AU4633989A

AU4633989A - Transgenic mice for the analysis of hair growth

Info

Publication number: AU4633989A
Application number: AU46339/89A
Authority: AU
Inventors: Paul S. Kaytes; Alistair R. Mcnab; Gabriel Vogeli; Linda S. Wood
Original assignee: Upjohn Co
Current assignee: Pharmacia and Upjohn Co
Priority date: 1988-11-30
Filing date: 1989-11-17
Publication date: 1990-06-26
Anticipated expiration: 2009-11-17
Also published as: NZ231502A; AU620477B2; JPH04501952A; EP0439553A1; CA2004156A1; WO1990006367A1

Description

_!_

TRANSGENIC MICE FOR THE ANALYSIS OF HAIR GROWTH

FIELD OF THE INVENTION

This invention relates to transgenic animals which contain recombinant genes comprising the regulatory elements involved in the expression of hair specific genes linked to at least one structural gene whose expression can.be detected. . pørdingly, this .-invention provides an animal model useful for sceening agents that influence the hair cycle. Additionally, this invention provides an animal.model useful in studying the expression of genes involved in hair growth as well as providing an animal model useful in studying structural genes which can be expressed in a time and space specific manner by a precisely characterized regulatory pattern.

_ BACKGROUND OF THE INVENTION

Transgenic animals are animals which have integrated foreign DNA in their somatic cells and germ cells. The most common way of intro¬ ducing the foreign DNA into the animal is by either microinjection or retroviral infection of the animal when it is in an embryonic state. The foreign DNA then integrates itself into the genetic material of the animal after which it is replicated along with the native genetic material of the animal during the development and life of the animal. Additionally, because the foreign DNA is integrated into the germ cell DNA, the offspring of such an animal will contain copies of the foreign DNA.

Much of the work done in the area of transgenic animals has been performed on rodents, specifically mouse species. Transgenic animals generally, and transgenic mice specifically, are especially useful as animal models to characterize and study regulation of gene expres- sion. Additionally, these models can be used to analyze the role of specific molecules in complex biological phenomena.

Of the two procedures commonly used to generate transgenic animals, microinjection has the advantage that there is no restraint on the size or sequence of the DNA that can be introduced. DNA that is microinjected at the one cell stage (i.e. fertilized egg) usually integrates at random sites prior to cleavage of the cell such that all cells of the embryo carry copies of the foreign DNA. More than one copy of the microinjected DNA usually integrates into the 367 - .₂.

chromosomal DNA of-the fertilized egg. In contrast, when retroviruses are used only single copies of the viral DNA integrate. In the microinjection protocol, DNA is loaded into a micropipet and expelled into one of the pronuclei of a fertilized egg. The injected embryos are implanted into the oviducts of pseudopregnant foster mothers, and some develop to term. A disadvantage of re roviral vectors is that the infection is usuall initiated at;, later embryonic stages, resulting in mosaic founder animals. The technical aspects of either of the two procedures are well known to those of ordinary skill in the art.

The foreign DNA which is introduced into the animal is called a transgene. Transgenes which have been expressed in mice code for a variety of proteins. Typical transgenic experiments introduce non- murine derived genes for structural proteins into mouse species. Detection of the non-mouse protein confirms the presence and expres¬ sion of the transgene. The phenotypic effect of the expression of some of these foreign DNA sequences includes oversized mice resulting from the expression of a rat or human growth hormone transgene. Viral protein genes have also been successfully introduced into mice. Recombinant genes have been constructed which contain promoter and structural protein regions derived from different sources. These recombinants include those using inducible promoter regions, enabling the control of expression of the structural gene by the presence or absence of the inducer needed to initiate gene expression. Foreign genes may be introduced into the genome of a particular animal which will be tissue-specific in their expression. That is, although the gene is present in all cells in the animal, it will only be expressed in tissues which normally induce the regulatory elements controlling expression of the particular foreign gene. Thus, introducing a gene with a tissue-specific regulatory element into an animal will provide an appropriate model for examining control of gene expression.

Mammalian hair is formed from epithelial cells that undergo terminal differentiation including complete cornification. Hair specific proteins accumulate within the mesh work of the cyto- keratins that form in the intermediate filaments of the epithelial cells. During hair growth, epidermal cells in the hair follicle shift their biosynthesis from skin-specific cyto-keratins and basement membrane components to hair specific proteins, under the inductive influence of the dermal papilla.

In young mice, waves of hair growth progress from head to tail along the body in a steady pattern. Accordingly, regulation of expression of the hair specific genes in the skin corresponds to the anterior-posterior temporal hair growth pattern. When the mice grow older, these waves give way to patchy, hair growth. __-, ._ ■ ■• ■

In the present invention, transgenic non-human mammals are generated which contain a recombinant transgene having the regulatory elements for hair specific proteins. These elements are fused to the coding region of a protein which can be detected. Thus, introduction of the transgene results in an animal having an identifiable charac¬ teristic or trait when expression of hair specific protein is induced.

INFORMATION DISCLOSURE U.S. Patent Number 4,736,866 issued April 12, 1988 to Leder et al discloses a transgenic non-human animal having a transgene comprising an activated oncogene sequence which increases the probability of development of neoplasms in the animal.

Strojek R.M. , et al, The Use of Transgenic Animal Techniques for Livestock Improvement, Genetic Engineering: Principles and Methods, J.K. Setlow, Ed. Vol. 10 New York (1988) reviews work in the area of transgenic mice. Methods are disclosed and various transgenic lines are described and discussed.

Skangos and Bieberich, Gene transfer into mice, Advances in Genetics, 24:285-322 (1987), provides a review of work in the area of transgenic mice. A list of reported transgenic mice species is included, listing various transgene constructs introduced into mice. Ornitz, D.M. et al., Nature (London) 313:600-603 (1985) reports a transgenic mouse in which the transgene introduced is a recombinant gene containing the rat Elastase I promoter and the codons for human growth hormone. In that species, the human growth hormone gene is expressed upon induction of the rat enzyme gene. Ornitz, D.M. et al., Cold Spring Harbor Symp. Quant. Biol. , 50:399-409 (1985) discloses an Elastase I/SV40 early region trans¬ genic mouse species wherein the early region of SV40 gene is ex¬ pressed upon induction of the rat Elastase I gene. . - - Overbeek,^ P'A et-al.,: Proc. Natl. Acad. Sci. U.S.A. 82:7815- 7819 (1985) discloses- an.¹ αA-crystallin/CAT transgenic mouse. The bacterial gene for chloramphenicol acetyltransferase (CAT) is expressed upon induction of the mouse gene of that construct.

