1. INTRODUCTION The present invention relates to transgenic non-human animals which can be used as model skin-testing systems. ⁵ Recombinant DNA techniques are used to produce novel genetic constructs which confer upon their transgenic hosts the ability to respond, in a quantifiable way, to physical or chemical stimuli applied to their skin. The transgenic skin-testing system offers a means for identifying effective ^skin-protective (i.e. ultraviolet light blocking) agents and also provides methods for evaluating the effects of ultraviolet radiation, chemical and cosmetic compounds on the skin, skin pathology, and aging.

¹⁵ 2. BACKGROUND OF THE INVENTION

2.1. BASIC SKIN PHYSIOLOGY The integument, or skin, consists of two major layers, the epidermis and the dermis (Reith and Ross, 1977, _in

"Atlas of Descriptive Histology," Third Edition, Harper and 0 Row, Publishers, New York, pp. 136-141) ; the character of these elements varies according to functional demands.

The epidermis consists of stratified squamous epithelium. Cells which originate in the deepest layer, or stratum germinativu , migrate to the surface, or uppermost 5layer, to replace cells which are continually sloughed off.

During migration, these cells accumulate keratin; at the skin surface, keratinized cells form what appears to be a fibrous, rather than a cellular, layer called the stratum corneum. Specialized epidermal cells, called melanocytes, 0 do not undergo keratmization but instead produce the skin pigment, melanin. Additional cell types found in the epidermis include Merkel cells, which serve a sensory function, and Langerhans cells, which are components of the immune system. The epidermis gives rise to nails, hair, 5

sebaceous glands, sweat glands, and the parenchyma of mammary glands.

The dermis consists largely of dense, irregular connective tissue. It contains nerve endings, blood vessels, and lymphatic vessels. 5

2.2. CONVENTIONAL SKIN-TESTING SYSTEMS A number of skin-testing methods are used, particularly in the sunscreen and cosmetic industries. Most methods utilize rodents, particularly the albino guinea pig ⁰ and hairless mouse.

2.2.1. EVALUATION OF ULTRAVIOLET LIGHT-PROTECTIVE AGENTS Two widely used methods for evaluating sunscreens include the American (FDA) and the German (DIN 67501) " ⁵ methods (Martini, 1986, Int. J. Cosmet. Sci. :215-224) . The sun protection factor (SPF) is a dimensionless ratio that estimates the protective efficacy of a sunscreen against acute damage. In the FDA method of determining SPF, successive skin sites are exposed to a constant, ultraviolet

20li.ght source (a xenon arc solar simulator) for progressively longer time periods, varying by 25 percent intervals; according to the DIN method, all skin sites are exposed simultaneously to a mercury vapor ultraviolet lamp which is sequentially occluded to provide exposure steps varying by

25 40 percent (Gabriel et al., 1987, J. Toxicol. Cutaneous

Oσul. Toxicol. ^:357-370) . To assess the photoprotective power of sunscreens, skin samples are evaluated for early uv-induced effects such as erythema, pigmentation, increased activity of ornithinine decarboxylase, altered DNA 30 metabolism, and the appearance of "sunburn" cells (Schauder,

1988, Z. Hautkr. 63:767-770) .

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2.2.2. COSMETIC TESTING SYSTEMS Animal skin testing methods used by the cosmetics industry range from the relatively benign topical application of compounds to animal skin, particularly to mouse ears or rabbit dorsal skin, to the much criticized TDraize eye irritancy test, in which cosmetic compounds are applied directly into rabbit eyes and then rated on the basis of the severity of conjunctivitis or corneal opacity produced. Two main difficulties have been identified in the development of alternatives to the Draize test, namely, lack '°of high correlation between in vitro alternative test results and _in vivo Draize test result, and the inability to present typical cosmetic formulations to these alternative test systems ( allin et al., 1987, J. Toxicol. Cutaneous

Ocul. Toxicol. jS:239-250) . However, a number of alternative 5cosmetic testing systems have been proposed. Some suggest that the Draize test itself be modified; Talsma et al.

(1988, Fundam. Appl. Toxicol. l):146-153) reports the results of a statistical study that concludes that a high level of test accuracy can be achieved in experiments which

Hitilize fewer animals; alberg (1983, Toxicol. Lett. 18:49-

56) recommends exfoliative cytology as a refinement of the

Draize eye irritancy test. In addition, numerous m vitro alternatives have been described, all of which bear questionable comparison to in vivo systems; these include the chick chorioallantoic membrane (McCormick et al., 1984,

Scanning Electron Microsc. 1984(4) :2023-2030) ; polysaccharide-coated lipsomes (Sunamoto et al., 1987, Chem.

