CA2332655C - Scce modified transgenic mammals and their use as models of human diseases - Google Patents

Scce modified transgenic mammals and their use as models of human diseases Download PDF

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CA2332655C
CA2332655C CA2332655A CA2332655A CA2332655C CA 2332655 C CA2332655 C CA 2332655C CA 2332655 A CA2332655 A CA 2332655A CA 2332655 A CA2332655 A CA 2332655A CA 2332655 C CA2332655 C CA 2332655C
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scce
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mammal
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skin
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Lennart Hansson
Torbjorn Egelrud
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Priority to JP2002562148A priority patent/JP4262479B2/en
Priority to DK02720389.2T priority patent/DK1367886T3/en
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Abstract

Genetic evidence that an imbalance in the activity of serine proteases can cause severe skin disease has recently been presented. The serine protease SCCE is preferentially expressed in cornifying epithelia. Increased expression of SCCE in psoriasis has previously been reported. Increased SCCE expression also in chronic lesions of atopic dermatitis is described herein. Transgenic mice expressing human SCCE in suprabasal epidermal keratinocytes were found to develop pathological skin changes with increased epidermal thickness, hyperkeratosis, dermal inflammation, and severe pruritus. The results strengthen the idea that SCCE may be involved in the pathogenesis of inflammatory skin diseases, and may offer a new therapeutic target.

Description

SCCE MODIFIED TRANSGENIC MAMMALS AND THEIR USE AS MODELS
OF HUMAN DISEASES

FIELD OF INVENTION

The present invention relates to transgenic scce mammals and mammalian embryos, their use as models of studying human diseases, to methods of using these models for identifying compounds and compositions effective for the treatment of these diseases, and to the compounds and compositions themselves. In particular, the invention relates to transgenic mammals overexpressing a scce gene in the skin.
These model animals display a major change in phenotype characterized by a severe skin disorder and are useful for identifying compounds and compositions for the treatment of various human diseases.

GENERAL BACKGROUND

The skin as an organ is of interest from biological, medical, and cosmetological points of view. There are a large number of skin diseases that are either organ-specific, e.g. psoriasis and eczemas, or are manifestations of general disease, such as general allergic reactions. The fact that there are skin-specific diseases can be considered as a proof of the existence of molecular mechanisms that are unique for the skin. Analogously, studies on skin-specific molecular processes are of importance for the understanding and treatment of skin disorders. It seems reasonable to assume that several of these processes in one way or another are related to the most specialized function of the skin, that is the formation of a physico-chemical barrier between body exterior and interior. The physico-chemical skin barrier is localized in the outermost layer of the skin, the stratum corneum.
The stratum corneum is the most specialized structure of the skin. It is the end product of the differentiation process of the epidermis, that is the stratified squamous epithelium which accounts for the outermost portion of the skin. The majority of the cells of the epidermis consist of keratinocytes in various states of differentiation. The lowermost keratinocytes, the basal cells, reside on a basal membrane in contact with the dermis, that is the connective tissue of the skin, and are the only keratinocytes that have dividing capability. A fraction of the basal cells continuously leaves the basal membrane and goes through a differentiation process which eventually makes the cells become building blocks of the stratum corneum. In this process the keratinocytes go through a number of adaptive changes. There is an increased content of cytoskeleton consisting of epidermis-specific cytokeratins. The intermediate filaments of contiguous cells are joined to a functional unit by an increased number of desmosomes. The most dramatic changes take place during the transition from the uppermost living cell layer, the stratum granulosum, to the non-viable stratum corneum in a process usually called keratinization.
Covalently cross-linked proteins are deposited close to the inner aspect of the plasma membrane, forming a very resistant cell envelope. Furthermore a lipid-rich substance, originating in a keratinocyte-specific cell organel, is secreted to the extracellular space and, by forming lipid lamellae which surround the cells of the stratum corneum, constitutes the permeability barrier to hydrophilic substances.
Finally all intracellular structures except the densely packed cytokeratin filaments disappear.

The cells of the stratum corneum, the corneocytes, are thus non-viable. This means that the regulation of various processes in the stratum corneum must be the result of a "programming" at a state where the keratinocytes are still viable. The turnover of the epidermis, which normally proceeds in about four weeks during which the cells are part of the stratum corneum for about two weeks, is ended by means of cell shedding from the skin surface in the process of desquamation. This process is an example of "programming" of the stratum corneum. A prerequisite for the function of the stratum corneum as a physico-chemical barrier is that its individual cells are held together by mechanically resistant structures, that is desmosomes. The degradation of desmosomes, which is a prerequisite for desquamation, must be regulated so as to give a cell shedding from the skin surface which balances de novo production of the stratum corneum without interfering with the barrier functions of the tissue.
Disorders of keratinization Under a large number of pathological conditions in the skin of varying severity, there are disturbances in the keratinization process. In psoriasis there is, in addition to a typical chronic inflammation, overproduction of an immature stratum corneum resulting in the typical scaling of this disease. There is a group of inherited skin diseases characterized by a thickened stratum corneum which leads to the formation of "fish scales", the so-called ichthyoses. In several of the ichthyoses there is a decreased rate of desquamation. Although less severe than the ichthyoses, "dry skin" (xeroderma) is also characterized by a stratum corneum from which corneo-cytes are shed, not as under normal conditions as single cells or as small aggregates of cells, but as large, macroscopically visible scales. This disorder is very common among elderly people and among atopics, that is individuals with a decreased resistance to skin irritants and a disposition to develop a characteristic form of endogenous eczema. In the acne diseases there is a disturbed keratinization in the ducts of the sebaceous glands which leads to the formation of comedones and plugging. The formation of comedones precedes and is believed to provoke the inflammatory acne lesion.
Proteolytic enzymes are involved in keratinization There are several stages in the keratinization process and during the turnover of the stratum corneum where proteolytic enzymes seem to play important roles.
Certainly the disappearance of all intracellular structures except for the cytokeratin filaments occurring during the transition between viable and cornified epidermal layers must involve proteolysis. The transformation of profilaggrin to filaggrin, a protein which is believed to function in the special type of aggregation of cytokeratin filaments during keratinization, may be catalyzed by a specific proteinase. In the stratum corneum filaggrin is further degraded to low-molecular weight components which are probably important as "natural moisturizers". Furthermore there are proteolytic modifications of cytokeratin polypeptides during the keratinization process. Finally, proteolytic events are likely to play crucial roles in the degradation of intercellular cohesive structures in the stratum corneum in processes eventually leading to desquamation.
Stratum corneum cell cohesion and desquamation. The role of desmosomes Intercellular cohesion in the stratum corneum as well as in the viable parts of the epidermis is mediated to a significant extent by desmosomes. A desmosome consists of two symmetrical halves, each of which is formed by two contiguous cells.
Each desmosomal half has one intracellular part linked to the cytokeratin filaments and one part made up by glycoproteins anchored intracellularly and with trans-membranal and extracellular parts. The extracellular parts of these proteins, the desmogleins, are adhesion molecules, and through their interaction with each other in the extracellular space a cohesive structure is formed. The degradation of desmosomes seems to follow somewhat different routes in the stratum corneum of palms and soles as compared to non-palmo-plantar stratum corneum. In the latter tissue around 85% of the desmosomes disappear soon after the cells have become fully comified. The remaining desmosomes, which are preferentially located at the villous edges of the extremely flattened cells, apparently remain intact up to the level where desquamation takes place. In palmo-plantar stratum corneum the corneocytes are much less flattened, and there is no extensive degradation of desmosomes in deeper layers of the tissue. In both tissues desquamation is associated with desmosomal degradation. In ichthyotic skin as well as in "dry skin", the number of desmosomes in the superficial layers of the stratum corneum has been shown to be increased.

Many of the tissue-specific molecular mechanisms of the skin are associated with the formation and turnover of the barrier-forming outermost layer of the epidermis, the stratum corneum, consisting of cornified epithelial cells surrounded by highly organized lipids. The stratum corneum is continuously being formed in the process of epidermal differentiation. In the efforts to understand the mechanisms by which a constant thickness of the stratum corneum is maintained via a continuos desquamation of surface cells, two human serine proteases, stratum corneum chymotryptic enzyme (SCCE) and stratum corneum tryptic enzyme (SCTE) have been identified (Hansson et al. 1994 and Brattsand et at. 1999). The cloning and expression of SCCE is described in W095/00651 hereby incorporated by reference.
Both enzymes belong to the kallikrein group of serine proteases, the genes of which are localized to a short stretch at chromosome 19g13.3-19g13.4 (Diamandis et at.
2000). The expression of SCCE and SCTE seems to be restricted to squamous epithelia undergoing cornification and in which there is a need for desquamation (Ekholm et al. 2000).

Common inflammatory skin diseases may result in severe handicap by causing 5 reduced function, stigmatisation, and almost unbearable sensory symptoms. A
dominating symptom of many of these diseases is itch, which in many instances may be extremely troublesome, causing severe disturbances in many aspects of every day life and sleeping patterns of sufferers. In atopic dermatitis, affecting more than 10% of children at some point of their childhood, pruritus is a major diagnostic criterion and always present in active disease. It has even been stated that "atopic dermatitis is an itch that when scratched erupts", and that "pruritus must be considered a quintessential feature of atopic dermatitis" (Beltrani, 1999).
The mechanisms of itch are poorly understood, and available treatments are often unsatisfactory. This may be due, at least in part, to lack of satisfactory animal models (Greaves and Wall, 1996).

In inflammatory skin diseases such as psoriasis and atopic dermatitis evidence in favour of a central role for the immune system in pathogenesis is overwhelming. It seems likely that the development of the various disease-specific skin lesions and signs is the result of interactions at the cellular and molecular level between the immune system and skin-derived structures and molecules. In most studies aimed at understanding these interactions focus has been on cytokines, growth factors, and adhesion molecules. Although many of these components are produced by skin cells, they are not unique for the skin, but are more or less generally present in cells and tissues throughout the body. This fact may cause problems in e.g. development of skin-specific therapies. The situation would be different if one could find a truly skin-specific structure or molecule with a central role in the pathophysiology of inflammatory skin diseases. The present invention present new evidence that the serine protease stratum corneum chymotryptic enzyme (SCCE) may belong to this category of skin-specific molecules.
SUMMARY OF THE INVENTION

The present invention relates to results from studies aimed at elucidation of the possible involvement of one of these proteases, SCCE, in skin pathology. The human and murine scce-genes were characterised, and transgenic animals overexpressing human scce mRNA produced. The only gross phenotypic changes observed in these animals were found in the skin, which showed histologic changes with several similarities to those seen in inflammatory skin diseases such as in the chronic stages of atopic dermatitis in humans. In addition, the transgenic animals showed signs of severe itch. Evidence of over-expression of SCCE in chronic lesions of atopic dermatitis in humans was also found corresponding to what has recently been shown in psoriasis (Ekholm et al. 1999). Taken together, the results give support for the idea that SCCE and related enzymes may be involved in the pathophysiology of itchy inflammatory skin diseases, and thus that SCCE may be a potential target for organ-specific treatment strategies. The transgenic animals of the invention may provide a new model for further studies of itch mechanisms and the testing of potential compounds and compositions for relieve of various skin diseases where itch is a component.

The human SCCE gene was isolated from a human leukocyte genomic library cat.
no. HL 1111 j lot # 3511 (Clontech, CA) by using cDNA probes derived from the human scce cDNA. Overlapping clones were isolated and the entire structural gene was sequenced by automated DNA sequencing and analysed by AB1377 (Applied Biosystems, Foster City, CA, USA). The entire sequence can be found using Gene Bank accession no AF 332583.
Table 1 Human SCCE [org=Homo sapiens] Homo sapiens stratum corneum chymotryptic enzyme gene, complete cds. (SEQ ID NO:3.) TACCACATTiTCTTAATCCAGTCTATCACTGATGGACATTTAGGTTGATTCCCTGT
GTTTGCTGTTGTCAATAGTTCTACAATGAACGTACGTGTCCATGTGTCTTTAAAC
AGAATGATTTATATTCCTTTGGGTACACACACTGGGGCTTATGAGAGGGTGGAG
AGTGGGAGGAAGGAGAGGATCAGAAAAAAATAACTAATGGGTACTAGGCTTAAT
ACCTGGGTGATTAAATAATCTGTATAACAAACCCCCATGGCGCACGTTCACCTA
CGCAACAAACCTGCACATCCTGCACATGTACCCCCGAACTGAAAAGTTAAAAAA
AGAAAAATAAATATTTGCTTATAAATTAATAAATGAAGCCCTCAAAAATGTTCTAT
TAGATAATGTTAAGTACAGACATTTTTGTTATAAATACATAATATACAAAGAAATC
TATGTATAACATGATTAAAATGACCATAAGAACATAGATCCTAAACATGGCAAAT
ATTAGTGG GGTGGGGTTAGGGAAAG CGTTGTTTTfAACTTACACCTCTCTGTTA
GAGTTGGGAATGGGTTCAGGCGTAATTACAGGCACGACTGGGATCAGCTTGGA
CAAGTTCCCCCAGGCGGGCCAGAATTAGGATGTAGGGTCTAGGCCACCCCTGA
GAGGGGGTGAGGGCAAGAAAATGGCCCCAGAAGCCGGGCGCAGTGGCTCACG
CCTGTAATCCCAGCACTTTGCGGGGCCGAGGCGGGCACATCATGAGGTCAGGA
GATCGAGACCATTCTGGCCAACATAGTGAAACCCGGTCTCTACTAAAAATACAA
AAATTATCTGGGAGTGGTGGTGCGTGCCTGTAATCCCAGGTACTCGGGAGGCT
GAGGCAGGAGAATCACTTGAACCTGGGAGGCGGAGCTGGCAGTGAGCCGAGA
TCGCGCCACCGCACTCCAGCCTGGCGATAGAGAGAGACTCCATCCAAAAAAAA
GAAAGGAAGGGAGGGAGGGAGGAGGGAAGAAAGAAAGAAAACCGCCCCAGAG