Overbeek, P.A. et al., Science 231:1574-1577 (1986) discloses RSV LTR/CAT recombinants introduced into mice. CAT is synthesized when expression is initiated by the Rous sarcoma virus long.-terminal repea .

Palmiter, R.D. et al. , Nature (London) 300:611-615 (1982) refers

sys em.

Khillan, J.S. et al., Proc. Natl. Acad. Sci. U.S.A. 83:725-729 (1986) discloses a transgenic mouse in which a oa(I)-collagen/CAT recombinant gene has been introduced. The CAT gene is expressed upon induction of the mouse gene promoter it is fused to.

Van Brunt, J. Bio/Technology, Vol. 6, No. 10, October 1988 generally describes transgenic animals and the future applications of transgenic animal technology. The publication notes on page 1149- 1154. The publication notes on 1151 that the most formidible technical challenge is to regulate the foreign gene's expression in the new host. The publication states that it is necessary to manipulate regulatory signals so that the genes spatial and temporal expression can be controlled at will. The present invention over¬ comes this noted main barrier by use of promoters from hair specific proteins so that the transgenes are expressed during the hair growth phase.

Wilmut, I. et al. , New Scientist 7, pp. 56-59, July 1988 briefly outlines genetic breeding of animals through the use of transgenic technology. The techniques used to incorporate a transgene into a fertilized embryo are described.

Rogers, G.E. Genes for hair and avian keratin. Ann. New York Acad. Sci 455:403-425 (1985) provides a review of investigations of genes coding for hair keratins. Nucleotide sequences of genes for hair specific proteins which have been isolated are provided and the amino acid sequences are derived therefrom.

Powell, B.C. and Rogers, G.E. Hair keratin: Composition, structure and biogenesis. In: Breiter-Hahn J., Matoltsy, A.G., Richards, K.S., eds. Biology of the integument. Vol. 2. Berlin: Springer-Verlag,,_, pp. 696-721, (1986) provides an inventory of hair proteins and a review of the the protein classes and location in fiber for the various proteins listed. The number of protein families within each of the classes is discussed and characteristics of the several families are noted.

Powell, B.C. et al., Nucleic Acids Research, 11 (No.6) :5327-5346 (1983) discloses genomic clones of high-sulphur keratin genes from sheep. Marshall, R.C. Characterization of the proteins of human hair and nail by electrophoresis. J. Invest. Dermat. 80:519-524 (1983) describes the characterization of low and high sulfur proteins from human hair. Differences in electrochemical and physical properties among members of both classes are disclosed. Heid, H.W. et al, The complement of native α-keratin poly- peptides of hair-forming cells: A subset of eight polypeptides that differ from epithelial cytokeratins. Differentiation 32:101-119 (1986) discloses the eight members of the group of proteins which make up the intermediate filament cytoskeleton of hair. The poly- peptides disclosed are characterized and distinguished from non-hair specific epidermal cytokeratins.

SUMMARY OF THE INVENTION

This invention is a transgenic, non-human mammal whose germ cells and somatic cells contain a recombinant transgene comprising a promoter sequence from a gene which codes for a hair specific protein and a reporter gene functionally linked to the promoter. This recombinant gene is introduced into the animal, or an ancestor of the animal, at an embryonic stage. In the present invention, the transgene introduced into the animal contains a tissue specific regulatory element and the DNA sequence for a detectable protein.

Thus, the transgenic animal of the present invention will express the reporter gene when induced under the control of the - .-regulatory mechanisms present in the animal. The expression of the _;reporter". ene will.'follow the pattern of expression of hair specific proteins and will likewise be tissue specific. Consequently, the transgenic animal of the present invention provides a system to study regulation of genes related to hair growth and a system which provides tissue specific and time specific limits on expression of structural transgenes.

Therefore, the present invention provides an animal model for investigating the expression of hair specific proteins by operably fusing the regulatory elements of hair specific genes with genes for stable, detectable proteins.

The present invention also provides an animal model useful for sceening agents that influence the hair cycle.

The present invention also provides a transgenic animal which contains a recombinant gene having a.structural gene whose activation is under the control of a hair specific gene which is expressed specifically during the hair growth cycle.

Additionally, the present invention also provides an animal model useful for sceening agents that influence wool production.

DETAILED DESCRIPTION OF THE INVENTION During the growth and differentiation of cells within the hair follicles, there is an accumulation of hair specific proteins. As the epithelial cells of the hair follicles differentiate and migrate up through the hair shaft to form hair, they express specific proteins in a tightly coupled fashion to their stage of differentia¬ tion within the developing hair follicles. Accordingly, genes coding for these hair specific proteins are expressed and this expression is strictly coupled with hair growth. By isolating the promoter region 0 from a hair specific protein gene and then operably linking it with a reporter gene, the reporter gene will be expressed when the hair specific promoter is activated. Thus, a transgenic animal having this recombinant gene incorporated into its genetic material will show tissue specific expression of the reporter gene during its hair 5 growth cycle. This results in an animal model useful for investigat¬ ing regulatory controls and temporal and spatial expression of hair specific genes. Additionally, the transgenic animal of the present invention provides an animal model in which structural genes of __?_

interest can be expressed at specific and determinable periods.

The transgene used in the present invention comprises a hair specific regulatory element and a reporter gene operably linked thereto. The regulatory element incorporated into the transgene is the promoter region of a hair specific protein. A hair specific protein is a protein which is expressed only during the hair growth cycle. Hair specific proteins are only produced in tissues which are involved in hair growth. Accordingly, the regulatory signals which induce expression of the hair specific proteins, act upon the promoters of the genes for hair specific proteins.

Hair specific proteins may belong to one of the following classes of proteins: ultra high sulfur keratins which help make up the matrix and cuticle of the hair follicle; high sulfur keratins which are also found in the matrix of the hair follicle; low sulfur keratins which make up the microfibrils, i.e. intermediate hair filaments; or high glycine/tyrosine proteins which are found in the matrix. Within each of these classes of proteins, several protein families exist. Each family is further comprised of several dif- ferent proteins. In each case, expression of the hair specific proteins is induced during the hair growth cycle within the cells in which hair follicles are produced.

The present invention uses the promoter region of a gene for a hair specific protein to regulate expression of a reporter gene. A promoter is a DNA region, usually upstream to the coding sequence of the gene, which binds RNA polymerase and directs the enzyme to the correct transcriptional start site. To isolate a hair specific promoter, it is necessary to locate the gene for a hair specific protein and then identify and isolate the portion upstream from the coding sequence which serves as the promoter. Promoter regions generally contain certain similarities in nucleotide sequence which are well known and readily recognizable to those of ordinary skill in the art. Thus, one skilled in the art could isolate the promoter region from a hair specific gene and use it to produce a recombinant transgene using well known techniques.

