; evaluating, in the live transgenic animal, the presence or absence of a reporter gene encoded by a transgenic sequence or under the control of a fransgenic confrol element, wherein said reporter gene encodes a luminescent or fluorescent product; thereby analyzing the expression of a fransgene.

16. The method of claim 15, wherein the reporter is GFP.

17. The method of claim 1, wherein the evaluation step is repeated at least once during the life of the animal.