TCCTGCCCCTCAGACCCAGCGCACCGGGTCCCTCCCCCGAGAGCAGCCAGGA
GGGACTGTGGGACCAGAATGTGCGGGGGCGCAGGAGCTGGGCACCGCCCGT
CCTTCGGAGGGAGGGTGGAGAGAGAGTGCAGTGGTGCCAATTGCTCTCGCTG
CGTCAGGGTTCCAGATAACCAGAACCGCAAATGCAGGCGGGGGTGTCCCAGAG
TCGGCTCCGCCTGCACCCCAGGGCGCTGGGGCCGGGCATGGGGCGGGGGGT
GATATAAGAGGACGGCCCAGCAGAGGGCTGAAGATTTTGGAGCCCAGCTGTGT
GCCAGCCCAAGTCGGAACTTGGATCACATCAGATCCTCTCGAGGTGAGAAGAG
GCTTCATCAAGGGTGCACCTGTAGGGGAGGGGGTGATGCTGGCTCCAAGCCTG
ACTCTGCTCTCGAGAGGTAGGGGCTGCAGCCTAGACTCCCGGTCCTGAGCAGT
GAGGGCCTGGAAGTCTGCAATTTG GGGCCTTTTAGGGAAAAACGAACTACAGA
GTCAGAAGTTTGGGTTCCACAGGGAAGGGCAAGATCGGAGCCTAGATTCCTGG
GTCTCTAGG GATCTGAAGAACAGGAATTTTGGGTCTGAGGGAGGAGGGGCTGG
GGTTCTGGACTCCTGGGTCTGAGGGAGGAGGGCCTGGGGGCCTGGACTCCTG
GGTCTGAGGGAGGAGGGGCTGGGGGTCTCGACTCCTGGGTCTGAGGGAGGAG
GGGCTGGGGGCCTGGACTCCTGGGTCTGAGGGAGGAGGGGCTGGGACCTGG
ACTCCTAGGTCTGAGGGAGGAGGAGCTGGGGCCTGGACTCCTGGGTCTGAGG
GAGGAGGGGCTGGGGCCTGGACTCCTGGGTCTGAGGGAGGATGGGCTGAGG
CCTAGACTCCTGGGTCTGAGGGAGGAGGGGCTGGGGCCTGGACTCCTGGGTC
TGAGGGAGGAGGGGCTGGAGCCTGGACTCCTGGGCCTGAGGGAGGAGGGAC
TGAGACCTGGACTCCTAGGTCTGAGGGAGGAGGGACTGGGACCTGGACTCCT
GGGTCTGAGGGAGGAGGAGCTGGGGGCCTGGACTCCTGGGTCTGAGGGAGG
CGGGGCTGGGGGCCTGGACTCCTGGGTCTGAGGGAGGAGGGGTTGGGGCCT
GGACTCCTGAGCCTGAGGGAGGAGGGACTTGGACCTGGACTCCTAGGTCTGA
GGGAGGAGGAGCTGGGGGCCTGGACTCCTAGGTCTGAGGGAGGAGGGGCTG
GGGGCCTGGACTCCTGGGTCTGAGGGAGGAAGGTGCTAGGGTCTGGACTCTT
GGGTATGAGGGAGGAGGAGGTTAGGGGTCTGGACTTCTGAGTGTAAGGAAGG
AGAGGCCAGAGAAAGGAATTTCTGGGTCTGAGGGAGGAGGGGCTGGGGTTCT
GGACCCCTAGGTCTGAGGGAGGAGGGGCTGGGGCCTGGACTCCTGGGTCTGT
GGGGGGAGGGGCTGGGGCCTGGACCCCTGGGTCTGAGTGGGGAGGGGCTGG
GCCTGAATGCTTTCTCCTTCTCAGCTCCAGCAGGAGAGGCCCTTCCTCGCCTG
GCAGCCCCTGAGCGGCTCAGCAGGGCACCATGGCAAGATCCCTTCTCCTGCCC
CTGCAGATCCTACTGCTATCCTTAGCCTTGGAAACTGCAGGAGAAGAAGGTGAA
AGCTGGACTGGGAAGTCTGACCTCACCTCAGGGCCCCCACTGACCCTCTCCAA
GGAGTCCCTGAGTCAGAACCCTTCCCTCCTCAAACAGCTTCCATCCTGGGAGG
ACCAGACTGTCGGCTGAAGCCCCCGCTCTTCCTGCTTCTGCTGACTCAGGGGG
TCTCTGTCCCCTCCAGGCCCTGCCTCCTGTGCTCAGGGTCTCTCTGTGGTTCCC
CAGATGAGATGCGCCTCCTGGGTTTCTGAGTGGGCTCCTTCTGTCTGTCTCTAT
CCCTATCTCTTGCTTTCTCTGTATTTCTCCACACATTTTCATCTGTCTCTGTCCAT
CTCTGACTCTGGGAATCCCTGAGGTGCAGCCTCAGCCTTCCCCTAATGCTAGCT
ACCCACATGCTCCTCCATGTCTCCATCCAGCCCAGGGTGACAAGATTATTGATG
GCGCCCCATGTGCAAGAGGCTCCCACCCATGGCAGGTGGCCCTGCTCAGTGG
CAATCAGCTCCACTGCGGAGGCGTCCTGGTCAATGAGCGCTGGGTGCTCACTG
CCGCCCACTGCAAGATGAAGTAGGTGCCACCCAAGTCTCTGCTGGAGGTGCGC
CAGCATCTCCAGCTCGCTATGGGGGTGGAAGGGCAGTCTTTCTGTGCCTACGG
CTCTATTCTCCTCTCTCTGGGTCTCTGTCCCCCTCTCTCTGGGCCTCTGTACCC
CCTCTCCCTGGGGCTCTGTCCCCCTCTCTCCCTGGCTCTCTGTCTCCCTCTCTC
TGGGTCTCTGTCCCCCTCTCTCTGGATCTCTGTTCCCCTCTCTCTGTGTCTCTGT
CCCCCATTCTCTCTAGGTCTCTGTTCCCCCTCCTCTCTCTCTGGGTCTCTGTCC
CTCTCTCTCTGGTCTCTGTCCCCCTCTCTCTCTGGATCTCTGTCCCCCTCTCCCT
GGGCCTCTGTACCCCCTCTCCCTGGGGCTCTGTCCCCCCTCTCTGGGTCTCTG
TCTGCCTTTCTCTCTGGATCTCTGTTCCCCTCTGTGTCTCTGTCCCCCTCTCTCT
CTGGGTCTCTGTTCCCCCTCCTCTCTTTCTGGGTCTCTGTCCCTCTCTCTCTGG
GTCTCTGTCCCCCTCTCTCTCTGGTCTCTGTTCCCCCTCCTCTCTCTCTGGTCTC
TGTCCCTCTCTCTCTGGGTCTCTGTCACCCTCTCTCTCTGGGTCTCTGTCACCC
TCTCTCTCTGGTCTCTGTTCCCCCTCCTCTCTCTGTGGGTCTCTGTCCCTCTCTC
TCTGGGTCTCTGTTCCCCTCTCTCTCTGGTCTCTGTTCCCCCTCCTCTCTCTCCG
GATCTCTGTCCCCCTCTCCCTGGGGCTCTGTCCCCCTCTCTCCCTGGCTCTCTG
TCTTCCTCTCTCTGGGGCTCTGTCCCCCTCTCTCTCTGGTCTCTGTTCCCCTCTC
TCTGGGTCTCTGTCCCTCTCTCTCTGGGTCTCTGTCCCTCTCTCTCTGGATCTCT
GTCCCCCTCTCCCTGGGCCTCTGTACCCCCTCTCCCTGGGGCTCTGTCCCCCT
CTCTCTGGGTCTCTGTCTGCCTTTCTCTCTGGATCTCTGTTCCCCTCTGTGTCTC
TGTCCCCCTCTCTCTCTGGGTCTCTGTTCCCCCTCCTCTCTTTCTGGGTCTCTGT
CCCTCTCTCTCTGGGTCTCTGTCCCCCTCTCTCTCTGGTCTCTGTTCCCCCTCC
TCTCTCTCTGGTCTCTGTCCCTCTCTCTCTGGGTCTCTGTCACCCTCTCTCTCTG
GGTCTCTGTCACCCTCTCTCTCTGGTCTCTGTTCCCCCTCCTCTCTCTGTGGGT
CTCTGTCCCTCTCTCTCTGGGTCTCTGTTCCCCTCTCTCTCTGGTCTCTGTTCCC
CCTCCTCTCTCTCCGGATCTCTGTCCCCCTCTCCCTGGGGCTCTGTCCCCCTCT
CTCCCTGGCTCTCTGTCTTCCTCTCTCTGGGGCTCTGTCCCCCTCTCTCTCTGG
TCTCTGTTCCCCTCTCTCTGGGTCTCTGTCCCTCTCTCTCTGGGTCTCTGTCCCT
CTCTCTCTGGATCTCTGTCCCCCTCTCTCTCTGGGTCTCTGTTCCCCTCTCTCTG
GGTCTCTGTCCCCTCTCCTCTCTCTGTGTCTCTCTCCCCCTCCTCTCTCTGTGTC
TCTGTCCCCCCTCCTATCTCTGTGTCTCTCTCCCCCCTCCTCTCTCTGGGTCTCT
GTCCCCCCCTCTCTGGGTCTCTGTCTCCCTCTCTCTGGGGCTCTGTCCCCCTCT
CTCTCTGGATCTCTGTTCCCCTCTCTCTGGGTCTCTGTCTCCCCTCCTCTCTCTG
TGTCTCTGTCCCCCCTCCTCTCTCTGGGTCTCTGTCCCCACCCCGTCCCCCAGG
TCTTTGCACACCCTCTCTGTCACAGTGTCTCTTCTGAATCTGTGAATGTCACTCC
TCGCAGTGAGTACACCGTGCACCTGGGCAGTGATACGCTGGGCGACAGGAGA
GCTCAGAGGATCAAGGCCTCGAAGTCATTCCGCCACCCCGGCTACTCCACACA
GACCCATGTTAATGACCTCATGCTCGTGAAGCTCAATAGCCAGGCCAGGCTGTC
ATCCATGGTGAAGAAAGTCAGGCTGCCCTCCCGCTGCGAACCCCCTGGAACCA
CCTGTACTGTCTCCGGCTGGGGCACTACCACGAGCCCAGATGGTAGGTGGCCT
CAGTGACCCAGGAGTGCAGGCCCCAGCCCTCCTCCCTCAGACCCAGGAGTCCA
GGCCCCCAGCCCCTCCTCCCTCAGACCCAGGAGTCCAGGCCTCAGCCCCTCCT
CCCTCAGACCCAGGAGTCCAGGCCCCCAGCCCCTCCTCCCTCAGACCCGCGA
GTCCAGACCCCAGCCCCTCCTCCCTCAGACCCAGCAGTCCTGGGCCCCAGACC
CTCCTCCCTCGGAACCAGGAGCCTGAACAACAGCCCTTCTGGTCCTCGCCCCC
ATCCTCTCTGACTGACAGCTCTCCCTGCTCCTCCCTGCAGTGACCTTTCCCTCT
GACCTCATGTGCGTGGATGTCAAGCTCATCTCCCCCCAGGACTGCACGAAGGT

TTACAAGGACTTACTGGAAAATTCCATGCTGTGCGCTGGCATCCCCGACTCCAA
GAAAAACGCCTGCAATGTGAGACCCTCCCCCCCAATTCCTCCCCAGTCCTGGG
TACCCTGTCTGCATGCCCCAGGGACAGAGCTTGACCCAAGTGACTGGGTACCA
AGCCCGGCCTTGCCCTCCCCCCAGGCCTGGCCTCCTCAGCTTTTTCCACCTCA