The techniques used to isolate a hair specific gene are also well known to those of ordinary skill in the art. A probe is used to screen a human genomic DNA library. The probe can be an oligo- ;- _₈.

;.* .nucleotide whic ; is...related, to, the sequence of the gene sought or . derived from; the cDNA; S:equeπce' of the gene transcript or amino acid sequence "protein. Probes must be substantially complimentary to the gene to be useful. Thus, probes designed from cDNA information are

5 most efficient.

To design a probe from the amino acid sequence, the portion of the protein must first be_ sequenced andiJthen the probe must be designed from the amino acid sequence with nucleotide sequences which are likely to be the same codons as are used by the gene to code for

10 the amino acid sequence. Due to the degenerative nature of the genetic code, it is often necessary to make several probes when using this strategy.

A genomic library is screened with the oligonucleotide probe. Clones which hybridize with the probe are then sequenced to confirm

15 they contain copies of the gene encoding the protein upon which the probe was designed. The promoter region of a gene may be isolated once the desired gene has been found as set forth. This isolation strategy is illustrative of those used by persons of ordinary skill in the art to isolate genes for proteins. Such persons would be able

20 to isolate genes for hair specific proteins using techniques well known in the art. The hair specific promoters described in the Examples below include Ultra High Sulfur Keratin (KER) , hair specific keratin MHKal (CYTO) , and Ultra High Sulfur Ser (UHS-Ser). Only the CYTO protein is a member of the keratin family. Both KER and UHS-Ser

25 are members of the keratin associated protein family.

Operatively linked to the promoter of a hair specific gene is a reporter gene. The term "operatively linked" as used herein means functionally fusing a promoter with a structural gene in the proper frame to express the structural gene under control of the promoter.

30 An operatively linked reporter gene will be expressed to produce the protein encoded thereby when the regulatory signals which normally induce the operon from which the promoter has been derived are present. Thus, during the hair growth cycle, the regulatory controls which act upon a hair specific promoter to initiate expression of a

35 hair specific gene will similarly act upon a hair specific promoter operatively linked to a reporter gene to facilitate expression of the reporter gene.

A "reporter gene", as used herein, means a gene which is trans- lated and transcribed into a protein that can be detected. The reporter gene codes for a protein which can be identified when it is expressed. Reporter genes include genes that code for proteins for which there are assays for detecting their presence. Alternatively, reporter genes can be genes which display an identifiable phenotype when expressed.

Several reporter genes have .been widely used as marker genes in a variety of diverse studies. The requirement necessary for a gene to be useful in such a capacity is that expression of such a gene may be detected by relatively simple means. These means are generally assays in which presence or absence of products of gene expression inves¬ tigated. Furthermore, expression of a reporter genes may result in an observable phenotype which is not demonstrated in animals lacking expression of such reporter gene. Reporter genes of this type include oncogenes as well as those which code for proteins which result in specific pigmentation or induce tail loss. Examples of reporter genes include chloramphenicol acetyltransferase, human placental alkaline phosphatase, firefly luciferase, beta-galac- tosidase, SV40 T-antigen, beta-glucuronidase, tissue plasmid ac- tivator, adenovirus ElA, beta-galactosidase, Harvey Ras oncogene and human growth hormone.

Non-human mammals which are suitable for production of trans¬ genic animals include mice, rats, sheep, cows and pigs. The present invention may be practiced with each of the above listed species but mice are the preferred species in applications involving the models useful in the screening of agents which promote human hair growth. Sheep are the preferred species for models useful in screening agents which promote wool production.

The homology and relationship of the genes from mammalian species is a topic of current scientific investigation. Since mammalian species have homologous structural and physiological characteristics, the biochemical mechanisms manifesting such traits, and therefore the genetic information of the various species, are thought to be similar in evolutionary predecessor and present condition. Genes encoding homologous phenotypic characteristics are thought to be structurally homologous and in many cases highly conserved, especially within mammals. A strong evolutionary argument has been made (Rogers., G.E., Genes for hair and avian keratins. Ann. NY Acad. Science 1985;455:403-25) that not only are the genes for all mammalian species closely related and regulated by homologous mechanism, but that there is a close relationship between the proteins of the epidermal appendages between mammals and birds. Findings in various fields indicates that the mechanisms which control the function of an organ system and the proteins produced in the organ system is closely related within the mammals, and-within the vertebrates in accordance with their evolutionary distance. The relationship between the hairs of different mammals, especially between sheep and other mammals, has been tested by several re¬ searchers.

The development of hair during embryonic development is the same in all mammalian species. Furthermore, a collection of the same proteins can be found in sheep as in rodents. For example, Bertolino et al. 1988 (Bertolino A.P. , Checkla, D.M., Notterman, R. , Sklaver, I., Schiff, T.A. , Freedberg, U.M. , DiDona, G.J., "Cloning and charac¬ terization of a mouse type I hair keratin cDNA," J. Invest. Dermatol. 1988 Dec:91(6) :541-6) show that by using sheep nucleic acid sequences as a molecular probe, it is possible to isolate the corresponding urine sequence. The homology reported is that 87% of the mouse sequence is the same as the sequence found in the sheep gene. In the present case, nucleic acid information from a high sulfur keratin of sheep has been used to isolate a related ultra high sulfur protein from a rodent genomic library. The conservation among homologous genes and their products has been shown in a variety of ways. It has been demonstrated by the use of sheep sequence for in situ hybridization that equivalents to gene products that are produced in the sheep are present in mice. Additionally, it has been reported that sheep genes can be expressed in mice under the control of the sheep gene regulatory sequences. Furthermore, using antibodies for a hair specific protein, Rothnagel and Rogers (Rothnagel, J.A.; Rogers, G.E.: "Trichohyalin, an inter¬ mediate filament-associated protein of the hair follicle," J. Cell. Biol. 1986 Apr:102(4):1419-29), showed that the antibody against the sheep protein also recognizes the same protein in other mammalian species (guinea pig and human) . It is therefore predictable that transgenes expressed in mice under the control of regulatory sequen¬ ces from human or murine hair specific genes would be expressed in ._n.

sheep. Furthermore, because of the similarity of the hair growth mechanism and the constituent components among all mammals, the specific examples which demonstrate the present invention in a murine model may be followed and applied to produce other transgenic animals according to the present invention, particularly sheep.