Pharm. Bull. 3_5:2958-2965) ; ^tne murine 3T3 cell-neutral red uptake assay (Hockley and Baxter, 1986, International 0 Conference on Practical In Vitro Toxicology, Berkshire,

England; Food Chem. Toxicol. ^4_:473-476) ; the corneal epithelium plasminogen activator (CEPA) test (Chan, 1987, J.

Toxicol. Cutaneous Ocul. Toxicol. £i :207-214) ; the agarose _j diffusion method (Wallin et al., supra) ; and Tetrahymena

thermophila motility tests (Silverman and Penniεi, 1987, J. Toxicol. Cutaneous Ocul. Toxicol. 6-:33-42), to name a few.

2.3. LIGHT-SENSITIVE TRANSCRIPTIONAL PROMOTERS It has recently been recognized that the transcription of certain genes is light-dependent. One such light- regulated system relates to the chlorophyll a/b, or "Cab", binding protein of the light-harvesting complex in plants (Lamppa et al., 1985, Mol. Cell. Biol. 5:1370-1378). A light-sensitive control region which contains both positive and negative regulatory elements has been identified (5' to the photoregulated Cab gene from Nicotiana plu baginifolia (Castresana et al., 1988, EMBO J. 2:1929-1936)). Nagy et al. (1986, EMBO J. 5:1119-1124) have shown that Cab RNA transcript levels in etiolated wheat leaves can be elevated

15 by red light and that the red enhancement can be partially reversed by far-red light. The Cab gene complex of wheat (Lamppa et al., supra) has been introduced into transgenic tobacco plants, in which Cab continued to be expressed in a light-regulated manner (Lamppa et al., 1985, Nature

20 316:750-752) . Si.mi.larly, the 5'-flankmg region of a member of the Pisu sativum (pea) gene family encoding ribulose 1,5-bisphosphate carboxylase, linked to the coding region of a bacterial chloramphenicol acetyltransferase gene, has resulted in light-inducible expression of the chimeric gene

25 when introduced into Nicotiana tabacύm via a Ti plasmid vector (Herrera-Estrella, et al., 1984, Nature 310:115-120). DNA sequences which promote photoregulated expression when fused to constitutive truncated promoters have been localized within promoter regions extending from -400 to - 100 bp from the cap site in the genes examined (Fluhr et al., 1986, Science 232:1106-1112; Simpson et al., 1986, Nature 323:551-554; Kuhlemeier et al., 1987, Genes Dev. , 1:247-255; Nagy et al. , 1987, EMBO J. :2537-2542) .

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Valerie et al. (1988, Nature 333:78-81) has reported that the human immunodeficiency virus (HIV) transcriptional control region, located in the viral long-terminal repeated sequence (LTR) , could be activated by uv light or mitomycin C. A genetic construct which placed the chloramphenicol aceytl transferase (CAT) reporter gene under the control of the HIV-LTR was introduced into the human Hela cell line. When cells were exposed to uv light or various chemical agents, expression of CAT was induced up to 150-fold; induction could be achieved by simply placing the cell lines ⁰ in sunlight.

2.4. TRANSGENIC ANIMALS The term "transgenic animals" refers to non-human animals which have incorporated a foreign gene into their 5 genome; because this gene is present in germ line tissues, it is passed from parent to offspring. Exogenous genes are introduced by microcapillary injection into single-celled embryos (Gordon et al., 1980, Proc. Natl. Acad. Sci. U.S.A.

77:7380-7384; Gordon and Ruddle, 1981, Science 214:1244- 0 ^u 1246; Gordon and Ruddle, 1983, Methods Enzymol. 101C:411-

433) . Transgenic mice have been shown to express globin

(Wagner et al., 1981, Proc. Natl. Acad. Sci. U.S.A.

28:6376-6380), transferrin (McKnight et al., 1983, Cell

3_4:335-341) , immunoglobulin (Brinster et al., 1983, Nature

306:332-336; Ritchie et al. , 1984, Nature 312:517-520;

Goodhardt et al., 1987, Proc. Natl. Acad. Sci. U.S.A.

8j4_:4229-4233; Stall et al., 1988, Proc. Natl. Acad. Sci.

U.S.A. 5:3546-3550), human major histocompatibility complex class I heavy and light chain (Chamberlain et al., 1988, 0

Proc. Natl. Acad. Sci. U.S.A. £5:7690-7694, functional human interleukin-2 receptors (Nishi et al., 1988, Nature

331:267-269) , rat myosin light-chain 2 (Shani, 1985, Nature

314:283-286) , viral oncogene (Small et al., 1985, Mol. Cell. 5

Biol. 5 _^ ι642-648) , and hepatitis B virus (Chisari et al., 1985, Science 230:1157-1163) genes, to name but a few.