GGATGGGACAGAGAGTTTAATAGGGGTGAGAAAGTGGGGGTGGGACCAGGGA
AGGAGACTGAGGTGCTGGCCTCAGGCCCAAACCCTAAGGGGGCACCAAAAACC
TCAGTGATTGAGATAAATCATAATGCAATATTTAAAAATAAAAATAAAAACTCATG
CAGAAGTCCATGATGGACAAAATGTCACATTTTAAATAAAGAGCAGGTGGATCTT
GCACTGTCTTCATTTAAAATGTGGATACCATGCCCATCATGCAGTTTTATGTATT
ACATTTGATTTCGTTAAGTACTGCATTGAAGTATTGTGTATTGCAGTTACTGAGAT
TTTGTGCCTGAAGCTGATGACTCACTCACCTGACCCTGGCCCTGGTCCCGGGG
AAAACACTCTTTCTCTCCACCTCCTCTCTGTTCCCTCTTTCTGGCCTTTTGTCATC
CCCTCTGTTTCTGAACAGTCTTCCCACATCTCTCTTTGTGACATAATTTCATTTCA
TTCTTTTCCTCTTTGTTTTTTCTCTGTGTTGAGCTAGCTTGCTCTCCCTCCCTTGT
TCTCTCTCCATGCCCTCCTCTCTGCTCTCTGTCTTCTCCCTCTTTCTCTTGCTTCT
CTCTCTCTCCTCCCCTCCCTCTCTCCTCTCCCTGCCCCCCTGCTCTCTCTTTTIT
CCTCTCTCTCTGTCTCCTCTCTGGCCCTCTCCTCTTTCTCTCTCTCCCCCACTTC
TCTGTCTCTCTTCATCTCTCTCCCTCATCTCTCCTTGCCCCCTCCTTTTTACTGTC
TCTCTCTTTCTCTTTCTTCTATCTCTCTCCTCTCCCCGCCGCTCCCCCATCTCTG
TCTTTCTTTCTCTCTCTTTATTCTCCTCCTCTCTTCCAGTCTCTCTCTCCTCTCCC
CACCCCCACCCCATCTCTCTCCCCACACCTTCCCCCCCTTTCTCTTTGTCTCTCT
CTTCTACCTCTTTCTTCTCCACCCCCATCTCTCTCTCTCTTCTCTTCCCACACCCT
CCCCATCTCCCTCATCTCTTTGTCTGTCTCTCTTCTCCCTCCTTCTTTTCCACCC
CCATCTCTCTGTCTCTCTCTCTCCCCATACCCTTTCCCTCTTCCTCATCTCTCTTT
GTCTCTCTCTCCTTTCCCTCTTTCTTCTCCACCTCCAACTCTCTCTGTCTCTCCA
CACCCATCCTCCTTGCTCACATCTGCACCTTCAGCTGTCAGGGGATGTGGGATG
GTGAGTGTTAGGGATAGAGGAGATGGGAGAGAGATGACTGTCCTAGAGAATAG
GGTGTTCCCCTTCTCCCCTGGTGAGGGCCAGTTTCATGAATGTGCAAGCTCTGC
ACGGACACAGAGCCCCACACTCAGAAGGGTCTCAAACTTAGTCTAATGCATTCC
TGCTGTTGTCTTGAAATTCTCAATAATTTTTGAACAAAGGGCCCTGCATTTTCGTT
TTGCACCAAGTCCTGTAAATTATGTAACTGGTCTTCACCCTGGTCTCCGAGACC
ATCGTGTCCCCCTTTCCTGCGCCACAGGGCACGCATCCACCCCTTGGAGATGA
TGTTCCTTCTCCCACTAGCTTGGAGCAGGGTCCTTAACATTGGAAAATAAAGAG
TGCTCTGATCCTGGAAGCCCCACCCCTTCTCTGCAATTGGTCTCATTGGCCAAG
GGTCAAACCAGTGTCTTCAAAGGACCTAGTGTGTCCCTAGCACTAGCTCTCCCA
TTAGTCCCCAGAGACAATGAGTCTCTTCTCATTGGCTATGGTGGAAGTCCATAAT
CTGCAAGACAAAGACCGATAACTGAGGAATGTATGAGAATGAGTTGGGCTTTGA
TCTGAAGCCAAAGTTAATCTCCGGCTCTATTCCCTCTAGGGTGACTCAGGGGGA
CCGTTGGTGTGCAGAGGTACCCTGCAAGGTCTGGTGTCCTGGGGAACTTTCCC
TTGCGGCCAACCCAATGACCCAGGAGTCTACACTCAAGTGTGCAAGTTCACCAA
GTGGATAAATGACACCATGAAAAAGCATCGCTAACGCCACACTGAGTTAATTAA
CTGTGTGCTTCCAACAGAAAATGCACAGGAGTGAGGACGCCGATGACCTATGA
AGTCAAATTTGACTTTACCTTTCCTCAAAGATATATTTAAACCTCATGCCCTGTTG
ATAAACCAATCAAATTGGTAAAGACCTAAAACCAAAACAAATAAAGAAACACAAA
ACCCTCAGTGCTGGAGAAGAGTCAGTGAGACCAGCACTCTCAAACACTGGAAC
TGGACGTTCGTACAGTCTTTACGGAAGACACTTGGTCAACGTACACCGAGACCC
TTATTCACCACCTTTGACCCAGTAACTCTAATCTTAGGAAGAACCTACTGAAACA
AAAAAAATCCAAAATGTAGAACAAGACTTGAATTTACCATGATATTATTTATCACA
GAAATGAAGTGAAACCATCAAACATGTTCCAAAAGTACCAGATGGCTTAAATAAT
AGTCTGGCTTGGCACAACGATGTTTTTTTTCTTTGAGACAGAGTCTCTGTTGCTT
GGGCTGCAATGCAGTGATGCAATCTTGGCTCACTGCAACCTCCGCCTCCTGGG
TTCAAGTGATTCTCGTGCTTCAGCCTCCCAAGTACCTGGGACTACAGGTGTGCA
CCACCACACCAGGCTAATTTTTTGTGTATTTTTACTAGAGACAGGGTTTCACCAT
GTTGGCCAGCGTGGTCTTGAACGCCTGACCTCAGATGATCCACCCACCTTGGC
CTCCCAAAGTGCTGGGATTACAGGCATGAGCCACCACGGCCAGCCCACAATGA
TATTACAAACCTATTAAAAATGATACTTAGACAGAATTGTCAGTATTATTCAAGAA
CATTTAGGCTATAGGATGTTAAATGACAAAAGGAAGGACAAAAATATATATGTAT
GTGACCCTACCCATAAAAAATGAAATATTCACAGAATCAGATCTGAAAACACATG
TCCCAGACTGCATACTGGGGTCGTCATGAGGTGTCTCCTTCCTTCTGTGTACTT
TTCCTTGAATGTGCACTTTTATAACATGAAAAATAAAGGTGGGGAAAAAAGTCTG
AAGATCTAAGATTGGAGAGAGGTGACCTTTCAGGAAGGGAGACTAGAAAGAAAT
ATGTGCCTGGTTTTGAGCCCTGGTCCTGCCGGCCCTGTTCCAGGGCATATTTCC
ATTTCCCAGATCTCAGTTTTTCCTGTCTGTAAAATGGGAGAGAGAGGAAAGGAT
GGAGAGAGGAAGAAGGAAGGGAGGAGGGAGGAGAGAACAGGCCAACTTCATC
AGCGTGGGAAGGGGTGTGAAAGTGTTTCTGAGCATCTCACGAGTGACAAGTGA
GGAGGGAGGCTGGCGGTTTTCAGAGGGATTGGGATGACAGTAGACAGGACAC
AGGGGTCCCACAGGGGTCTGCCAGAAGTAAGCAAACAGTGCCGGAGGAAGAT
GGTGGCACCTGCTCCCCAAGAAGGGAGGGAAAGGAACCTCGGGAAGCGGGTA
GGATGAGGGAGGAGTCCTCTGTGACTCAGAGCCTGGCCACAGCCCCAGCCATC
TAACATCAAAGATCCTCTGTGTGGTCACACCTCAGACGCTGCTGACCGAGGAGC
CACTCCAGCCCAGGACACGCCCTCCTACCTGTTCTTCCTGTTTTTCTCCCAGAA
TTC

To isolate the murine scce gene cDNA probes derived from the murine scce cDNA
(Ekholm et al. 1999) were used to screen and isolate clones from a 129SVJ
Lambda Fix II genomic library cat. no. 946306 (Stratagen, La Jolla, CA). The entire gene sequence was determined and analysed as described above. The entire sequence can be found using Gene Bank accession no AF 339930 and is not shown here.
The amino acid sequences (as deduced from cDNA) of human and murine SCCE
show around 80% similarity (Hansson et al. 1994 and Backmann et al. 1999).
The genomic organization of the human and murine scce structural genes are schematically shown in Figure 1. The most apparent difference between the structural genes from the two species is that the introns are longer in the human scce gene. As seen in Figure 1 the scce genes from man and mouse both contain six exons, here indicated as black boxes, and have the translational start located in exon 2, and the stop codon in exon 6. Overall the organization of the exon-intron structures of the two genes is similar but due to shorter introns, the murine gene is smaller, approximately 4kb as compared to 8 kb. In the human gene, the translation initiation site is found 60 nt downstream the 5'-end of exon 2, and a potential TATA-box approximately 35 bp upstream of exon 1. Similarly, the murine initiation codon is positioned within the second exon, 39 nt downstream of the intron-exon junction.

To generate transgenic mice with a modified regulation of expression compared to the endogenous scce, recombinant human scce gene under control of the SV40 early enhancer and promoter element was constructed as described in example 2.
Three founders shown to be transgenic for SV40e-hscce integrated at a single site were obtained and lines were established by further breeding in C57BU6JxCBA
mice. As expected, initial characterization of the three lines revealed very large i differences in levels of recombinant scce expression (see below). In line #1010, which has the highest hscce transcript levels, skin abnormalities were apparent, whereas in the two other lines no skin changes or other gross phenotypic deviations could be observed. For further detailed comparative studies of the #1010 transgenics one of the lines with apparently normal phenotype (#107) and non-transgenic littermates were included as controls.

The importance of the transcriptional regulation of the recombinant scce gene was demonstrated by the results achieved from other variants of transgenic mouse models. In these experiments different regulatory elements were inserted upstream of a genomic fragment comprising the human scce structural gene. For example, the mouse/human keratin 14 promoter (Vassar et al.) was utilised with the idea to target the expression of recombinant scce to more basal cell layers than is the normal distribution for endogenous SCCE. Also, a long genomic fragment containing the native human scce upstream regulatory sequence including the promoter was tested and evaluated. In these experiments the resulting transgenic mice neither showed any signs of altered skin morphology nor signs of itch. The detailed construct for recombinant scce expression comprising the sv4O early enhancer and promoter elements resulted in a surprisingly restricted distribution of expression and a transgenic mouse having very interesting changes in skin biology and clear signs of itch. This phenotype and expression pattern were surprising since the sv4O
early regulatory sequences normally mediates high level transcription in proloferative cells whereas here the strongest expression in differentiated corneocytes was observed.

To the knowledge of the present inventors, this is the first report of a mouse model for itchy inflammatory skin diseases produced by genetic manipulation of an enzyme which may be skin specific. The SV40-scce transgenic mice are likely to give new insights into the pathophysiology of itchy human skin diseases and provide a new animal model for development of treatments directed at an organ-specific target. At the RNA-level expression of SCCE can be detected in several organs, although not at levels comparable to skin (Hansson et al. 1994 and Brattsand et al. 1999).
In non-malignant tissues SCCE protein has so far been found only in high suprabasal cells in squamous epithelia undergoing cornification and with a need for desquamation (Ekholm et at. 2000 and Ekholm et al 1998). The present inventors show here that over-expression of SCCE in mice at a site close to where it is normally expressed leads to a condition which to some extent simulates common, often debilitating human skin diseases such as atopic dermatitis and psoriasis.

In SV40-scce transgenic mice with phenotypic skin changes expression of transgenic SCCE, RNA as well as protein, was found also in other organs, especially small and large intestine, and lungs. The fact that no pathological changes were seen in these organs may be explained either by a resistance or unresponsiveness to effects mediated by SCCE, or by a lack of SCCE-activating enzymes in unaffected organs.
SCCE, human as well as murine, is produced as an inactive precursor which is converted to active protease by tryptic cleavage at a conserved site (Hansson et al.
1994 and Backmann et al. 1999). The enzyme responsible for SCCE-activation in the epidermis has not yet been identified.

The SV40-scce transgenic mice had a somewhat unexpected expression pattern of SCCE in the skin. Since the transgene construct contained the SV40 promoter it was expected to find the highest expression at sites with proliferating keratinocytes, i.e. in the basal layer of the epidermis and in hair follicles. On the contrary, no evidence of SCCE-expression was found in basal cells. Instead, as found by immunohistochemistry, there was expression in suprabasal cells, the intensity of which continuously increased with distance from the basal layer. This pattern is similar to that seen in psoriasis (Ekholm et al. 1999) lesions and chronic lesions in atopic dermatitis in humans. A possible explanation may be that the human scce-gene contains internal regulatory elements which suppress its expression in undifferentiated keratinocytes in the epidermis.

The mechanisms by which SCCE can cause a thickened epidermis with hyperkeratosis, a dermal inflammatory infiltrate, and itch remain to be elucidated.
According to the current view the SCCE precursor is synthesised in high suprabasal epidermal keratinocytes and stored in lipid rich lamellar bodies. In the process in which a terminally differentiated keratinocyte is transformed from a viable cell to a corneocyte, i. e. a building block of the cornified surface layer of the epidermis - the stratum comeum - the contents of the lamellar bodies, including SCCE-precursor, are secreted to the extracellular space, where conversion of pro-SCCE to active protease is taking place (Sondell et al. 1995). One possibility is that SCCE, which has been activated as postulated, diffuses through the epidermis to the superficial parts of the dermis, thereby inducing epidermal thickening as well as dermal inflammation and activation of itch-mediating nerve endings. In previous studies on proteases as 5 potential mediators of itch the enzymes were injected intradermally in human volunteers. Injection of trypsin and mast cell chymase caused itch by a mechanism believed to involve release by mast cells of histamine, whereas the itch caused by intradermally injected kallikrein appeared to be mediated by a mechanism not involving histamine (Hagermark et al. 1972 and Hagermark (1974). Treatment with 10 an antihistaminic drug appeared not to relieve the itch seen in SV40-scce transgenic mice (A. Ny and T.Egelrud, unpublished observation). The fact that SCCE
detected by immunohistochemistry in skin of SV40-scce transgenic mice was confined to superficial parts of the epidermis, suggests that the dermal inflammation and the pruritus observed in these mice were not direct effects of active SCCE. In addition, 15 signs of itch were not seen before the age of around 5 weeks, whereas overexpression of SCCE was found also in younger animals. An alternative explanation to the changes and signs caused by over-expression of SCCE in the epidermis could be that an increased proteolytic activity in the transition zone between viable epidermal layers and the stratum corneum may lead to release of mediators, which diffuse to other parts of the skin where they cause epidermal changes, dermal inflammation, and pruritus. A third possibility is that the epidermal hyperkeratosis and achantosis, dermal inflamation and pruritus are results of adaptive respones to a deterioration of the barrier function of the stratum corneum caused by increased proteolytic degradation of structures responsible for intercellular cell cohesion in the cornified layer. The proliferative response of the epidermis could be a result either of a direct effect of the released mediators on keratinocytes, or an effect which is secondary to the dermal inflammation.

Recently a direct association between a defective epidermal barrier function and aberrant proteolysis in an inherited human condition with severe skin disease was described. Strong evidence was presented that the disease-causing mutations in Netherton's syndrome are localized to a gene coding for a precursor of serine protease inhibitors (Chavanas et al. 2000). These results, together with the present results, suggest that increased activity of serine proteases in the skin may indeed play a significant role in skin pathophysiology. They also provide incitaments for further exploring of possible new therapeutic principles for skin diseases.
In accordance with an aspect of the present invention, there is provided a transgenic mammal cell, having integrated within its genome a nucleotide sequence (SCCE-construct) comprising a heterologous nucleotide sequence coding for a human stratum corneum chymotryptic enzyme (SCCE), operably linked to a SV40 early promoter that drives expression of said heterologous nucleotide sequence in skin, wherein a transgenic mammal comprising the transgenic mammal cell exhibits epidermal hyperplasia and hyperkeratosis and a mild cellular inflammatory reaction of the skin.

In accordance with an aspect of the present invention, there is provided a transgenic mammal cell, having integrated within its genome a nucleotide sequence (SCCE-construct) comprising a heterologous nucleotide sequence of SEQ ID NO:1 coding for a human stratum corneum chymotryptic enzyme (SCCE), operably linked to a SV40 early promoter that drives expression of said heterologous nucleotide sequence in skin, wherein a transgenic mammal comprising the transgenic mammal cell exhibits epidermal hyperplasia and hyperkeratosis and a mild cellular inflammatory reaction of the skin.
SUMMARY OF THE DRAWINGS

Figure 1.

Organization of the human and murine structural genes and the recombinant sv40e/hscce gene. The six exons are indicated as black boxes. The translational start sites, located in exon 2, are indicated with "ATG", and the stop codons in exon 6 with "TAA". Also the position of the sv4Oe transcriptional regulatory element in the construct used to generate transgenic is indicated by an arrow.
Figure 2 pS99.

16a Figure 3.

A: Real time quantitative PCR analyses of recombinant human scce mRNA in various tissue preparations from the transgenic lines #1010 (black bars) and #107 (empty bars). Analyses in triplicate were carried out on RNA samples comprising pooled material from three animals from each line. The murine acidic ribosomal phosphoprotein PO was used as internal standard. Mean and SD.

B. ELISA-analyses of SCCE-protein in various tissues from the transgenic lines #1010 (black bars) and #107 (empty bars), and non-transgenic siblings (gray bars). Analyses in triplicate were carried out on pooled extracts from three animals from each line and controls. Mean and SD.