. DESCRIPTION-OF THE PREFERRED -EMBODIMENTS - »

Example 1 KER-CAT TRANSGENIC NON-HUMAN MAMMAL In the preferred embodiment, the promoter region from the gene for ultra high sulfur keratin protein is combined with the CAT gene. The gene for ultra high sulfur keratin has been isolated and charac¬ terized. The gene is expressed in the skin during the hair cycle and this expression is coupled with hair growth. Chloramphenicol acetyltransferase is a bacterial enzyme. The enzyme is stable and it is not found in mammalian cells. The regulatory elements of a gene for a hair specific protein are operatively linked to the CAT gene which may be easily detected by techniques well known to those skilled in the art. The resulting recombinant gene is then introduced into an animal at an embryonic stage by techniques well known to- those skilled in the art. The resulting transgenic animal carries the recombinant gene in which expression of the reporter protein occurs during the hair growth cycle. Accordingly, the resulting transgenic animal may be used as a model for investigating factors that control or in¬ fluence hair growth. Additionally, the resulting transgenic animal contains genes which will be expressed specifically during limited and discrete periods in the animal's life, the hair growth cycle. ISOLATION OF THE ULTRA HIGH SULFUR KERATIN GENE The Ultra High sulfur keratin gene is isolated and cloned as follows. A murine genomic library is screened with a synthetic oligodeoxynucleotide (AMC-16) that has been derived from position 854 to 890 of a published DNA sequence of the B2A high sulfur keratin from sheep. The sequence of oligonucleotide AMC-16 is 5'-GCAG- GTGGGCTGGCAGCAGCAGGCTGGGCGGCAGCA. (Powell, et al. , Nuc. Acids Res. 11:5327-5346, 1986). The methods used for plating and screening of the genomic DNA library, isolation and purification of phage DNA have been published. (Vogeli, et al., Methods Enzymol. 152:407-415, 1987 -12-

and- Maniatis^ e l, Molecular. Cloning: A Laboratory Manual, .Cold Spring Harbor, .Laboratory,^NY 1982). A total of 10,000 λ Charon 4A recombinant clones are plated onto 15 cm dishes and transferred to NEN colony hybridization membrane (Dupont). The filters are treated following the manu acturer's recommenda¬ tions. They are pre-hybridized and hybridized at low stringency in 0.9 mM NaCl, 0.2 M Tris-HCl pH .7.4, 20 mM EDTA pH 1.5, 30% fqrmamide, 20 mM Na-phosphate buffer pH 7.4, 1% SDS, 50 Mg/ml each of poly C and poly A (Collaborative Research) , 1 mM ATP (Sigma), 50 μg/ml denatured Salmon testis DNA (Sigma) and 100 μg/ml denatured E. coli DNA (Sigma, EC-DNA) . The hybridization mixtures also contain 10% Dextran sulfate. A maximum of 20 filters is transferred singly into the pre- hybridization and the hybridization mixture before being sealed into plastic bags. Hybridization with 5' end-labelled oligonucleotide probe (Gamma ATP crude from ICN: „ 10⁸-10⁹ cpm/ g, 500,000 cpm [³²P]/ml) is at 42°C for 16 hrs. All filters -are washed in 0.1 x SSC (15 mM NaCl, 1.5 mM Na-Citrate, pH 7.2), 0.1% SDS at room temperature for low stringency. High stringency washes are in 0.1 x SSC, 0.1% SDS at 51°C. The filters are exposed at -70^βC with Lightening Plus Screens (Dupont) to XAR-5 X-ray film (Kodak) that has been pre- flashed. The areas of the positive colonies are cut out from the filter, eluted with 1 ml LB broth and dilutions are plated again to purify the positive clones. The positive genomic charon 4A clone, gUHK-704, is isolated, analyzed and sequenced. The λ phage gUHK-704 is grown in E. coli strain LE392 (ATCC) and the phage DNA is purified by standard protocols. The subclone gUHK- 704Eco-pUC (-M13) is made by ligating a 2 Kb EcoRl fragment of gUHK- 704 into the plasmid pUC 13 (BRL) and the sequencing phage M13mpl8 (BRL) , respectively. The bacteria infected with the recombinant plasmid are grown to stationary phase in modified super-broth containing per liter 24 g Yeast extract, 12 g tryptone, 5 ml glyce- rol, 2 g uridine, 25 mg of ampicillin and 0.1 M K-phosphate buffer pH 7.5. No chloramphenicol amplification is done. The plasmid DNA is isolated using CsCl density gradients. The subclone gUHSK-704Eco, sequenced using the dideoxy proce¬ dure, has a gene with no intervening sequences. There are 558 nucleotides which code for 186 amino acids with 37% cysteine, 13% serine, 11% proline and 9% glutamine. A Cys-Cys-Gln-Pro repeat is found 12 times within the coding region. DNA sequence analysis shows at the 5' end of the coding sequences the elements (TATAA and CAAT box) expected from an eukaryotic promoter region. At the 3' side of the gene, after the translation termination, is a poly A addition site. The foremost feature of the gene is the high cysteine content

(37%), the presence of a 4 amino acid repeat unit (Cys-Cys-Gln-Pro) and the complete absence of any introns. ...÷ι* •- -.. *, .*■^•_-

CONSTRUCTION OF THE KER-CAT TRANSGENE

Recombinant DNA techniques used to construct the recombinant transgene are generally known and are as described in Maniatis, et al, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, (1982) . Structures 1-5 show formulas of the plas ids and fragments used for the construction of the vector pKER-CAT. The structures represent both circular and linear double-stranded DNA with initiation or transcription occurring from left to right (5' to 3'). Asterisks (*) represent the bridging of nucleotides to complete the circular form of plasmids. Fragments do not have asterisk marks because they are linear pieces of double-stranded DNA. Endonuclease restriction sites are indicated below the line by arrows and the name of the specific digestion enzyme. Capitol letters below the line are used to represent the fragment of interest for the various strands of DNA depicted.

The recombinant promoter-reporter gene construct, pKER-CAT, is produced in two steps. First, the reporter gene, CAT, is subcloned into the multiple cloning site of the vector pGEM to generate the vector pBlue-CAT. Second, the promoter region from the ultra high sulfur keratin gene pUHSK-704Eco is ligated in front of the CAT gene of pBlue-CAT.