3. SUMMARY OF THE INVENTION The invention is directed to a transgenic non-human animal skin-testing system. Briefly described, recombinant DNA techniques are used to produce novel genetic constructs which confer upon their transgenic hosts the ability to respond, in a quantifiable way, to physical or chemical stimuli applied to their skin. The genetic constructs of the invention may encode a variety of promoter and/or enhancer sequences in combination with a number of reporter genes. For example, and not by way of limitation, a recombinant DNA construct may comprise a light sensitive, topically-activated or skin-specific promoter in combination with a colorimetric or tumorigenic reporter gene. Transgenic non-human animals which have incorporated such a construct into their germ line would provide powerful systems for evaluating the effects of physical (i.e., u.v. light) or chemical agents and the efficacy of protective agents (i.e., sunscreen, anti-aging formulations) on the skin, and may also supply model systems for various skin pathologies including malignancy (i.e., melanoma) , psoriasis, allergy, and aging. Importantly, the reliability of such transgenic systems would decrease the number of animals necessary to ensure the accuracy of experimental results, and may also diminish the discomfort of animals utilized for research.

4. DESCRIPTION OF THE FIGURES

FIGURE 1. Plasmid pHIV-LTRl/lac Z carries the β- galactosidase gene under the control of the HIV-LTR promoter in addition to the gene for ampicillin resistance.

5. DETAILED DESCRIPTION OF THE INVENTION The present invention is directed to transgenic non- human animal skin-testing systems in which recombinant genetic constructs are used to confer upon transgenic animals the ability to respond, in a quantifiable way, to physical or chemical stimuli applied to their skin. This capability arises from the use of promoter elements that may be selectively activated in skin, either due to tissue- specificity or induction by physical (i.e., ultraviolet light) or chemical topically active substances. According '"to the invention, these skin selective promoters are combined with a reporter gene associated with a readily identifiable product.

For purposes of clarity of disclosure, and not by way of limitation, the detailed description of the present

15i.nvention is di.vided into the following subsections:

(a) Creation of Transgenic Animals

(b) Skin-Selective Promoters

(c) Reporter Genes

(d) Utility of Transgenic Skin-Testing Systems 0

5.1. CREATION OF TRANSGENIC ANIMALS

The preparation of animals used in the bioassay of the invention will utilize the recently developed technology of introducing foreign genes into the germ line (inheritable 5trait) of an animal. The animal carrying in its germ line a foreign piece of DNA, termed a transgene, is called a transgenic animal. One method for the creation of transgenic mice for producing test animals is described below. 0 In general, the scheme presently employed to produce transgenic mice involves the following: male and female mice are mated at midnight. Twelve hours later, the female is sacrificed and the fertilized eggs are removed from the uterine tubes. At this time, the pronuclei have not yet 5

fused and it is possible to visualize them in the light microscope. Foreign DNA is then microinjected (100-1000 molecules per egg) into a pronucleus. Shortly thereafter fusion of the pronuclei (a pronucleus or the male pronucleus) occurs and, in some cases, foreign DNA inserts ^Lnto (usually) one chromosome of the fertilized egg or zygote. The zygote is then implanted into a pseudo-pregnant female mouse (previously mated with a vasectomized male) where the embryo develops for the full gestation period of 20-21 days. The surrogate mother delivers these mice and by %our weeks the pups are weaned from the mother. To test these mice for the presence of foreign DNA, a portion of the tail (a dispensable organ) is removed and the DNA extracted. DNA-DNA hybridization (in a dot blot, slot blot or Southern blot test) is employed to determine whether the mice carry he foreign DNA. Of the eggs injected, on average 10% develop properly and produce mice. Of the mice born, on average one in four (25%) are transgenic for an overall efficiency of 2.5%. Once these mice are bred they pass along the foreign gene in a normal (Mendelian) fashion linked to a mouse chromosome. Mating two homozygous mice with the transgenic DNA means 100% of the offspring carry two copies of the transgene.

When this is done it is common that the mice carry tandemly repeated copies of the foreign gene (from 3-80 copies) at one chromosomal location or site.

The present invention is not limited to any one species of animal, but provides for any non-human animal species which may be appropriate for skin testing. For example, mice, including hairless mice; guinea pigs, 0 including albino guinea pigs; rabbits and pigs, to name but a few, may provide useful transgenic skin-testing systems.