Figure 4 Pro-SCCE and active SCCE in skin from #1010 scce-transgenic mice. Hu =
extract of human plantar stratum corneum; Tg = extract of skin from #1010 transgene; Wt = extract of skin from wild type littermate. Approximately 0.1 mg of mouse skin was homogenized in 10 ml of 1 M acetic acid and extracted over night at 4 C. After clearing by centrifugation extracts were aliquoted, lyophilized, and resolubilized in electrophoresis sample buffer.

A: Immunoblot with SCCE-specific antibodies, reduced samples. Arrowheads denote, from top to bottom, glycosylated pro-SCCE, mixture of unglycosylated pro-SCCE
and glycosylated SCCE, and unglycosylated SCCE. Amount of sample applied corresponding to 0.1 mg and 4.5 mg of skin for Tg and Wt, respectively.

B: Zymography in 12.5% acryalmide gel with 1% casein; non-reduced samples.
Amount of sample applied corresponding to 0.4 mg and 4.5 mg of skin for Tg and Wt, respectively. Arrow denotes SCCE.
To the far left (marked by asterisks) molecular weight markers; from top 106, 81, 47.5, 35.3, 28.2, and 20.8 kDa respectively Figure 5.

Scratching behavior of scce-transgenic (#1010) mice. Twenty one mice, (11 transgenes, 5 females; 10 wild type litter mates, 2 females) were observed every fifth.
day for 45 days, starting when the mice were 5-6 weeks of age. At each observation point mice were transferred to individual cages, and episodes of scratching with hind or front paws were counted during three 5-min periods with 2.5 min lapsing from the transfer to the cage to the first counting, and between counting periods. The results for the three observation periods were pooled and the number of episodes of scratching per min calculated. In A the number of episodes of scratching (mean and SEM for all animals in each group) is shown, in B the percentage of animals with at least one episode of scratching per min is given. ^ (square) = #1010 transgenic mice; A (triangle) = wild type litter mates.

Figure 6.

Histology and SCCE-immunohistology of skin from scce #1010 transgenic mouse and control; comparison with normal human skin and chronic lesion of atopic dermatitis. Formaldehyde fixed and paraffin embedded samples. A-B stained with hematoxylin and eosin. C-F immunoperoxidase staining with SCCE-specific antibodies, contra-staining with hematoxylin. A and C: #1010 transgenic mice, weeks of age. B and D: non-transgenic litter mate. E: Atopic dermatitis. F:
Normal human skin. Bar= 50 m.

Figure 7.

The effect on itch in scce-transgenic mice of the glucocorticoid triamcinolone acetonide. Squares = triamcinolone acetonide, n = 4; triangles = controls (saline), n =
6.
* = statistically significant difference (p < 0.05) between controls and treated group.

Figure 8.

The effect on itch in scce-transgenic mice of the antihistamine loratidine.
Black bars = loratidine (n = 7); White bars = controls ( n = 7); mean and SE.. There were no statistically significant differences in frequency of scratching between treatment group and control group.
Figure 9 Deduced amino acid sequences of SCCE from five species. The sequences for cow, pig, and rat are not complete in the C-terminal parts. See Example 6 for further information. Seq 2 (cow) in the figure is (SEQ ID NO:46), Seq 3 (pig) in the figure is (SEQ ID NO:47), Seq 1 (homo) in the figure is (SEQ ID NO:48), Seq 4 (rat) in the figure is (SEQ ID NO:49) and Seq 5 (mouse) in the figure is (SEQ ID NO:50).

DETAILED DISCLOSURE OF THE INVENTION

The present invention relates to a transgenic mammal or mammalian embryo having integrated within its genome a nucleotide sequence comprising at least a significant part of a nucleotide sequence coding for a stratum corneum chymotryptic enzyme (SCCE) or a variant thereof operably linked to a promoter that drives expression of scce in skin.
By the term "a human stratum corneum chymotryptic enzyme (SCCE)" is meant a serine protease having the amino acid sequence SEQ ID NO:2 described in W095/00651 and shown in the enclosed sequence listing. By the term "a SCCE
variant" is meant a variant of said sequence not having exactly the amino acid sequence shown in SEQ ID NO:2, it may e.g. be a SCCE protease from another species, such as from a cow, pig, rat or mouse, or a synthetic polypeptide comprising a part of SEQ ID NO:2. The SCCE variant will generally react with antibodies raised against purified native or recombinant human SCCE and will generally have significant "SCCE activity", i.e. be a serine proteinase which can be inhibited by the same inhibitors as the spontanceous cell dissociation that can be induced in model systems with samples of cornified layer of skin incubated at neutral or near neutral pH at physiological temperature, i.e. about 37 C, as described in W095/00651.

As can be seen from the following tables, there are significant similarities between SCCE from different species:

Table 2. Alignment of partial deduced amino acid sequences from different species, corresponding to residues 162-184 of human SCCE (Hansson et al.1994).
In bold are shown the residues Asn-170 and Ser-176.

Cow SCCE NH2...AGIPNSRTNACNGDSGGPLMCKG...(SEQ ID NO:4) Pig SCCE NH2...AGIPNSKTNACNGDSGGPLVCKG... (SEQ ID NO:5) Hum SCCE NH2...AGIPDSKKNACNGDSGGPLVCRG... (SEQ ID NO:6) Rat SCCE NH2...AGIPDSKTNTCNGDSGGPLVCND... (SEQ ID NO:7) Mouse SCCE NH2 AGIPDSKTNTCNGDSGGPLVCND... (SEQ ID NO:8) The bottom of the primary substrate specificity pouch (see Hansson et al., 1994) in SCCE from different species (residue no 170 in Table 2 above) contains a conserved asparagine residue, which is unique among known serine proteases.
Also the sequence between this residue and the active serine residue (no. 176 in Table 2) is highly conserved. This suggests that the function, e.g. specialized catalytic properties, of SCCE is critically dependent on the mentioned asparagine residue.

Table 3 Alignment of partial deduced amino acid sequences from different species, corresponding to residues (-)7 - 27 of human SCCE (Hansson et al.1994). In bold are shown the residues adjacent to activation site (C-terminal of Lys-(-1) of Arg (-1).

Cow SCCE .. QEDQGNKSGEKIIDGVPCPRGSQPWQVALLKGSQLHCG... (SEQ ID NO:9) Pig SCCE .. EGQDKSGEKIIDGVPCPGGSRPWQVALLKGNQLHCG... (SEQ ID NO: 10) Hum SCCE ..EEAQGDKIIDGAPCARGSHPWQVALLSGNQLHCG... (SEQ ID NO:11) Rat SCCE ..QGERIIDGYKCKEGSHPWQVALLKGDQLHCG... (SEQ ID NO:12) 10 Mouse SCCE ...QGERIIDGYKCKEGSHPWQVALLKGNQLHCG... (SEQ ID NO:13) Active human SCCE is formed by cleavage C-terminal of K in the sequence KIIDG
etc.. This activation can be catalysed by trypsin in vitro (Hansson et al., 1994).
Examining the amino acid sequence adjacent to this cleavage site reveals a high 15 degree of conservation between species. The consensus sequence (SEQ ID
NO:14) is G-X,-X2-I-I-D-G, where X, is either aspartate (D) or glutamate (E), and X2 is either lysine (K) or arginine (R). Aspartate and glutamate are functionally similar, both having negatively charged functional groups. The same holds true for lysine and arginine, which both have positively charged functional groups and forms sites for 20 cleavage catalysed by enzymes with trypsin-like primary substrate specificity. The consensus sequence adjacent to the activation site is unique among known serine proteases, suggesting an important function. It also suggests that there may exist enzymes in tissues (e.g.) epidermis, the specific function of which is SCCE-activation.
More specifically, the invention relates to a transgenic mammal or mammalian embryo having integrated within its genome a nucleotide sequence comprising at least a significant part of a nucleotide sequence coding for a protein with an amino acid sequence which has a sequence identity of at least 75% to the amino acid sequence shown in SEQ ID NO:2 and which contains the partial sequence (SEQ ID NO: 14):
glycine-X,-X2- isoleucine-isoleucine-asparagine-glycine, wherein X, is aspartate or glutamate and X2 is lysine or argininine, operably linked to a promoter that drives expression in skin.
Preferably, the invention relates to a transgenic mammal or mammalian embryo having integrated within its genome a nucleotide sequence comprising at least a significant part of a nucleotide sequence coding for a protein with an amino acid sequence which has a sequence identity of at least 75% to the amino acid sequence shown in SEQ ID NO:2 and which contains the partial sequence (SEQ ID NO:15):

asparagine-X4-X5-X6 X7-X8-serine, wherein X3 is any amino acid residue, X4 is any amino acid residue, X5 is a cystein residue X6 is any amino acid., X7 is a glycine residue, X6 is an aspartate residue, and the serine is the active serine residue characteristic of serine proteases, operably linked to a promoter that drives expression in skin.

In alternative embodiments, the encoded polypeptide has a sequence identity of at least 80% with the amino acid sequence shown in SEQ ID NO:2, such as at least 90%, e.g. at least 95%, preferably at least 98%, e.g. at least 99%. Sequence identity can be calculated by the BLASTP program ((Pearson W.R and D.J. Lipman (1988) PNAS USA 85:2444-2448) in the EMBL database (www.ncbi.nlm.gov/cgi-bin/BLAST). Generally, the default settings with respect to e.g. "scoring matrix" and "gap penalty" will be used for alignment.
By the term "at least a significant part of a nucleotide sequence coding for SCCE" is meant a nucleotide sequence (i.e. a DNA sequence or a RNA sequence) encoding a polypeptide having at least a part of the amino acid sequence shown in SEQ ID
NO:2 and preferably resulting in an abnormal phenotype as described in the following. It is contemplated that it is useful and maybe even necessary to include intron sequences when preparing a nucleotide sequence coding for a SCCE or a variant thereof, i.e.
one or more of the introns present in the human scce shown in Table 1 or one or more of the murine introns which may be deduced from the murine sequence. It is likely that not all of the intron sequences are necessary and that intron sequences from SCCE from other species or intron sequence from genes coding for other proteins may also be suitable and should be inserted in the nucleotide sequence coding for SCCE in a suitable manner.
It is contemplated that only a minor part of SCCE is necessary in order to obtain the abnormal phenotype. By the term "a significant part" is meant a nucleotide sequence encoding at least 50 amino acids of SEQ ID NO:2, e.g. at least 70 amino acids, at least 100 amino acids, at least 150 amino acids or at least 200 amino acids.
These lengths are considered to be "a significant part of the peptide shown in SEQ
ID
NO:2". The polypeptides encoded may be longer than the above stated lengths which will then indicate the parts which are common between the polypeptides encoded and SEQ ID NO:2. Generally, however, such nucleotide sequences will comprise the major part of the nucleotide sequence shown in SEQ ID NO:1 described in W095/00651 and shown in the enclosed sequence listing, such as at least 500 nucleotides, e.g. at least 600 nucleotides, at least 650 nucleotides, at least 700 nucleotides, e.g. 750 nucleotides.

Such nucleotide sequences will generally hybridize with the complementary sequence to nucleotide sequence SEQ ID NO: 1 or a part thereof under stringent hybridization conditions. Within the concept of the present invention is thus a transgenic mammal or mammalian embryo having integrated within its genome a nucleotide sequence which hybridizes with the complementary sequence to the nucleotide sequence SEQ ID NO: 1 or a part thereof under stringent hybridization conditions, preferably under highly stringent conditions, said sequence comprising at least a significant part of a nucleotide sequence coding for a stratum corneum chymotryptic enzyme (SCCE) or a variant thereof operably linked to a promoter that drives expression of scce in skin. The term "stringent" when used in conjunction with hybridization conditions is as defined in the art, i.e. 15-20 C under the melting point Tm, cf. Sambrook et al, 1989, pages 11.45-11.49. Preferably, the conditions are "highly stringent", i.e. 5-10 C under the melting point Tm. However, due to the degeneracy of the genetic code also nucleotide sequences which have only minor resemblance to SEQ ID NO:1 may be able to encode a SCCE.

The vectors for expressing the nucleic acids having nucleotide sequences coding for a SCCE require that the nucleic acid having a nucleotide sequence coding for a human SCCE be "operatively linked." A nucleic acid is operatively linked when it is placed into a functional relationship with another nucleic acid sequence. For instance, a promoter or enhancer is operatively linked to a coding sequence if it affects the transcription of the sequences. The promoter and enhancer may be the same or two different entities. The SV40 early promoter is an example of an integrated promoter and enhancer. Operatively linked means that the DNA
sequences being linked are contiguous and, where necessary to join two protein-coding regions, contiguous and in reading-frame. By the term "a SCCE
construct" is meant a nucleotide sequence comprising at least a significant part of a nucleotide sequence coding for a stratum corneum chymotryptic enzyme (SCCE) or a variant thereof operably linked to a promoter that drives expression of scce in skin.

In a preferred embodiment according to the present invention, the promoter is a ubiquitous promoter. By the term "ubiquitous promoter" is meant a promoter that is active in many different cell types of the host organism in contrast to a promoter whose expression is specific for one or a few target cell types (a tissue-specific promoter). An example of "ubiquitous" promoter" is the SV40 promoter and variations thereof such as the SV40 early promoter. Other examples of ubiquitous promoters are other viral promoters such as polyoma early promoter, retroviral long terminal repeats (5'-LTR) adenovirus promoters, and house keeping cellular genes such as (3-actin, and ribosomal protein promotors, The promoter is preferably a heterologous promoter. It is contemplated that constitutive viral promoters, such as polyoma early viral promoter, Ebstein Barr virus promoter and retroviral long term repeat LTR promoters will be useful in the construction of transgenic mammals according to the invention.

An important embodiment of the invention relates to a transgenic mammal or mammalian embryo selected from the group consisting of rodents, such as mice, rats and rabbits, cats and dogs. A preferred embodiment of the invention is a transgenic mammal or mammalian embryo which is selected from the group consisting of mice.
Preferably, the transgenic mammal or mammalian embryo according to the invention comprises a nucleotide sequence comprising a significant part of DNA sequence coding for human SCCE as shown in SEQ ID NO:1. The transgenic mammal or mammalian embryo according to the invention preferably comprises a nucleotide sequence coding for a significant part of the peptide shown in SEQ ID NO. 2 as defined above. In preferred embodiments, the DNA sequence codes for the peptide corresponding to amino acid no. -7 through no. 224 of the amino acid sequence shown in SEQ ID NO. 2, the peptide corresponding to amino acid no. 1 through no.
224 of the amino acid sequence shown in SEQ ID NO. 2 or the peptide shown in SEQ ID NO. 2. Presently preferred embodiments relate to transgenic mammals or mammalian embryos according to the invention, wherein the DNA sequence comprises the DNA shown in SEQ ID NO. 1 or the DNA sequence is SEQ ID NO: I.
In an important embodiment of the invention, the transgenic mammal or mammalian embryo according to the invention exhibits an abnormal phenotype, such as an abnormal skin phenotype and/or a predisposition for cancer, e.g. a predisposition for ovarian cancer. Preferably, the mammal or mammalian embryo according to the invention exhibits an abnormal skin phenotype resembling one or more inflammatory skin diseases characterized by epidermal hyperkeratosis, acanthosis, epidermal and/or dermal inflammation and/or pruritus, e.g. inherited skin diseases with epidermal hyperkeratos, ichthyosis vulgaris, psoriasis, chronic atopic dermatitis or chronic eczema. The mammal or mammalian embryo according to the invention may thus exhibit epidermal hyperkeratosis, achantosis, epidermal/dermal inflammation and/or pruritus.