To construct the pKER-CAT, the plasmid pSVOCAT (ATCC) is used. Recombinant genomes which express chloramphenicol acetyltransferase in mammalian cells. Mol. Cell Biol. 2(9):1044-51 (Sept. 1982). As shown in Structure 1, pSVOCAT is digested first with the restriction enzyme Hind III and then partially digested with EcoRI. The pSVOCAT is only partially digested with EcoRI so that some of the plasmids are only digested at the EcoRI site at the end of the SV40 sequence while leaving an internal EcoRI site undigested. Thus, among the fragments generated are segments which extend from the Hindlll site upstream from the CAT gene to the EcoRI site located downstream of the SV40 gene.,, that* is_^-a. fragment comprising the CAT-SV40 genes. This fragment,,is designated by BBBBBBBBBB in Structure 1.

STRUCTURE 1 pSVOCAT

CAT - SV40 5' * \ • ^■ I - * 3' - tBBBBBBBBBBBBBBBBBBBBt Hindlll EcoRI

The fragments generated are isolated from a 10 ml analytical 0.8% agarose gel by spinning them through a filter unit. After one ethanol precipitation, the fragments are ligated into the Blue script plus vector (pGEM [Stratagene Cloning Systems]). The Blue script plus vector which is an M13 sequencing vector which has a region that includes multiple cloning sites, i.e. a series of unique restriction enzyme sites. Genes to be cloned in the sequencing vector are inserted as fragments into the vector at sites in the multiple cloning sites region. As shown in Structure 2, pGEM is cut with EcoRI and Hind III within the Multiple Cloning Sites region (MCS).

The resulting plasmid, called pBlue-Cat and shown in Structure 3, is transfected into E. coli JM101 and grown as single colonies.

STRUCTURE 3 pBlue-CAT

MCS CAT-SV40 MCS 5' * 1 1 j I * 3' tBBBBBBBBBBBBBBBBBBBBBB| Hindlll The methods used for plating and screening of the recombinant plasmids have been published (Vogeli, et al., Methods Enzymol. 152:407-415, 1987). The recombinant plasmids are plated onto NEN colony hybridization membrane (Dupont) and the filters are treated following the manufacturer's recommendations. The colonies are hybridized with oligonucleotide GV-92 to isolate the colonies that contain the CAT-SV40 fragment. The^ssequence_*of oligonucleotide GV-92 is 5'-GTCTTTCATTGCCATACGGAATTCCGGATGAGCATTCATCAG. GV-92 contains the internal EcoRI restriction site found in the CAT gene. Sequence analysis by priming with the oligonucleotides GV-1 and GV-2 and GV-99 is done to confirm that the correct fragment has been cloned into the new vector pBlue-CAT. The sequence for oligonucleotide GV-1 is 5'- GTAAAACGACGGCCAGT. GV-1 hybridizes to a sequence in the Bluescribe region of the cloning vector downstream from the CAT-SV40 insert. The sequence for oligonucleotide GV-2 is 5' -AACAGCTATGACCATG. GV-2 hybridizes to a region of the cloning vector upstream of the CAT-SV40 insert. The sequence for oligonucleotide GV-99 is 5' -TTTCTCCAT- TTTAGCTTC. GV-99 hybridizes to a region of the CAT-SV40 insert which contains the CAT protein intiation codon. The oligonucleotides described may by synthesized using methods well known in the art or from commercial sources which supply custom made products.

Next, pBlue-CAT is digested with Hindlll to open the plasmid at a site upstream from the CAT gene. The ends are treated with Klenow and free nucleotides to form blunt ends. The UHS-keratin subclone gUHK-704Eco, shown in Structure 4, is digested with the restriction enzyme Fokl to generate a fragment containing the promoter region of that gene, designated as DDDDDDDDD in Structure 4. The ends are treated with Klenow and free nucleotides to form blunt ends.

STRUCTURE 4 gUHSK-704Eco

KER 5' I I 3' tDDDDDDDDDDDDDDDt Fokl Fokl

The FokI/UHK-704Eco fragment is then blunt end ligated into the blunt end cut pBlue-CAT vector. The resulting plasmids, called pKER- CAT; and shown* In Structure- 5, are sequenced using the oligonucleotide primer GV--99 to : make sure." the TATAA box region of the promoter is contained within the construct.

STRUCTURE 5 pKER-CAT ,MCS . ,MCS -^"

5'* I I I I I * 3' t |DDDDDDDDDDDDD|BBBBBBBBBBBBB| . t Clal Bgll

PRODUCTION OF TRANSGENIC MICE CONTAINING THE KER-CAT TRANSGENE The recombinant fragment containing the keratin promoter, the CAT enzyme and portions of SV40 is removed from the plasmid pKER-CAT with the two enzymes, Cla I and Bgl I, that cut within the multiple cloning site (MCS) of pGEM. The fragment is isolated and purified. This fragment is introduced into mouse embryos using the methods described in Wagner, T.E. et al, Microinjection of a rabbit 3-globin gene into zygotes and its subsequent expression in adult mice and their offspring. Proc. Natl. Acad. Sci. USA Vol. 78, No. 10 pp.6376- 6380, (Oct. 1981). Between 200 and 400 copies of the recombinant KER- CAT fragment were microinjected into each male pronuclei. Several lines of transgenic mice are generated. The copy number for the pKER-CAT construct range between 5 to 10 copies per genome. ANALYSIS OF CAT ACTIVITY IN SKIN SAMPLES

Four day old mice are shaved and sacrificed by cervical disloca¬ tion and the whole skin is removed. The skin is cut into small pieces and suspended in 0.25 M Tris pH 7.8, 10 mM EDTA at 1 mg of tissue per ml of buffer. Samples are then homogenized on ice to obtain a even suspension. Aliquots of this suspension are taken and the cells are lysed by repeated cycles of freeze-thawing. Samples are then spun in a micro centrifuge for 1 min and the supernatant is assayed for CAT activity. Sample preparations of internal organs are made as described and assayed for CAT activity to confirm tissue specificity of expression of the transgene.

ISOLATION OF HAIR FOLLICLES Using hair tweezers, clumps of at least 20 hair follicles are removed from mice. The hairs are pulled out of the skin so that traces of skin tissue, the hair bulb, remain at the root of the follicle. The bases of the removed hair are observed with magnif¬ ication to assure that the hair bulbs are present and that the hairs were not merely broken off. The hair follicles including the cells of the skin fragments are added to 100 μl of 0.25M Tris pH 7.8, lOmM EDTA buffer and disrupted by sonication. Samples are then spun in a microcentrifuge 1 .minute and the supernatant is removed- and assayed for CAT activity.