Likewise, any method known in the art may be used to produce transgenic animals, including but not limited to, 5

microinjection, transfection of DNA, sperm transfer and electroporation.

5.2. SKIN-SELECTIVE PROMOTERS According to the present invention, a skin-selective ⁵ promoter is combined with a suitable reporter gene (see infra) to create a genetic construct for introduction into transgenic animals. The term "skin selective promoter" should be construed to mean any DNA sequence that will control the expression of its associated reporter gene in '"skin and it may or may not also control the expression of its reporter gene in other tissues; this includes promoter sequences which are skin-selective because they are induσible by stimuli which may be applied to skin. For example a light-inducible promoter could be activated in any

15ti.ssue that i.s exposed to li.ght; i.f only the skm. i.s exposed to light, the promoter behaves as a skin-selective promoter. Activity of the skin selective promoter is not restricted to any particular cell type, so that the term "skin-selective promoter" can apply to any one, or any combination, of the 0cell types present i.n skm. (includi.ng, but not li.mi.ted to the cells of the epidermis and the deπrtis, such as epithelial cells, keratinocytes, melanocytes, or cells of the immune system) .

According to the present invention, the skin selective 5promoter can be manipulated to result in either an increase, or decrease of reporter gene transcription.

Appropriate promoter elements would include light- inducible promoters, including, but not limited to the HIV- LTR (Valerie et al., 1988, Nature 333:78-81) the Cab-1- 0 associated regulatory region (Lamppa et al., 1985, Nature

316:750-752) , and the ribulose 1,5 bisphosphate carboxylase regulatory region (Herrera-Estrella et al., 1984, Nature 310:115-120) . 5

In addition, promoters which are inducible by chemical stimuli, which may be topically applied, may be used according to the invention, for example, the HIV-LTR

(Valerie et al., supra) . A variety of promoters are responsive to phorbol esters, for example, the c-jun

° promoter responds to 12-0-tetradecanoyl phorbal 13-acetate

(TPA) (Lamph et al. , 1988, Nature 3_3_4:629-631) . Such a promoter, if expressed in the epidermis, could be used to monitor compounds for tumor promotion activity when applied to the skin.

'0 Likewise, promoter elements which are endogenously skin-specific may be used according to the invention.

Eukaryotic cellular and viral promoter-elements, and even portions of prokaryotic promoter-operator elements may be employed. 15

5.3. REPORTER GENES The skin-selective promoter may be combined with any reporter gene that has a readily identifiable product according to the invention.

* 90 In vari.ous embodiments, the sk. -selecti.ve promoter may be combined with a reporter gene with a visually or autoradiographically detectable product. In a preferred specific embodiment of the invention, the HIV-LTR is combined with the _/ 9-galactosidase (jø-gal) gene in transgenic

25animals; the sk of the resulting transgenic animals, in the presence of -gal substrate (X-gal) , will turn blue after exposure to light. In another embodiment of the invention, a skin-selective promoter may be associated with the gene for luciferase (Ow et al., 1986, Science 234:856- 30

859 such that luciferase is selectively produced in the transgenic animals skin; exposure to luciferin (i.e. topically applied) may result in emission of light.

In other embodiments of the invention, the skin- selective promoter may be combined with a reporter gene

associated with a chemically detectable product including, but not limited to, ,9-glucuronidase (Jefferson et al., 1986, Proc. Natl. Acad. Sci. U.S.A. £3:8447-8451) ; chloramphenicol acetyltransferase (Gorman et al., 1982, Mol. Cell. Biol. 2 1044-1051) , and xanthine-guanine phosphoribosyltransferase ⁵ (Mulligan and Berg, 1980, Science 209:1422-1427) .

In further embodiments of the invention, the skin- selective promoter may be combined with a reporter gene with a biologically active product. In particular embodiments of this invention, this product may be tumorigenic, and the O reporter gene may be an oncogene. Any oncogene whether previously described or yet to be discovered could potentially be utilized in the assays described in the subsections below. Known oncogenes which could be utilized include but are not limited to those derived from DNA tumor

15viruses (e.g., T antigen genes from SV40 or polyoma viruses,

E1A and E1B genes from adenoviruses, and other genes known to have tumorigenic potential derived from papillomaviruses and herpes viruses; reviewed in Bishop, 1985, 1985, Cell