The invention further relates to a method for making a transgenic mammal or mammalian embryo having integrated within its genome a nucleotide construct comprising at least a significant part of a nucleotide sequence coding for a human stratum corneum chymotryptic enzyme (SCCE) or a variant thereof as defined above operably linked to a promoter that drives expression of scce in skin. In a preferred embodiment, the invention relates to a method for making a transgenic mammal according to the invention, where the mammal exhibits an abnormal phenotype as defined above. The method comprises introducing the SCCE-construct into an ovum or embryo of the mammal by physical, chemical or viral means, e.g. by electroporation, transfection, microinjection or viral infection. In a preferred embodiment of the invention, the SCCE-construct is microinjected into an ovum or embryo of the mammal or into embryonal stem cells of the mammal. In a preferred embodiment, the method according to the invention comprises microinjecting the SCCE-construct into C57BU6JxCBA-f2 mice ovum or embryos. The method preferably further comprises breeding the resulting mice with C57BU6JxCBA or with C57BU6J to obtain transgenic litter and stable mouse lines. Such stable cell lines derived from the transgenic mammals comprising a SCCE construct as described above are contemplated to be useful for e.g. high through-put screening of suitable compounds as described in the following.

Another aspect of the invention relates to the use of the transgenic mammal or mammalian embryo according to the invention as a model for the study of disease with the aim of improving treatment, relieve or ameliorate a pathogenic condition, for development or testing of a cosmetic or a pharmaceutical formulation or for the 10 development of a diagnostic method. A preferred use according to the invention of said transgenic mammal or mammalian embryo is as a model for a skin disease or a model for cancer such as ovarian cancer.

An important aspect of the invention relates to a method of screening for a compound 15 or composition effective for the prevention or treatment of an abnormal or unwanted phenotype, the method comprising (a) administering a compound or composition to a transgenic mammal having integrated within its genome a nucleotide sequence coding for at least a significant 20 part of SCCE operably linked to a promoter that drives expression of the scce in an organ, wherein the rodent exhibits an abnormal phenotype, (b) evaluating the appearance of the relevant organ and/or the behaviour of a mammal treated according to step (a), and (c) comparing the appearance of the relevant organ and/or the behaviour of a treated rodent with an untreated control mammal.

In preferred embodiments, the organ is the ovaries or the skin. A presently preferred embodiment of the invention relates to a method of screening for a compound or composition effective for the prevention or treatment of itchy inflammatory skin diseases such as ichthyosis vulgaris, prurigo nodularis, neurodermatitis, lichen planus. Other preferred embodiments of the invention relate to a method of screening for a compound or composition effective for the prevention or treatment of chronic atopic dermatitis and psoriasis. Also, the invention relates to a method according to the invention for screening of a cosmetic composition.

In particular, the invention relates to a cosmetic or pharmaceutical composition that has been discovered or developed by use of the above methods comprising use of a transgenic mammal or mammalian embryo as described above. In this respect the invention relates to pharmaceutical formulations for systemic treatment as well as for cosmetic and pharmaceutical formulations for topical application on the skin or epithelium.
EXAMPLES

The following examples are provided for illustration and are not intended to limit the invention to the specific examples provided.

EXAMPLE 1.

Isolation and cloning of the human SCCE gene.
The human SCCE gene was isolated from a human leukocyte genomic library cat.
no. HL 1111 j lot # 3511 (Clontech, CA) by using cDNA probes derived from the human scce cDNA. A 253 bp cDNA fragment was amplified from pS500 (Hansson et al., 1994) by PCR using SYM3300 (5'-GGTGGCCCTGCTCAGTGGCA-3') (SEQ ID
NO: 16) and SYM3301 (5'-CACCATGGATGACACAGCCTGG-3') (SEQ ID NO: 17), 32P-labelled by random priming using oligo-labelling kit (Amersham, UK) and used as a probe for screening. The fragment covers bases 149 to 401 of the published human SCCE cDNA sequence (Hansson et al., 1994). Approximately 5x105 plaques were screened. Filters were prepared, prehybridized and hybridized at 65 C, and washed at 65 C and 25 C in accordance with the membrane manufacturers recommendations (Colony/Plaque ScreenTM hybridization transfer membranes DuPont NEN, MA). Filters were exposed to HyperfilmTM-MP (Amersham, UK). After three rounds of screening, individual positive clones were selected, and phage DNA
was isolated using standard techniques (Sambrook et al., 1989). Phage DNA were digested with several restriction enzymes and Southern blotting was performed using three different probes. First, the 253 bp 5"-fragment described above was used.

Second, a 618 bp 3'-noncoding cDNA fragment was used as a probe. The fragment was amplified by PCR using pS501 as template, forward primer SYM3302 (5'-AATAAAGAAACACAAAACCC-3") (SEQ ID NO: 18) and reverse primer SYM3418 (5"-TGTAATATCATTGTGGGC-3') (SEQ ID NO: 19). pS501 is a plasmid containing 1888bp human SCCE cDNA isolated from a ? gt11 keratinocyte cDNA library ligated into EcoRl site of pUC19 and covers cDNA with coding sequence from amino acid four over the stop codon and contains 868 bp extra untranslated 3' sequence.
Finally, a 897 bp fragment containig the entire coding SCCE cDNA sequence was isolated from EcoRl/Dral digested pS500 (Hansson et al., 1994) and used as a probe.
Probes were labelled and hybridization was performed as described above. Two positive clones were digested with Sall and cloned into pUC19 generating pS772 and pS773.
In order to determine the DNA sequence of the human SCCE gene, several overlapping subclones of pS772 and pS773 were generated in pUC19. Subclones were sequenced using the dideoxy chain termination method (T7 sequencing kit, Pharmacia, Sweden or the Dye Terminator Cycle Sequencing Ready Reaction kit, PE Applied Biosystems, CA) with M13 forward and reverse primers as well as specific primers..

Isolation and cloning of the mouse SCCE gene.
To isolate the murine SCCE gene, a 430 bp cDNA fragment was isolated from Hindill/Sall digested pS506 (Backman et al., 1999). The fragment was 32P-labelled by random priming using oligo-labelling kit (Amersham, UK), and used as probe to screen a 129SVJ Lambda Fix IITM genomic library (Stratagen, CA). Approximately 1xi06 plaques were screened. The blots were prepared, prehybridized and hybridized at 65 C as described by the manufacturer (Colony/Plaque Screen TM
hybridization transfer membranes DuPont NEN, MA). Washing was also performed as described in the hybridization protocol and membranes were exposed to Hyperfilm-MP (Amersham, UK). Individual positive clones were selected after three rounds of screening. A few positive plaques were further investigated by PCR
using SYM4118 (5"-GGATGTGAAGCTCATCTC-3') (SEQ ID NO: 20) and SYM4121 (5'-TGGAGTCGGGGATGCCAG-3') (SEQ ID NO: 21). Obtained PCR products were analysed by Southern blotting using the probe and conditions described above.
Phage DNA was isolated from confirmed positive clones using standard techniques.
Southern analysis was performed on phage DNA digested with a panel of restriction enzymes using the probe and conditions described above. One of the positive clone was digested with Sacl, and a fragment of -15.5 kb was isolated and cloned into pUC19 generating pS714. Several overlapping subclones of pS714 were generated in pUC19. DNA sequencing of the subclones were performed as described for the human SCCE gene.
Primer extension analysis.
Two exon 1-specific oligonucleotides; one human and one mouse, were used to determine the 5'-prime ends of the human and murine SCCE transcripts. To determine the start of the human transcript (Ausubel et al.) a PCR fragment of 346 bp was amplified from plasmid pS779 (A subclone covering 5'-untranslated sequence, exons 1-3, 5'-end of exon 4 and introns 1-3) using forward primer SYM4720 (5'-GGGAGGGTGGAGAGAGA GTGCAGTG) (SEQ ID NO: 22) and reversed primer SYM4899(5'-AGTCTAGGCTGCAG CCCCTAC-3') (SEQ ID NO: 23). To prepare a 245 bp 32P-dCTP labelled single stranded probe, primer hEXON1 (5'-CTCGAGGGATCTGATGTGATCC-3') (SEQ ID NO: 24) was annealed to the amplified fragment and labelling was performed using the Prime-A-ProbeTM DNA
labelling kit (Ambion, Austin, Texas, USA). 106 cpm labelled probe was mixed with 50 g total RNA from human skin. Hybridisation and S1 treatment was performed using S1-AssayTM (Ambion, Austin, Texas, USA). The final product was analysed on a sequencing gel. Dideoxy sequencing reactions of pS779 primed with oligo hEXON1 were used as size markers.

The start of the murine transcript was determined using Sacl linearised pS721 (A
subclone covering 5'-untranslated sequence, exons 1-3, introns 1-2 and 5'-end of intron 3). A 225 by 32P-dCTP labelled single stranded probe was prepared by annealing of primer mEXON1 (5'-CTGGGAGTGACTTGGCGTGGCTCT-3') (SEQ ID
NO: 25) to the linear plasmid and labelling was performed using the Prime-A-ProbeTM
DNA labelling kit (Ambion, Austin, Texas, USA). 106 cpm labelled probe was mixed with 50 g total RNA isolated from mouse tail. Hybridisation and S1 treatment was performed using S1-AssayTM (Ambion, Austin, Texas, USA). The obtained product was analysed as described above using sequencing reactions of pS721 primed with oligo mEXON1 as size markers.

i RESULTS

(Nucleotide sequences in Gene Bank: Human scce(hSCCE): accession number AF332583; Murine scce (mSCCE): Acession number AF339930.) A human leukocyte EMBL3?, genomic library was screened using a probe made from the coding region of human ssce cDNA (Hansson et al., 1994) individual positive clones were identified. Based on restriction analysis and Southern blotting two overlapping clones, 12 and 15.5 kbp in size respectively, were selected. These clones were spanning the entire scce cDNA. The genomic structure of the human scce structural gene comprises six exons and spans approximately 8 kb. The organisation and sizes of exons and introns are shown in fig 1. The translation initiation site (designated +1) is found 60 nt downstream the 5'-end of exon 2.

To isolate the murine scce gene, a SVJ129 genomic XFIX'M II library was screened using a probe corresponding to the coding region of murine scce cDNA (Backman et al.). Among the isolated clones one harboring about 15.5 kb was shown to contain the entire murine structural gene. A major part comprising 11770 nt was sequenced and the murine structural scce gene was shown to be shorter than the human gene.
However, the overall organisation reveals several similarities with the human homolog and also consist of six exons (figure 1). Since the polyadenylation site of the murine cDNA have not been identified so far, the exact size of exon 6 could not be determined. However, a putative poly A site was localised 136 bp 3'-prime of the stop codon. The translation initiation site (designated +1) is found in exon 2, 39 nucleotides 3' of the intron 1 3'-intron-exon junction.
To determine the 5' ends of the human and murine transcripts primer extension studies were performed. Sequence analysis of the human cDNA (exon1, unpublished results) revealed that the major human primer extension product extends to the nucleotide identified at the 5' end of the human cDNA sequence.(Hansson et al).
Analysis of the two major products obtained from the murine gene by primer extension reveal two different transcription starts. One products extends to one nucleotide 5' of the murine SCCE cDNA 5' end (Backman et al.). The other product extends to one nucleotide 3' of the cDNA 5' end.

Generation and gross phenotypic characterization of of scce transgenic mice with the hscce gene under control of the SV40e promoter Construction of transgene.
In order to overexpress the human genomic scce structural gene under transcriptional regulation of the simian virus 40 early, SV40e, enhancer and promoter, an expression vector was constructed. The scce genomic DNA was 10 modified by insertion of Hindlll linkers 20 bp upstream of the start codon and 4.8 kb downstream of the stop codon, respectively. The resulting Hind Ill scce fragment was the ligated to a 325 bp BamHl/Hindlll fragment of pS99 (Figure 2) containing the SV40e enhancer and promoter elements and cloned into pBluescriptTM SK+/-(Stratagene) resulting in pAM119. For gene transfer, the plasmid pAM119 was 15 digested with BamHl and Clal and the SV40e/scce fragment of about 10.7 kb was isolated and purified by electro-elution before microinjection into one-cell stage mouse ova.

Transgenic mice were generated in C57BU6JxCBA-f2 embryos by standard 20 microinjection procedures (Hogan et al, 1986). The 10,7 kb SV40e/scce fragment to be injected was excised from the pAM119 plasmid by restriction enzyme cleavage with BamHl and Clal, separated by gel electrophoresis through an agarose gel, cut out, isolated using isotachophresis and preciptated with ethanol.
25 Identifying transgenic animals.
To identify transgenic animals, DNA was extracted from tail biopsies of 3-wk old mice and the DNA was analysed either by Southern blot analyses or with PCR as described in Ausubel et al. The PCR analysis was performed using primers specific for human scce (IE2: 5'-GCT CTC CCA TTA GTC CCC AGA GA-3' (SEQ ID NO:
30 26),MJ2: 5'-CCA CTT GGT GAA CTT GCA CAC TTG-3'(SEQ ID NO: 27)). Briefly, the PCR was performed with an initial denaturation at 95 C for 10 min., followed by 28 cycles of denaturation at 95 C for 30 sec, annealing at 65 C for 30 sec, elongation at 72 C for 45 sec and finally by a 10 min elongation at 72 C.
The resulting PCR products were analyzed by standard agarose gel electrophoresis using a 1 % agarose gel and visualising the DNA with Ethidium bromide as described in Ausubel et al., 1992. Three transgenic lines (#103, #107 and #1010) were established by breeding heterozygous mice with C57BU6JxCBA.