CHLORAMPHENICOL ACETYL TRANSFERASE (CAT) ASSAY Chloramphenicol acetyl transferase (CAT) is a bacterial enzyme which acetylates chloamphenicol. Radiolabeled chloramphenicol in the presence of CAT is converted into enzyme products which are distin¬ guishable from unacetylated by using autoradiography. CAT activity, if existing in a sample, can be detected by combining samples with -^Carbon, labeled chloramphenicol and allowing sufficient time to elapse for the reaction to occur. If CAT is present, the chloram¬ phenicol will be acetylated. Using Thin Layer Chromotography (TLC) , acetylated chloramphenicol, if present, can be seperated from chloramphenicol which has not been acetylated. By exposing the TLC plate to X Ray film, the presence of acetylated chloramphenicol can be determined and thus the presence of CAT in the sample can be established.

The assay used to determine CAT activity is similar whether the samples are derived from whole skin or hair bulbs. In both cases the supernatant recovered from the microcentrifuged lysed cells is incubated at 60°C for 10 min, to inactivate endogenous acetylase. Combining 25 μl of extract (0.25 x 10⁷ cells), 5 μl of 4 mM acetyl coenzyme A, 18 μl of water, and 5μl of ^•'^•^^'C chloramphenicol (Amersham- 54 mCi/mmol CAF754) , samples are incubated at 37°C for 2 hrs. The reaction is stopped by the addition of 400 μl of ethyl acetate. The samples are then mixed and spun for 1 min in a micro centrifuge. The upper organic phase is removed to a fresh tube and dried under vacuum with rotary evaporation. The samples are resuspended in 25 μl of ethyl acetate and spotted onto TLC plate (Whatman PE SIL G TLC plates) . Unreacted ~ C chloramphenicol is spotted on the plate as a control. The TLC plate is placed in pre equilibrated tank with 200 ml of chloroform:me hanol (19:1) and chromatographed until the solvent front is two-thirds of the way up the plate. -■;_.,;. , After^*. air drying, -the plate is exposed to X-ray film for 18 hours, and then developed. The control lane will contain a single spot which is unreacted 14C chloramphenicol. The presence of active CAT activity will yeild a second acetyl chloramphenicol spot running further than the unreacted ~ C Chloramphenical spot. Accordingly, expression of the CAT gene in the transgenic animal may be detected. TISSUE SPECIFIC AND DEVELOPMENTAL SPECIFIC EXPRESSION OF KER-CAT The results obtained demonstrate that the KER-CAT gene is expressed in the skin during the hair cycle in the same anterior- posterior temporal pattern as the normal murine hair cycle. As the expression of the ultra high sulfur keratin gene is strictly coupled with hair growth, the promoter region of the KER gene is an ideal tool to construct a KER-CAT fusion gene to investigate the develop¬ mental regulation of this hair specific gene in transgenic mice. The elements that control the promoter act on the recombinant plasmid by synthesizing chloramphenicol acetyl transferase enzyme in a develop¬ mental and tissue specific manner.

To demonstrate that the KER-CAT construction is being expressed in the correct tissue, CAT activity of whole skin samples from the transgenic mice are compared to that in the internal organs of the same mouse. From the results of such CAT assays, it can clearly be seen that in six day old mice the only detectable activity is in the skin and the partially purified hair follicles, demonstrating the correct tissue localization of the reporter enzyme. In order to evaluate the sensitivity of our assay system, the level of CAT activity in whole skin samples was compared to that of isolated hair follicles from the same animal. Hair follicles were isolated by plucking. CAT activity from 20 μg of whole skin protein is compared to that from approximately 50 plucked hair follicles (plucked hair samples were quantitated microscopically to determine the number of follicles) . From the result^", it is clear that the CAT enzyme can easily be detected in as few as 50 follicles.

This result also further strengthens the evidence for the tissue localization of the reporter enzyme. The plucked hair follicle 5 sample contains only follicular material, with no contaminating skin tissue.

The expression of the KER genes in mouse vibriεsa is localized to the shaft area. It was therefore necessary to confirm the presence of CAT enzyme activity in isolated mouse vibrissa^'e to demonstrate unequivocally the tissue specific expression of the reporter enzyme. Results show that the CAT activity is localized to the inner shaft area of dissected vibrissae. Twenty vibrissa were dissected from 3 day old mice and the outer capsular materail removed by microdissection. A comparison of the CAT activity between the shaft and the capsular material show the localization of expression to the hair shaft cells of the vibrissa. Additional experiments demonstrate that CAT activity can be detected in single vibrissa samples from transgenic mice.

Validation of transgenic mice as a possible animal model for investigating agents that influence hair growth is done by showing that the synthesis of the CAT enzyme is regulated in the correct developmental manner; CAT activity correlates with hair growth. Two transgenic mice are assayed during the first and second hair growth phase (anagen) . Samples are also obtained during the "down phase" (telogen) . The results confirm that the reporter enzyme is indeed being expressed at the correct developmental time.

Male mice with evidence of an impaired hair cycle have also been analyzed. In these animals, the normal hair cycle seen in young mice converts to a "patchy" growth phase. The mice are shaved to deter¬ mine areas of hair growth and skin samples were taken from areas of active hair growth and compared to non-growth areas from the same mice. The results of this comparison show that the CAT activity is only found in areas of active hair growth, demonstrating that the pattern of CAT expression correlates with areas of hair growth even during this later stage of hair growth in mice.

The expression of the CAT gene is directed by 650 bp of 5' flanking sequences from the previously characterized KER-704 UHSK matrix gene. The reporter gene is expressed in the same anterior- posterior temporal pattern seen in the normal murine hair cycle.

Results confirm the tissue specificity and developmental regulation of the CAT enzyme activity in the transgenic mice carrying the KER-CAT construct. The results demonstrate that the CAT gene is expressed specifically in the skin and in partially purified hair follicle preparations. The localization of expression of the CAT enzyme to the hair follicle is demonstrated using a relatively small number of plucked hair follicles. This result is further substanti- ated by the.-*analysis o-f- isolated mouse vibrissae which shows the CAT activity is confined torthe inner shaft area of the vibrissa.

The expression of the KER-CAT construction correlates with the normal murine hair cycle. The expression of the CAT enzyme is seen during the first and second anagen phases (growth phases) , while no activity is detected during the intervening telogen phase (resting phase). This result confirms that the KER-CAT construction was being regulated in the same manner as the endogenous KER matrix gene. The KER-CAT construct contains all the necessary controlling elements for the correct tissue and developmental expression of the reporter CAT gene in our transgenic mice.