£2 _^ :23-38); retroviruses (e.g., v-abl, v-fes, v-fps, v-fgr, * 0v-src, v-erbA, v-erbB, v-fms, v-ros, v-yes, v-mos, v-ras, v-fos, v-myb, v-myc, v-ski, v-sis, v-rel, v-kit, v-jun, v- ets; reviewed in Bishop, 1985, Cell 4^:23-38) ; cellular proto-oncogenes corresponding to the viral oncogenes, activated cellular oncogenes corresponding to viral 5oncogenes, or cellular oncogenes for which no viral counterpart has yet been described (e.g., c-neu, Hung et al., 1986, Proc. Nat. Acad. ^" Sci. U.S.A. 83_:261-264) . In other embodiments, the reporter gene product may act as a cytokine or lymphokine. 0

5.4. UTILITY OF TRANSGENIC SKIN-TESTING SYSTEMS The present invention provides methods for evaluating the effects of physical or chemical agents on the skin, offers reliable methods for determining the efficacy of

skin-protective compounds or devices, and allows for the development of novel animal model systems for various human skin pathologies.

In various embodiments, the present invention can be used to test the efficacy of skin-protective agents. In a ° preferred embodiment, the invention can be used to quantitate the ability of sunscreen agent to protect skin against the deleterious effects of ultraviolet radiation. According to this embodiment, an ultraviolet light-inducible promoter DNA sequence may be combined with the β- ' galactosidase reporter gene and introduced into transgenic mice. If these mice were exposed to ultraviolet light and then to X-gal, a chromogenic substrate for 9-galactosidase, their skin, including their tails, would turn blue; an effective sunscreen would prevent the development of blue

15color. Si.milarly, promoters m. ducible by compounds that damage the skin (hereafter referred to as stress-inducing chemicals) coupled to appropriate reporter genes could be introduced into transgenic animals which could be used to determine the effectiveness of protective agents against

2 *0 ^w stress-inducing chemicals (i.e., DNA damaging agents).

In related embodiments of the invention, transgenic skin-testing systems may be used as reliable tests to identify skin-damaging agents. For example, transgenic animals which carry exogenous DNA comprising a stress-

25chemical mducible promoter and suitable reporter gene could be used to screen cosmetics for stress-chemical, i.e. DNA damaging, activity. In addition, mutations could be introduced into the promoter/reporter gene construct prior to introduction into transgenic animals. These mutated 30 constructs would require reverse mutation in order to express reporter gene product, and thus would provide a reliable method for testing for mutagenizing agents. A complete description of transgenic testing systems for mutagens and carcinogens is contained in U. S. patent

application serial No. 132,383, filed December 15, 1987, which is incorporated by reference in its entirety herein.

In further embodiments, the present invention provides model systems for various skin diseases. In a particular embodiment, a skin-selective promoter sequence may be combined with an oncogene and introduced into transgenic animals. Animals carrying this transgene may develop skin tumors in response to promoter induction; such animals may also exhibit proliferative diseases of the skin, i.e. psoriasis. In a specific embodiment of the invention, the ' HIV-LTR may be combined with the gene encoding simian virus 40 T antigen.

Additionally, a skin selective promoter may be combined with a biologically active substance, such as a lymphokine, and introduced into transgenic animals which may

15serve as models for cutaneous immune diseases. In a related embodiment, regulatory sequences which are active only in cells of the immune system (i.e. the immunoglobulin gene control region as described by Grosschedl et al., 1984, Cell 3_8:647-658; Adames et al., 1985, Nature 318:533-538; ⁰ Alexander et al. , 1987, Mol. Cell. Biol. 2:1436-1444) could be combined with a light-inducible promoter and a reporter gene; thus the immune cells of the transgenic animals would produce reporter gene product only in response to light, thereby, only when localized to the skin. 5 Further, a considerable utility of the present invention resides in its ability to engender reliable, quantifiable assays for skin testing. Many of the present methods which utilize animal models for skin testing are based on the measurement of tissue damage, i.e., sunscreen 0 tests and the Draize test. The present invention provides more sensitive assays which may detect a harmful stimulus before damage is done; for example, if a light-activated promoter were combined with ^-galactosidase ( -gal) in a transgenic animal, exposure to ultraviolet light could D

induce β-gscl before a painful sunburn could arise. In addition, the quantitative nature of transgenic skin-testing systems would be likely to reduce the number of animals presently used in research, in which large numbers are necessitated by wide fluctuations in experimental results.

Therefore, the present invention provides for an accurate time-efficient, cost-effective, and hopefully more humane method of animal skin-testing.

The present invention is not to be limited in scope by the various embodiments described supra. Indeed, various modifications of the invention in addition to those shown and described herein will become apparent to those skilled in the art from the foregoing description and accompanying drawings. Such modifications are intended to fall within the scope of the appended claims. 5

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