RESULTS

As expected, initial characterization of the three lines revealed very large differences in levels of recombinant scce expression (see example 3). In line #1010, which has the highest hscce transcript levels, skin abnormalities were apparent, whereas in the two other lines no skin changes or other gross phenotypic deviations could be observed. For further detailed comparative studies of the #1010 transgenics one of the lines with apparently normal phenotype (#107) and non-transgenic littermates were included as controls.
Macroscopic phenotypic changes in transgenic #1010 animals were noted as a loss of hair from a narrow zone around the eyes in mice 4-5 weeks of age. In older mice there was an apparent thinning of body hair in general, and a luster-less appearance of the coat. On the back the skin surface was sometimes covered with fine scales.
From the age of 5-6 weeks and onwards several of these transgenic animals showed signs of itch with scratching, the frequency of which increased with time.
Diagnostic necropsies with routine histological analyses were carried out on transgenic mice of the #1010 and #107 C57BU6JxCBA lines, and of littermate controls. Tissues examined were brain, cerebellum, intestines (duodenum/jejunum, ileum, colon, rectum), and skin. In some animals 3 weeks of age heart, liver, lung, salivary gland, spleen, thymus and thyroid were also examined. In littermate controls (for #1010: 3 weeks, n = 5; 5 weeks, n = 5; for #107 5 weeks, n = 3) and transgenic mice of the #107 line (5 weeks n = 3) no significant macro- or microscopic abnormalities were observed. In transgenic animals from line #1010 abnormalities were found in the skin, but in no other organs or tissues. In mice 3 weeks of age (i.e.
before phenotypic changes could be observed by inspection of living animals) skin changes were found in all animals examined (n = 4). These changes included a mild to moderate epidermal hyperplasia and hyperkeratosis and a mild cellular inflammatory reaction with mixed leukocytes in the upper dermis. In animals 5 weeks of age (n = 4) the skin abnormalities were of the same type but more pronounced with a marked acanthosis-like hyperplasia and an hyperkeratosis of the epidermis which was mainly orthokeratotic. In addition, the number of mast cells in the dermis was increased in some of the animals. Leukocyte invasion of the epidermis was occasionally found and then manifested as small groups of granulocytes within the thickened cornified layer which at these sites was parakeratotic.
EXAMPLE 3.
Determining the expression of scce-mRNA, SCCE proteingene in mice and catalytically active SCCE in SV40e-scce-transgenic mice.

Isolation of tissues.
Tissue specimens were collected at different ages and immediately frozen and stored in liquid nitrogen until analyzed.

RNA Isolation and cDNA synthesis and Real Time Quantitative PCR.
From 50-300 mg of the isolated tissues liver, skin, lung, brain, small intestine, colon, and ear, total RNA were prepared using RNA STAT-60TM (Tel-Test "B", Inc., Friendswood,TX, USA) according to the manufacturer. 50pg of each RNA
preparation were DNase treated using RQ1 DNase (Promega, Madison, WI, USA) according to Ausubel et al . About 1,6 pg total RNA from each tissue was used for cDNA synthesis. Three RNA samples from animals with same genetic background and tissue were mixed and cDNA synthesis was made using Superscript1M
Preamplification System for First Strand cDNA Synthesis (Life Technologies, Inc.
Gaithersburg, MD, USA) according to the manufacurer. The cDNA synthesis was primed using Oligo d(T)12_18 primer. The synthesized cDNA were diluted 100x in water prior to real time quantification. Real time quantification were performed three times on each cDNA. Primer and probe for real time quantification of transgenic human SCCE were designed over exons four and five where the sequence between human and murine SCCE show little(less) homology. The forward primer (5'-GCGAACCCCCTGGAACAA-3') (SEQ ID NO: 28) covers the position 427 - 444 of the human cDNA sequence (ref. Hansson et al) in exon four. The reverse primer (5'-ACATCCACGCACATGAGGTCA-3') (SEQ ID NO: 29) covers the position 490 - 510 of the human cDNA sequence in exon five. The real time amplification probe (5'-CCTGTACTGTCTCCGGCTGGGGCACTACC- 3') (SEQ ID NO: 30) covers the position 445 - 473 of the human cDNA sequence in exon four, and was labeled with the reporter fluorecent dye FAM in the 5'- end and the quencher fluorescent dye TAMRA in the 3'-end. The amplification of PCR products and real time detection were performed in ABI Prism 7700 Sequence Detecttion System (PE Applied Biosystems, Foster City CA, USA). Amplification of a part of murine acidic ribosomal phosphoprotein PO (ACC# X15267) was used as endogenous control for the real time quantitation studies. The relative quantitation was calculated according to the formula 2- T, where ACT is the difference in CT values between the target and the endogenous control (User Bulletin #2, PE Applied Biosystem).

SCCE-specific polyclonal antibodies.
Polyclonal antibodies to recombinant human SCCE were prepared and affinity purified as described by Sondell et al.(Sondell et al. 1996). These antibodies are reactive towards human SCCE and pro-SCCE, as well as murine SCCE..

Tissue preparation, ELISA, immunoblotting and zymography..
Tissue extracts for ELISA were prepared by homogenization of 200-400 mg frozen tissue in 1 ml dH2O containing a mixture of protease inhibitors (Complete TM
Protease Inhibitor Cocktail Tablets cat. no. 1836153, Boehringer Mannheim, Germany), followed by centrifuging at 20 000 x g for 30 min at 4 C. Protein concentrations was determined by reaction with bicinchoninic acid with bovine serum albumin as standard For SDS-polyacrylamide gel electrophoresis approximately 0.1 mg of mouse skin was homogenized in 10 ml of 1 M acetic acid and extracted over night at 4 C.
After clearing by centrifugation extracts were aliquoted, lyophilized, and resolubilized in electrophoresis sample buffer for zymography. SDS-polyacrylamide gel electrophoresis, zymography, and immunoblotting were carried out as described (Ekholm et al. 2000).
For ELISA polystyrene microtiter plates were coated with 100 l of SCCE-specific rabbit polyclonal antibodies at a concentration of 7 g/ml prepared in coating buffer (0.1 M Na2CO3, 0.02 % NaN3 (w/v), pH 9.6). After incubation over night at 4 C
on a wobbling table, the plate was washed once with washing buffer (10 mM NaH2PO4, 0.15 M NaCl, 0.05% (v/v) Tween 20, pH 7.2). Thereafter, 200 l blocking buffer (10 mM NaH2PO4, 0.15 M NaCl, 0.1 % (w/v) Bovine Serum Albumine (BSA), pH 7.2) was added to each well and the plate was incubated at 37 C for 1 h. The plate was washed three times with washing buffer, 50 l of sample (or standard) in dilution buffer (10 mM NaH2PO4, 0.15 M NaCl, 0.1% (w/v) BSA, 0.05% (v/v) Tween 20, pH
7.2) was added to each well and the plate was incubated for 1 h at 37 C.
Plates were washed three times with washing buffer, and further prepared by adding 100 l/well of SCCE-specific antibodies (7 g/ml) labelled with alkaline phosphatatse Plates were incubated for 1 h at 37 C before washing three times with washing buffer.
Development was performed by addition of 100 l freshly prepared substrate solution (2 tablets of phosphatase substrate (Sigmal04 phosphatase substrate tablets) dissolved in 10 ml 0.1 diethanol amine-HCI, 0.5mM MgCI2, pH 9.8). Plates were incubated in the dark for 30 min at room temperature. Finally, 25 l stop solution was added to each well and the absorbance was read at 405 nm. For quantitation recombinant human pro-SCCE (Hansson et al) was used as standard.

RESULTS
Real Time Quantification of human SCCE transcribed in transgenic mice.
In order to investigate if the difference in skin phenotype between #1010 ABD
#107 transgenic lines expression of hscce mRNA in various tissues was analyzed by quantitative RT-PCR. The results are shown in Fig. 3A.
Six different tissues were analyzed. The analyses showed significantly higher expression of hscce in all tissues examined for transgenic mice of the #1010 line as compared to mice of the #107 line and non-transgenic littermates. The highest relative hscce mRNA levels were found in the intestines and lungs, but the difference in hscce expression between the two transgenic lines was most pronounced for skin, in which the relative level of hscce mRNA was about 24 times higher in #1010 mice than in #107 mice.

ELISA
Analyses of SCCE protein with ELISA (Fig. 4B) showed values close to or below the detection limit for tissues from transgenics of the #107 line and normal controls. In #1010 transgenics SCCE protein was readily detectable in several tissues including skin, intestines, and lung, the relative level (ng/mg) being highest in the skin.

Immunoblotting and zymography Immunoblotting with SCCE-specific antibodies corroborated the ELISA-results.
In extracts of skin of control mice small amounts of a component with molecular mass similar to human SCCE was detected, whereas a component with the same relative 5 molecular mass detected in high amounts in skin extracts from #1010 transgenic mice (Fig. 4A). Zymography in casein-containing acrylamide gels showed that the extracts of skin from #1010 transgenics contained a proteolytic enzyme with the same electrophoretic mobility as human SCCE. A corresponding enzyme could not be detected in control extracts (Fig. 4B; the amounts of active murine SCCE
are too 10 low to be detected under the experimental conditions used). These results suggest that a fraction of the human pro-SCCE produced in skin of #1010 transgenics is converted to proteolytically active enzyme. This was supported also by the immunoblotting experiments (Fig. 3A), where a component corresponding to active human SCCE was labeled with the antibodies. In addition to SCCE, the skin extracts 15 of #1010 transgenics contained increased amounts of a proteolytic enzyme not related to SCCE. The nature of this enzyme is presently not known.
CONCLUSION
The expression of hscce in various tissues at the RNA level was higher in #1010 20 transgenic mice than in the #A107 transgenic mice. The difference between transgenics from the two lines was even more pronounced as regards expression of SCCE-protein. In skin of #1010 transgenic mice high amounts of SCCE protein could be detected with immunoblotting. The majority of this protein appeared to be pro-SCCE, but also active SCCE could be detected in increased amounts.

Scce-transgenic mice as models for studies of inflammatory skin diseases and itch Three male transgenic #1010 mice were mated with wild type C57BU6J females, resulting in 6 litters with a total of 40 mice. Of these 19 (8 transgenics) were sacrificed at the age of 7-8 weeks and 21 (11 transgenics) were followed to the age of 13-14 weeks. In the latter group scratch movements with the legs were quantified.

Macroscopic phenotypic changes in transgenic #1010 animals were noted as a loss of hair from a narrow zone around the eyes in mice 4-5 weeks of age. In older mice there was an apparent thinning of body hair in general, and a luster-less appearance of the coat. On the back the skin surface was sometimes covered with fine scales.
From the age of 5-6 weeks and onwards several of these transgenic animals showed signs of itch with scratching, the frequency of which increased with time.

Itching behavior Of the 11 transgenic mice followed for 13-14 weeks 8 animals (73%) showed signs of itch (at least one period of scratching with hind or fore paws per minute) at the age of 10-11 weeks. The frequency of scratching varied among the observed animals;
whereas some animals showed weak or moderate signs of itch, other animals spent most of their time scratching (Fig. 5). Up to the age of 3 weeks there was no statistically significant difference in weight between transgenic and normal animals.
With increasing age there was a tendency towards lower weights among transgenics.
At the age of 14-15 weeks there was a 7-10% reduction in weight in transgenics as compared to wild-type litter mates (mean for males 27.0 gm versus 30.0 gm; p =
0.022; mean for females 21.7 gm versus 23.5 gm; p = 0.033).

Histological analysis For histology and immunohistochemistry (Ekholm et al. 1998 and Sondell et al.
1996) samples were either formaldehyde fixed and paraffin embedded according to routine protocols or frozen after fixation for 2 h in formaldehyde.
Upon sacrifice of the animals tissues (dorsal skin, large and small intestines, and lung) were prepared for microscopic analyses. The preliminary microscopic examination of routinely processed skin samples was carried out blindly (the examiner was not informed about genotype or scratching behavior). In all cases but one, transgenics could be differed from wild type controls, the most prominent difference being the thickened epidermis in transgenic animals. Epidermal thickness was 55 m (SD = 21 m; n = 19) for transgenic animals, and 15 m (SD = 2.6 m;
n = 21; p < 0.001) for controls. There was no statistically significant difference in epidermal thickness between younger (7 - 8 weeks) and older (13-14 weeks) transgenic animals. Other prominent and frequent histologic findings in skin of transgenic animals as compared to controls (Fig. 6 A-B) were a marked hyperkeratosis, an increased cellularity of the dermal part of the skin, and increased epithelial thickness of adnexal structures (hair follicle walls and sebaceous glands and ducts). The increase in number of cells in the connective tissue was only partially due to lymfocytes and granulocytes; there appeared to be an increase also in the number of fibroblasts and/or histiocyte-like cells. Tolouidine blue staining showed increased number of dermal mast cells in some transgenic animals (results not shown). In routine stained sections no differences could be found between transgenics and controls for any of the other organs examined (results not shown).
Immunohistochemistry Immunohistologic analyses of skin samples from #1010 transgenic animals and littermate controls with SCCE-specific antibodies showed strong labeling of keratinocytes in suprabasal parts of interfollicular epidermis in transgenics, including the thickened cornified layer. In hair follicles and sebaceous ducts only luminal parts, including the cornified lining of follicles and ducts, were stained (Fig. 6C).
This was in marked contrast to basal cells of interfollicular epidermis and the major parts of hair follicles and sebaceous ducts and glands, where no or very weak labeling by the antibodies was seen. In controls there was a relatively weak labeling of a narrow zone of interfollicular epidermis close to the transition to the stratum corneum, of the stratum corneum, and of luminal parts of hair follicles (Fig. 6D). This pattern was similar to that previously described for normal human epidermis (Ekholm et at 1998).
With immunofluorescence microscopy on formaldehyde fixed frozen samples similar results (not shown) were obtained.

In the intestines SCCE-specific labeling was seen only in transgenics and in irregularly distributed epithelial cells. Stained cells were more numerous at the tips of villi in the small intestine and in the luminal parts of colonic epithelium.
In the lungs of transgenics apical parts of bronchiolar epithelia cells were weakly labeled.
At higher antibody concentrations there appeared to be a diffuse labeling also of the alveolar epithelium (results for intestines and lung not shown).
Comparison with diseased human skin.
Skin biopsies from human volunteers and patients were taken after informed consent and with the approval of the Human research ethics committee, Umeb University.

Biopsies were taken from chronic eczematous lesions on the flexural sides of lower arms of five adults with atopic dermatitis and processed for microscopy as above.
Biopsies from corresponding sites were obtained from volunteers. In routine stained sections (not shown) the lesions showed, as expected, marked acanthosis, hyperkeratosis, and a sparse dermal infiltrate consisting mainly of lymphocytes.
Immunohistology with SCCE-specific antibodies showed a drastic increase in the number of labeled suprabasal cell layers as compared to controls (Fig. 6 E-F).
As regards the acanthosis, hyperkeratosis, and pattern of SCCE-specific staining the differences seen between lesional and normal skin were strikingly similar to those seen between skin of #1010 transgenic mice and controls.