Example 2

PRODUCTION OF TRANSGENIC SHEEP CONTAINING THE KER-CAT TRANSGENE As described in Example 1, the KER-CAT transgene is constructed. This fragment is introduced into sheep embryos using the methods described in Wagner, T.E. et al, Microinjection of a rabbit /3-globin gene into zygotes and its subsequent expression in adult mice and their offspring. Proc. Natl. Acad. Sci. USA Vol. 78, No. 10 pp.6376- 6380, (Oct. 1981). Between 200 and 400 copies of the recombinant KER- CAT fragment were microinjected into each male pronuclei. Several lines of transgenic sheep are generated. The copy number for the KER-CAT construct range between 5 to 10 copies per genome. Assays to detect CAT activity are performed using the protocols described in Example 1.

Example 3 ISOLATION OF CYTOKERATIN GENE

A library of Balb/C genomic DNA, digested partially with Alul and Haelll, and cloned into lambda Charon 4A by EcoRI linkers, was screened with oligonucleotide GV143, a 42-mer corresponding to region of nucleotides 157-198 of hair-specific keratin MHKal (CYTO)(Ber¬ tolino et al., J. Inv. Derm. 91(6):541-6 Dec. 1988). The actual sequence of oligonucleotide is a reverse complement. Three positive phage were plaque purified from the library, gMHK113, gMHK124, and gMHK137.

The three recombinant phage were digested separately with Rsal, Alul, and Haelll, and each assortment of fragments was cloned separately into the Smal site of ml3mp8 or ml3mpl0. Subclones containing inserts of exons were identified by plaque hybridization using restriction fragments of CYTO. These subclones were sequenced by the dideoxy method using the Klenow polymerase with reagents from the kit from Amersham. This analysis indicated that gMHK113 con- tained the gene for CYTO. The clone gMHK124 and gMHK137 contains of proteins which are also members of the acidic hair keratin family.

An EcoRI restriction map of gMHK113 (Structure 6) was obtained by the cos oligonucleotide hybridization method (Rackwitz, H. et al, GENE 30:195-200, 1984).

Structure 6 gMHK113

3.5kb 4.4kb 6.5kb .95kb

J I ! I L t t t t t

EcoRI EcoRI EcoRI EcoRI EcoRI

Southern blot analysis indicates that the clone gMHK113 contains two discreet sections which hybridize to oligonucleotides derived from cDNA sequences of the protein CYTO. This suggests that the clone contains two genes or gene fragments for CYTO. The other two clones were also shown by Southern analysis to contain multiple genes or gene fragments. All EcoRI fragments from gMHK113 were subcloned into the EcoRI site of pGEM. Sequencing was accomplished on these double-stranded subclones using T7 DNA polymerase and the reagents kit from Pharmacia-PL. Sequencing of the 4.4 EcoRI fragment in plasmid pMHK113-4.4R-8 showed that it contains the entire structural gene corresponding to CYTO with the exception of those sequences contained to the 5' side of the EcoRI site in the 5' untranslated region (nucleotides 32-37 in Bertolino et al) . Those sequences are found in the 3.5 kb fragment immediately to the left in the above restriction map contained in the plasmid subclone pMHK113-3.3-K. Twenty-eight nucleotides farther to the 5' end of the beginning of the cDNA sequence is found a TATAAA box, characteristic of eukaryotic promoters. Thus, the promoter for CYTO is found in the 3.3kb fragment. This fragment is linked to a reporter gene to construct the transgene.

Plasmid pBlueCAT (Structure 3) was digested with Hindlll, rendered ihlύnt-ended'"by-, the Klenow fragment of DNA polymerase I in --the presence¹;* of- all four dNTPs, dephosphorylated with CIAP, and ligated to the 3.5 kb EcoRI fragment excised from pMHK113-3.3-K with

EcoRI and rendered blunt-ended by the Klenow fragment. Two resulting clones, containing the promoter fragment in the correct and reverse orientation, were isolated and named respectively, pkerll3-CAT

(pCYTO-CAT) and pkerll3-CATR (pCYTO-CATR) . For the construction of transgenics, the insert containing the promoter, CAT gene, and 3' stabilizing sequences were isolated by digestion of pCYTO-CAT with Xhol and Xbal.

Example 4 ISOLATION OF ULTRA HIGH SULFUR SER GENE

The Ultra High Sulfur Ser gene is isolated and cloned as follows. A murine genomic library is screened with a synthetic oligodeoxynucleotide (LW-199) that has been derived from cDNA clone F533, a Cys-Arg-Pro ultra high sulfur clone isolated by differential screening. The sequence of oligonucleotide LW-199 is 5' -GAGACTGGCAG- CACTGGGGTCTGCAGCTGGACACACAGC. The methods used for plating and screening of the genomic DNA library, isolation and purification of phage DNA have been published. (Vogeli, et al., Methods Enzymol. 152:407-415, 1987 and Maniatis, et al, Molecular Cloning: A Laborat¬ ory Manual, Cold Spring Harbor Laboratory, NY 1982). A total of 10,000 λ Charon 4A recombinant clones are plated onto 15 cm dishes and transferred to NEN colony hybridization membrane (Dupont) . The filters are treated following the manufacturer's recommenda¬ tions. They are pre-hybridized and hybridized at low stringency in 0.9 mM NaCl, 0.2 M Tris-HCl pH 7.4, 20 mM EDTA pH 7.5, 30% formamide, 20 mM Na-phosphate buffer pH 7.4, 1% SDS, 50 μg/ml each of poly C and poly A (Collaborative Research) , 1 mM ATP (Sigma) , 50 μg/ml denatured Salmon testis DNA (Sigma) and 100 μg/ml denatured E. coli DNA (Sigma, EC-DNA) . The hybridization mixtures also contain 10% Dextran sulfate. A maximum of 20 filters is transferred singly into the pre- hybridization and the hybridization mixture before being sealed into plastic bags. Hybridization with 5' end-labelled oligonucleotide probe (Gamma ATP crude from ICN: 10⁸-10⁹ cpm/μg, 500,000 cpm [³²P]/ml) is at 56°C for 16 hrs. All filters are washed in 0.1 x SSC (15 mM NaCl, 1.5 mM Na-Citrate, pH 7.2), 0.1% SDS at 51° for high stringency. High stringency washes are in 0.1 x SSC, 0.1% SDS at 51*C. The filters are exposed at -70^βC with Lightening Plus Screens (Dupont) to XAR-5 X-ray film (Kodak) that has been pre-flashed. The areas of the positive colonies are cut out from the filter, eluted with 1 ml LB broth and dilutions are plated again to purify the positive clones. The positive genomic charon 4A clone, gUHS-Ser-M16, was isolated, analyzed and sequenced.

The λ phage gUHS-Ser-M16 is grown in E. coli strai ^" E392 (ATCC) and the phage DNA is purified by standard protocols. The subclone gUHS-Ser-HindM16 is made by ligating a.2 Kb EcoRI fragment of gUHS- Ser-M16 into the plasmid pBluescript (pGEM) . The bacteria infected with the recombinant plasmid are grown to stationary phase in modified super-broth containing per liter 24 g Yeast extract, 12 g tryptone, 5 ml glycerol, 2 g uridine, 25 mg of ampicillin and 0.1 M K-phosphate buffer pH 7.5. No chloramphenicol amplification is done. The plasmid DNA is isolated using CsCl density gradients.

The subclone gUHS-Ser-M16, sequenced using the dideoxy proce¬ dure, has a gene with no intervening sequences. There are 690 nucleotides which code for 230 amino acids with 40% cysteine, 23% serine, 15% glycine and 6.5% proline. DNA sequence analysis shows at the 5' end of the coding sequences the elements (TATAA and CAAT box) expected from an eukaryotic promoter region. At the 3' side of the gene, after the translation termination, are two poly A addition sites. The foremost feature of the gene is the high cysteine content (40%) and the complete absence of any introns.

Example 5

CONSTRUCTION OF VARIOUS TRANSGENES Constructions have been made using different reporter genes and different hair specific promoters. Three hair specific promoter have been isolated and are described above (Ultra high sulfur keratin- KER; hair specific keratin MHKal - CYTO; and Ultra high sulfur Ser - UHS-Ser). Reporter genes used include chloramphenicol acetyl transferase (CAT), Harvey Ras oncogene (RAS), SV40 T-antigen (T-ant) , Adenovirus protein E1A (ElA) and /9-galactosidase (beta-Gal). Thus, a total of three promoters and five reporter genes were used to construct fifteen transgenes each useful in generating transgenic non human mammals which express the reporter genes under the control of the hair specific promoter in a spatial and temporal manner consis- _₂₄_

tent those observed-in. hair specific proteins.

In order-. to construct each of the fifteen transgenes, the promoters are first identified and cloned -using the strategies outlined in-Examples 1,- 3 and 4. Copies of each of the five reporter gene are widely available. The gene for CAT is available as de^'scri-. bed in Example 1. The construct from which the RAS gene is supplied is described by Quaife, CELL 48:1023-34 (1987). The fragment used is removed from that construct by digestion with EcoRI and BamHl. The T antigen gene fragment was removed from the vector paA366T, described In Mahon, et al., Science 233:1622-1628 (1987). The gene fragment of E1A is removed from the plasmid pGC212, Chinnadurai, CELL 33:759-66 (1983), using EcoRI and Hindlll.

The strategy used to make each of the constructions is basically the same. First, the cloning plasmid pGEM2 is linearized by diges- tion with Smal and blunt ended. The cloning vector pGEM2 is identi¬ cal to pGEM described in Example 1 except pGEM2 contains BssHII sites on both ends of the region containing the multiple cloning sites. The promoter to be used in a construct is removed from the plasmid it is cloned in with appropriate restriction enzymes (KER - Fokl; CYTO- EcoRI; UHS-Ser - Hindlll/BspM) and blunt ended. The blunt ended promoters are ligated with the blunt ended linearized vector. The construction is then sequenced to determine the orientation of the promoter. After the orientation is determined, the reporter gene is inserted downstream from the promoter at an appropriate restriction site in the multiple cloning site region.

Reporter gene fragments are removed from the cloning plasmids by appropriate restriction enzyme digestions and the fragments are blunt ended. The pGEM2 construction containing the promoter is digested with an enzyme that will open the plasmid downstream from the promoter. The linearized plasmid is blunt ended and ligated to the blunt ended reporter gene fragment. The resulting construction is sequenced to confirm proper orientation. To use for generating transgenic animals, the promoter/reporter gene construction (trans¬ gene) is isolated from the pGEM2 construction by digestion with BssHII. The transgene can then be incorporated into the host animal genome as described in Examples 1 and 2.

Claims

1. A transgenic non-human mammal having a recombinant transgene comprising a promoter sequence from a gene which codes for a hair specific protein operatively linked to a reporter gene.

2. A transgenic non-human mammal according to claim 1 wherein said promoter sequence is selected from a group consisting of a promoter sequence from a gene for KER, a promoter sequence from a gene for CYTO, and a promoter sequence from a gene for UHS-Ser.

3. A transgenic non-human mammal according to claim 2 wherein said promoter sequence is from a gene for KER.

4. A transgenic non-human mammal according to claim 1, wherein said reporter gene is selectect form the group which consists of a CAT gene, a RAS gene, a T-antigen gene, an E1A gene, a betaGal gene.

5. A transgenic non-human mammal according to claim 4, wherein said reporter gene is a CAT gene.

6. A transgenic non-human mammal according to claim 5 wherein said promoter sequence is from a gene for KER.

7. A transgenic non-human mammal according to claim 1 wherein expression of said reporter gene may be determined by an assay which detects the presence of protein products of said expression.

8. A transgenic non-human mammal according to claim 1, wherein said transgenic non-human mammal is a rodent.

9. A transgenic non-human mammal according to claim 8, wherein said rodent is a mouse.

10. A transgenic non-human mammal according to claim 1, wherein said transgenic non-human mammal is a sheep.

11. A transgenic non-human mammal according to claim 6, wherein said transgenic non-human mammal is a sheep. 7 __26„

__-.- ■_ &. transgenic mouse having a recombinant transgene comprising a promoter sequence from a KER gene operatively linked to the coding^* sequence for a CAT gene.

13. A recombinant DNA molecule comprising a promoter sequence from a gene which codes for a hair specific protein operatively linked to a reporter gene.

14. A recombinant DNA molecule according to claim 13 wherein said promoter sequence is selected from a group consisting of a promoter sequence from a gene for KER, a promoter sequence from a gene for CYTO, and a promoter sequence from a gene for UHS-Ser.

15. A recombinant DNA molecule according to claim 14 wherein said promoter sequence is from a gene for KER.

16. A recombinant DNA molecule according to claim 13, wherein said reporter gene is selectect form the group which consists of a CAT gene, a RAS gene, a T-antigen gene, an ElA gene, a betaGal gene.

17. A recombinant DNA molecule according to claim 16, wherein said reporter gene is a CAT gene.

18. A recombinant DNA molecule according to claim 13 wherein expres¬ sion of said reporter gene may be determined by an assay which detects the presence of protein products of said expression.

19. A recombinant DNA molecule according to claim 17 wherein said promoter sequence is from a gene for KER.

20. A recombinant DNA molecule according to claim 17 wherein said promoter sequence is from a gene for CYTO.