EXAMPLE 5.

Scce-transgenic mice for testing of antipruritic agents Transgenic mice, 18-22 weeks of age, mean weight 24.2 mg, were given subcutaneous injections of either 250 g of the glucocorticoid triamcinolone acetonide in a total volume of 100 l on day 0, and 100 g triamcinolone acetonide in a total volume of 100 l on days 7, 14 and 21, or 100 l of physiological saline at the same time points. Episoldes of scratching were counted in the morning and injections were given in the afternoon. To prepare solutions for injections 25 l or 10 l or Kencort -TTM suspension, 10 mg/ml (Bristol-Myers Squibb), was mixed with 75 l or 90 l of physiological saline. The results are shown in Fig. 7. Triamcinolone acetonide was highly efficient in diminishing scratching.
Transgenic mice, 20-21 weeks of age, mean weight 24.5 mg, were given either loratidine in a total volume of 100 l, or 100 l of a control solution by means of tube feeding. Episodes of scratching were counted immediately before feeding (0 hours), and then at time points as indicated. Feeding solutions were prepared by mixing either 30 l of loratidine 1 mg/ml, sucrose 600 mg/ml (Clarityn mixture TM, Schering-Plough), or, for control solutions, 30 l of sucrose 600 mg/ml, with 70 l of physiological saline. The results are shown in Figure 8A. The same mice were then treated 7 days later with 90 l of loratidine mixture of sucrose solution mixed with 10 l of physiological saline. The results are shown in Figure 8B. As seen from figures 8A and 8B there was no significant difference in frequency of scratching between treatment group and control group. This indicates that the itching behavior of the SCCE mouse is not relieved by treatment with an antihistamine.
The two experiments show that scce-transgenic mice can be used for evaluation of drugs with potential effects on itch (anti-pruritic drugs). The glucocorticoid triamcinolone acetonide appeared to be highly effective in relieving itch, whereas the antihistamine loratidine had no statistically significant antipruritic effect.
It thus appears that the pruritus in SCCE-transgenic mice respond to treatment with a glucocorticoid but not to treatment with an antihistamine. A similar situation can be found for human patients suffering from pruritus associated with e.g atopic dermatitis, eczema, and psoriasis.

Determination of nucleotide sequences of homologues to hscce-cDNA from cow, rat and pig.

Skin biopsies from cow, pig and rat were obtained, immediately frozen in liquid nitrogen and homogenized, using a Mikro-Dismembranator U (B.Braun Biotech International GmbH, Melsungen, Germany) at 2000 rpm for 45 s. The homogenate was transferred to RNA was isolated using 1 ml of Trizol Reagent (Life Technologies AB, T6by, Sweden) according to the manufacturer's instructions, DNase treated, extracted with Phenol:CHCl3, and precipitated with LiCI according to the Boehringer Mannheim protocol (Nonradioactive In Situ Hybridisation application Manual, Boehringer Mannheim, Mannheim, Gemany).
RT-PCR was performed as described (Lindstrom et al. with oligo d(T)16 primers (Perkin Elmer, Foster City, CA, USA) in the RT reaction. In each RT reaction 100 ng of total RNA was used.

For PCR five primers were designed from conserved sequences found in hscce and mscce cDNA resulting in primers mS3, 698,696,H2 and mS4 (Table 4). PCR
products were cloned into pCR II vector using the TOPO TATM cloning kit (Invitrogen/NOVEX, Groeningen, The Netherlands) as recommended by the 5 manufacturers. Plasmid DNA was isolated using the QlAprepTM Spin Miniprep Kit (Qiagen, Chatsworth, CA). Nucleotide sequencing was performed using the DYEnamicTM ET Terminator Cycle Sequencing Kit (Amersham Pharmacia Biotech Sverige, Uppsala, Sweden) and an AB1377 automated DNA sequencer (Perkin-Elmer).
To obtain the 5'cDNA end the SMART Race cDNA Amplification Kit (Clontech Laboratories, Inc., Palo Alto, Ca) were used according to the manufacturers instructions. Species specific primers were designed from the cDNA sequences obtained in previous steps (Table 4).

Table 4 Oligomer primers used in RT-PCR, 5'-RACE and nested 5'-RACE
Oligomers a - e were designed from conserved sequences found when comparing SCCE and mSCCE cDNA sequences. Positions are derived from the mSCCE
cDNA(Backman et al., Oligomers f - j were designed based on nucleotide sequencing data from the preceding species specific cloning reactions.

Oligomer Sequence, 5' to 3' a) mS3 CAAGGAGAAAGGATTATAGATGGCT (SEQ ID NO: 31) b) 698 AAGGCTCCGCACCCATGGCAG (SEQ ID NO: 32) c) 696 TGCAATGGTGACTCAGGGGGGCCCTT (SEQ ID NO: 33) d) H2 GACCCAGGCGTCTACACTCAAGT (SEQ ID NO: 34) e) mS4 GAGACCATGAAAACCCATCGCTAAC (SEQ ID NO: 35) f) KO0905 TGACTTTCTTCACACTGGACGACAGC (SEQ ID NO: 36) g) GR0905 CTTCACACTGGCTGATAGCCTGGCCG (SEQ ID NO: 37) h) Ngr CAGGGTGGCGGAATGACCTCATGGCCCT (SEQ ID NO: 38) i) RA1016 CTACTCCACAAGGACCCATGTCAATGAC (SEQ ID NO: 39) j) nRA1016 GCTGTGTGCTGGCATTCCCGACTCTAAG (SEQ ID NO: 40) First strand cDNA was prepared from total RNA using SMART II oligonucleotide (5'-AAGCAGTGGTAACAACGCAGAGTACGCGGG-3') (SEQ ID NO: 41) and 5'-RACE cDNA synthesis primer (5'-(T)25 N_,N-3') (N = A, C, G, or T; N_, = A, G, or C) (SEQ ID NO: 42). 5'-RACE was performed using Universal primer mix (UPM) containing Long (0.02 M) (5'-CTAATACGACTCACTATAGGGCAAGCAGTGGTAACAACGCAGAGT-3') (SEQ ID
NO: 43) and Short (1 M) (5'-CTAATACGACTCACTATAGGGCC-3') (SEQ ID NO:
44) universal primer and a specific primer for each species (KO 0905, GR 0905 and RA 1016). Cyclic parameters for the PCR reaction were adapted from the manufacturers recommendations for a Perkin-ElmerTM DNA Thermal Cycler 480 but with 25 cycles in the last step. 5'-RACE PCR products from reactions with specific primers for pig and rat were subjected to nested PCR using Nested Universal Primer (NUP) (5'-AAGCAGTGGTAACAACGCAGAGT-3') (SEQ ID NO: 45) and nested specific primers for pig (nGR0905) and rat (nRA1016) respectively. The nested PCR
reactions were performed according to the manufacturers instructions with 20 cycles of amplification. Products from 5'-RACE and nested 5'-RACE were checked on agarose gel. For characterization products were cloned and sequenced as described above.
The results are shown in Fig. 9 as deduced amino acid sequences. Sequences for human (Hansson et al., 1994) and mouse SCCE (Backman et al., 1999) are included for comparison.

i REFERENCES

Ausubel et al. (1992). Current protocols in Molecular Biology. John Wiley &
Sons Brattsand, M. & Egelrud, T. Purification, molecular cloning, and expression of a human stratum corneum trypsin-like serine protease with possible function in desquamation. J Biol Chem 274, 30033-30040 (1999).

Backman, A., Stranden, P., Brattsand, M., Hansson, L. & Egelrud, T. Molecular cloning and tissue expression of the murine analog to human stratum corneum chymotryptic enzyme. J Invest Dermatol 113, 152-155 (1999).

Chavanas, S. et al. Mutations in SPINK5, encoding a serine protease inhibitor, cause Netherton syndrome. Nat Genet 25, 141-142 (2000).

Diamandis, E.P., Yousef, G.M., Liu-Ying, L., Magklara, A. & Obiezu, C.V. The New Human Kallikrein Gene Family - Implications in Carcinogenesis. Trends in Endocrinology and Metabolism 11, 54-60 (2000).
Ekholm, E. & Egelrud, T. Stratum corneum chymotryptic enzyme in psoriasis.
Arch Dermatol Res 291, 195-200 (1999).

Ekholm, E. & Egelrud, T. The expression of stratum corneum chymotryptic enzyme in human anagen hair follicles: further evidence for its involvement in desquamation-like processes. Br J Dermatol 139, 585-590 (1998).

Ekholm, I.E., Brattsand, M. & Egelrud, T. Stratum corneum tryptic enzyme in normal epidermis: a missing link in the desquamation process? J Invest Dermatol 114, (2000).

Hansson, L. et al. Cloning, expression, and characterization of stratum corneum chymotryptic enzyme. A skin-specific human serine proteinase. J Biol Chem 269, 19420-19426 (1994).

Hogan, B., Constanini, F. & Lazy, E. 1986 In Manipulating the mouse embryo: A
Laboratory Manual. Cold Spring Harbor, NY, Cold Spring Laboratory Press. (Cold Spring Laboratory Press, Cold Spring HarborNew York, 1986).
Hagermark, D., Rajka, G. & Berqvist, U. Experimental itch in human skin elicited by rat mast cell chymase. Acta Derm Venereol (Stockh) 52, 125-128 (1972).
Hagermark, O. Studies on experimental itch induced by kallikrein and bradykinin.
Acta Dem, Venereol (Stockh) 54, 397-400 (1974).
Lindstrom et al, Prostate 29:209-218, 1996 Lusky, M. and Botchan. Inhibition of sv4O replication in simian cells by specific pBR322 DNA sequences. Nature, 293, 55-84 (1981) Sambrook et al. (1989) Molecular Cloning: A laboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. USA

Sondell, B., Thornell, L.E. & Egelrud, T. Evidence that stratum corneum chymotryptic enzyme is transported to the stratum corneum extracellular space via lamellar bodies. J Invest Dermatol 104, 819-823 (1995).

Sondell, B., Dyberg, P., Anneroth, G.K., Ostman, P.O. & Egelrud, T.
Association between expression of stratum comeum chymotryptic enzyme and pathological keratinization in human oral mucosa. Acta Derm Venereol (Stockh) 76, 177-181 (1996).

Vassar et al (1989) Tissue-specific and differentiation-specific expression of a human K14 keratin gene in transgenic mice. Proc Natl Acad Sci U S A.86, 1563-7.

SEQUENCE LISTING
(1) GENERAL INFORMATION:

(i) APPLICANT: Egelrud, Torbjorn Hansson, Lennart (ii) TITLE OF INVENTION: SCCE modified transgenic mammals and their use as models of human diseases (iii) NUMBER OF SEQUENCES: 50 (iv) CORRESPONDENCE ADDRESS:
(A) ADDRESSEE: Sim & McBurney (B) STREET: 6th Floor, 330 University Avenue (C) CITY: Toronto (D) PROVINCE: Ontario (E) POSTAL CODE: M5G 1R7 (v) COMPUTER READABLE FORM:
(D) SOFTWARE: FastSEQ for Windows Version 4.0 (vi) CURRENT APPLICATION DATA:
(A) APPLICATION NUMBER: 2,332,655 (B) FILING DATE: 2001-02-09 (vii) PATENT AGENT INFORMATION:
(A) NAME: Patricia A. Rae (Dr.) (B) REFERENCE NUMBER: 2103-19/PAR
(2) INFORMATION FOR SEQ ID NO: 1:

(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 986 (B) TYPE: nucleic acid (C) STRANDEDNESS: unknown (D) TOPOLOGY: unknown (vi) ORIGINAL SOURCE:
(A) ORGANISM: Homo sapiens (ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: (25) ... (786) (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1:

gaattccgcg gatttccggg ctcc atg gca aga tcc ctt ctc ctg ccc ctg 51 Met Ala Arg Ser Leu Leu Leu Pro Leu cag atc cta ctg cta tcc tta gcc ttg gaa. act gca gga gaa gaa qcc 99 Gln Ile Leu Leu Leu Ser Leu Ala Leu Glu Thr Ala Gly Glu Glu Ala cag ggt gac aag att att gat ggc gcc cca tgt gca aga ggc tcc cac 147 Gln Gly Asp Lys Ile Ile Asp Gly Ala Pro Cys Ala. Arg Gly Ser His cca tgg cag gtg gcc ctg ctc agt ggc aat cag ctc cac tgc gga ggc 195 Pro Trp Gln Val Ala Leu Leu Ser. Gly Asn Gln Leu His Cys Gly Gly gtc ctg gtc aat gag cgc tgg gtg ctc act gcc gcc cac tgc aag atg 243 Val Leu Val Asn Glu Arg Trp Val Leu Thr Ala Ala His Cys Lys Met aat gag tac acc gtg cac ctg ggc agt gat acg ctg ggc gac agg aga 291 Asn Glu Tyr Thr Val His Leu Gly Ser Asp Thr Leu Gly Asp Arg Arg 75 80 85i get cag agg atc aag gcc tcg aag tca ttc cgc cac ccc ggc t.ac tcc 339 Ala Gln Arg Ile Lys Ala Ser Lys Ser Phe Arg His Pro Gly Tyr Ser aca cag acc cat gtt aat gac ctc atg ctc gtg aag ctc aat agc cag 387 Thr Gln Thr His Val Asn Asp Leu Met Leu Val Lys Leu Asn Ser Gln 110 115 1.20 gcc agg ctg tca tcc atg gtg aag aaa gtc agg ctg ccc tcc cgc tgc 435 Ala Arg Leu Ser Ser Met Val. Lys Lys Val Arg Leu Pro Ser Arg Cys gaa ccc cct gga acc acc tgt act gtc tcc ggc tgg ggc act acc acg 483 Glu Pro Pro Gly Thr Thr Cys Thr Val Ser Gly Trp Gly Thr Thr Thr agc cca gat gtg acc ttt ccc tct gac ctc atg tgc gtg gat gtc aag 531 Ser Pro Asp Val Thr Phe Pro Ser Asp Leu Met Cys Val Asp Val Lys ctc atc tcc ccc cag gac tgc acg aag gtt tac aag gac tta ctg gaa 579 Leu Ile Ser Pro Gln Asp Cys Thr Lys Val Tyr Lys Asp Leu Leu Glu aat tcc atg ctg tgc get ggc atc ccc gac tcc aag aaa aac gcc tgc 627 Asn Ser Met Leu Cys Ala Gly Ile Pro Asp Ser Lys Lys Asn Ala Cys aat ggt gac tca ggg gga ccg ttg gtg tgc aga ggt acc ctg caa ggt 675 Asn Gly Asp Ser Gly Gly Pro Leu Val Cys Arg Gly Thr Leu Gln Gly ctg gtg tcc tgg gga act ttc cct tgc ggc caa ccc aat gac cca gga 723 Leu Val Ser Trp Gly Thr Phe Pro Cys Gly Gln Pro Asn Asp Pro Gly gtc tac act caa gtg tgc aag ttc acc aag tgg ata aat gac acc atg 771 Val Tyr Thr Gln Val Cys Lys Phe Thr Lys Trp Ile Asn Asp Thr Met aaa aag cat cgc taa cgccacactg agttaattaa ctgtgtgctt ccaacagaaa 826 Lys Lys His Arg atgcacagga gtgaggacgc cgatgaccta tgaagtcaaa tttgacttta cctttcctca 886 aagatatatt taaacctcat gccctgttga taaaccaatc aaattggtaa agacctaaaa 946 ccaaaacaaa taaagaaaca caaaaccctc aacggaattc 986 (2) INFORMATION FOR SEQ ID NO: 2:

(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 253 (B) TYPE: amino acid (C) STRANDEDNESS: unknown (D) TOPOLOGY: unknown (vi) ORIGINAL SOURCE:
(A) ORGANISM: Homo sapiens (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2:

Met Ala Arg Ser Leu Leu Leu Pro Leu Gin Ile Leu Leu Leu Ser Leu Ala Leu Glu Thr Ala Gly Glu Glu Ala Gin Gly Asp Lys Ile Ile Asp Gly Ala Pro Cys Ala Arg Gly Ser His Pro Trp Gln Val Ala Leu Leu Ser Gly Asn Gln Leu His Cys Gly Gly Val Leu Val Asn Glu Arg Trp Val Leu Thr Ala Ala His Cys Lys Met Asn Glu Tyr Thr Val His Leu Gly Ser Asp Thr Leu Gly Asp Arg Arg Ala Gln Arg Ile Lys Ala Ser Lys Ser Phe Arg His Pro Gly Tyr Ser Thr Gln Thr His Val Asn Asp Leu Met Leu Val Lys Leu Asn Ser Gln Ala Arg Leu Ser Ser Met Val Lys Lys Val Arg Leu Pro Ser Arg Cys Glu Pro Pro Gly Thr Thr Cys Thr Val Ser Gly Trp Gly Thr Thr Thr Ser Pro Asp Val Thr Phe Pro Ser Asp Leu Met Cys Val Asp Val Lys Leu Ile Ser Pro Gln Asp Cys Thr Lys Val Tyr Lys Asp Leu Leu Glu Asn Ser Met Leu Cys Ala Gly Ile Pro Asp Ser Lys Lys Asn Ala Cys Asn Gly Asp Ser Gly Gly Pro Leu Val Cys Arg Gly Thr Leu Gln Gly Leu Val Ser Trp Gly Thr Phe Pro Cys Gly Gln Pro Asn Asp Pro Gly Val Tyr Thr Gln Val Cys Lys Phe Thr Lys Trp Ile Asn Asp Thr Met Lys Lys His Arg (2) INFORMATION FOR SEQ ID NO: 3:

(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 9729 (B) TYPE: nucleic acid (C) STRANDEDNESS: unknown (D) TOPOLOGY: unknown (vi) ORIGINAL SOURCE:
(A) ORGANISM: Homo sapiens (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3:

taccacattt tcttaatcca gtctatcact gatggacatt taggttgatt ccctgtgttt 60 gctgttgtca atagttctac aatgaacgta cgtgtccatg tgtctttaaa cagaatgatt 120 tatattcctt tgggtacaca cactggggct tatgagagag tggagagt:gg gaggaaggag 180 aggatcagaa aaaaataact aatgggtact aggcttaata cctgggtcat taaataatct 240 gtataacaaa cccccatggc gcacgttcac cta.cgcaaca aacctgcaca tcctgcacat 300 gtacccccga actgaaaagt taaaaaaaga aaa:ataaata tttgcttata aattaataaa 360 tgaagccctc aaaaatgttc tattagataa tgttaagt:ac agacattttt gttataaata 420 cataatatac aaagaaatct atgtataaca tgattaaaat gaccataaga acatagatcc 480 taaacatggc aaatattagt ggggtggggt tagggaaagc gttgtttt:ta acttacacct 540 ctctgttaga gttgggaatg ggttcaggcg taattacagg cacgactggg atcagcttgg 600 acaagttccc ccaggcgggc cagaattagg atgtagggtc taggccaccc ctgagagggg 660 gtgagggcaa gaaaaggacc ccagaaggcg ggcgcagtgg ctgacgcctg taatcccagc 720 actttgcggg gccgaggcgg gcacatcatg aggtcaggag atcgagacca ttctggccaa 780 catagtgaaa cccgctctct actaaaaata caaaaattat ctgggagt:gg tggtgcgtgc 840 ctgtaatccc aggtactcgg gaggctgagg caggagaatc acttgaacct gggaggcgga 900 gctggcagtg agccgagatc gcgccaccgc actccagcct ggcgatagag agagactcca 960 tccaaaaaaa agaaaggaag ggagggaggg agaaaggaag aaagaaagaa aaccgcccca 1020 gagaaggacc cgagccagag cctattctct gagctcagcg actgcttgaa tcccgctcct 1080 gcccctcaaa cccagcgcac cgggtccctc ccccgagagc agccaggagg gactgtggga 1140 ccagaatgtg cggaggcgca ggagctgggc acc:gcccgtc cttcggaggg agggtggaga 1200 gagagtgcag tggctccaat tgctctcgct gcgtcagggt tccagataac cagaaccgca 1260 aatgcaggcg ggggtgtccc agagtcggct ccgcccgcac cccagggcgc tggggccggg 1320 catggggcgg ggggtgatat aagaggacgg ccc:agcagag ggctgaagat tttggagccc 1380 agctgtgtgc cagcccaagt cggaacttgg atcacatcag atcctctcga ggtgagaaga 1440 ggcttcatca agggtgcacc tgtaggggag ggggtgatgc tggctccaag cctgactctg 1500 ctctcgagag gtaggggctg cagcctagac tcccgctcct gagcagtgag ggcctggaag 1560 tctgcaattt ggggcctttt agggaaaaac gaactacaga gtcagaagtt tgggttcaac 1620 agggaagggc aagatcggag cctagattcc tgggtcccta gggatctgaa gaacaggaat 1680 tttgggtctg agggaggagg ggctggggtt ctggactcct gggtctgagg gaggagggcc 1740 tgggggcctg gactcctggg tctgagggag gaggagctgg gggtctcgac tcctgggtct 1800 gagggaggag gggctggggg cctggactcc tgcgtctgag ggaggagggg ctgggacctg 1860 gactcctagg tctgagggag gaggagctgg ggcctggact cctgggtctg agggaggagg 1920 ggctggggcc tggactcctg ggtctgaggg agaatcggct gaggcctaga ctcctgggtc 1980 tgagggagga ggggcggggg cctggactcc tgggtctgag ggaggagggg ctggagcctg 2040 gactcctggg cctgagggag gagggactga gacctggact cctaggtctg agggaggagg 2100 gactgggacc tggactcctg ggtctgaggg aggaggagct ggggagctgg actcctgggt 2160 ctgagggagg cggggctggg ggcctggact cctgggtctg agggaggagg ggttggggcc 2220 tggactcctg agcctgaggg a.ggagggact tggacctgga ctcctaggtc tgagggagga 2280 ggagctgggg gcctggactc ctaggtctga gggaggaggg gctgggggcc tggactcctg 2340 ggtctgaggg aggaaggtgc tagggtctgg aci:cttgggt atgagggagg aggaggttag 2400 gggtctggac ttctgagtgt aaggaaggag aggccagaga aagaaatttc tgggtctgag 2460 ggaggagggg ctggggttct ggacccctag gtctgaggga ggaggggctg gggcctggac 2520 tcctgggtct gtggggggag gggctggggc ctggacccct gggtctgagt ggggaggggc 2580 tgggcctgaa tgctttctcc ttctcagctc cagcaggaga ggcccttcct cgcctggcag 2640 cccctgagcg gctcagcagg gcaccatggc aagatccctt ctcctgcccc tgcagatcct 2700 actgctatcc ttagccttgg aaactgcagg agaagaaggt gaaagctgga ctgggaagtc 2760 tgacctcacc tcagggcccc cactgaccct ctccaaggag ttcctgagtc agaacccttc 2820 cctcctcaaa cagcttccat cctgggagga ccagactgtc ggctgaagcc cccgctcttc 2880 ctgcttctgc tgactcaggg ggtctctgtc ccctccaggc cctgcctcct gtgctcaggg 2940 tctctctgtg gttccccaga tgagatgcgc ctc:,ctgggtt tctgagtggg ctccttctgt 3000 ctgtctctat ccctatctct tgctttctct gta.tttctcc acacattttc atctgtctct 3060 gtccatctct gactctggga atccctgagg tgcagcctca gcctccccct aatgctagct 3120 acccacatgc tcctccatgt ctccatccag cccagggtga caagattatt gatggcgccc 3180 catgtgcaag aggctcccac ccatggcagg tgcccctgct: cagtggcaat cagctccact 3240 gcggaggcgt cctggtcaat gagcgctggg tgctcactgc cgcccactgc aagatcaagt 3300 aggtgccacc caagtctctg ctggaggtgc gccagcatct ccagctcgct atgggggtgg 3360 aagggcagtc tttctgtgcc tacggctcta ttc:tcctctc tctgggtctc tgtccccctc 3420 tctctgggcc tctgtacccc ctctccctgg ggc:tctgtcc ccctctct:cc ctggctctct 3480 gtctccctct ctctgggtct ctgtccccct ctctctggat ctctgttccc ctctctctgt 3540 gtctctgtcc cccattctct ctaggtctct gtt.ccccctc ctctctctct gggtctctgt 3600 ccctctctct ctggtctctg tccccctctc tctctggatc tctgtccccc tctccctggg 3660 cctctgtacc ccctctccct ggggctctgt cccccctctc tgggtctctg tctgcctttc 3720 tctctggatc tctgttcccc tctgtgtctc tgtccccctc tctctctggg tctctgttcc 3780 ccctcctctc tttctgggtc tctgtccctc tctctctggg tctctgtccc cctctctctc 3840 tggtctctgt tccccctcct ctctctctgg tctctgtccc tctctctctg ggtctctgtc 3900 accctctctc tctgggtctc tgtcaccctc tctctctggt ctctgttccc cctcctctct 3960 ctgtgggtct ctgtccctct ctctctgggt ctctgttccc ctctctct:ct ggtctctgtt 4020 ccccctcctc tctctccgga tctctgtccc cctctccctg gggctctgtc cccctctctc 4080 cctggctctc tgtcttcctc tctctggggc tct:gtccc_cc tctctctctg gtctctgttc 4140 ccctctctct gggtctctgt ccctctctct ctaggtctct gtccctctct ctctggatct 4200 ctgtccccct ctccctgggc ctctgtaccc cct:ctccctg gggctctgtc cccctctctc 4260 tgggtctctg tctgcctttc tctctggatc tctgttcccc tctgtgtctc tgtccccctc 4320 tctctctggg tctctgttcc ccctcctctc ttt:ctgggtc tct9tccctc tctctctggg 4380 tctctgtccc cctctctctc tggtctctgt tccccctcct ctctctctgg tctctgtccc 4440 tctctctctg ggtctctgtc accctctctc tct:gggtcctc tgtcaccctc tctctctggt 4500 ctctgttccc cctcctctct ctgtgggtct ctgtccctct ctctctgggt ctctgttccc 4560 ctctctctct ggtctctgtt ccccctcctc tct:ctccgga tctctgtccc cctctccctg 4620 gggctctgtc cccctctctc cctggctctc tgt:cttcctc tctctggggc tctgtccccc 4680 tctctctctg gtctctgttc ccctctctct gggtctctgt ccctctctct ctgggtctct 4740 gtccctctct ctctggatct ctgtccccct ctctctctgg gtctctgttc ccctctctct 4800 gggtctctgt cccctctcct ctctctgtgt ctctctcccc ctcctctctc tgtgtctctg 4860 tcccccctcc tatctctgtg tctctctccc ccctcctctc tctgggtctc tgtccccccc 4920 tctctgggtc tctgtctccc tctctctggg gctctgtccc cctctctctc tggatctctg 4980 ttcccctctc tctgggtctc tgtctcccct cctctctctg tgtctctgtc ccccctcctc 5040 tctctgggtc tctgtcccca ccccgtcccc caggtctttg cacaccctct ctgtcacagt 5100 gtctct.tctg aatctgtgaa t.gtcactcct cgcagtgagt acaccgtgca cctgggcagt 5160 gatacgctgg gcgacaggag agctcagagg atcaaggcct cgaaatcatt ccgccacccc 5220 ggctactcca cacagaccca tgttaatgac ctcatgctcg tgaagctcaa tagccaggcc 5280 acgctgtcat ccatggtgaa gaaagtcagg ctgccctccc gctgcgaacc ccctggaacc 5340 acctgtactg tctccggctg gggcactacc ac(3agcccag atggtaggtg gcctcagtga 5400 cccaggagtg caggccccag ccctcctccc tcagacccag gagtccaggc ccccagcccc 5460 tcctccctca gacccaggag tccaggcctc agaccctcct ccctcagacc caggagtcca 5520 ggcccccagc ccctcctccc tcagacccgc gagtccagac cccactccct cctccctcag 5580 acccagcagt cctggccccc agaccctcct ccctcgaaac caggagcctg aacaacagcc 5640 cttctggtcc tcgcccccat cctctctgac tgacagctct ccctgctcct ccctgcagtg 5700 acctttccct ctgacctcat gtgcgtggat gtc,7aagctca tctcccccca ggactgcacg 5760 aaggtttaca aggacttact ggaaaattcc at!gctgtgcg ctggcacccc cgactccaag 5820 aaaaacgcct gcaatgtgag accctccccc cccattctcc cccattcctg ggtaccctgt 5880 ctgcattccc cagggacaga gcttgaccca agtgactggg taccaagccc ggccttgccc 5940 tccccccagg cctggcctcc tcagcttttt ccacctcatt ctctccctag gtcaggggtg 6000 ggagtttact taggggccga tgtggccctg gg,gatgggac agagagttta ataggggtga 6060 gaaagtgggg gtgggaccag ggaaggagac tgaggtgctg gcctcagtcc caaaccctaa 6120 gggggcacca aaaacctcag tgattgagat aaatcataat gcaatattta aaaataaaaa 6180 taaaaactca tgcagaagtc catgatggac aaaatgtcac attttaaata aagagcaggt 6240 ggatcttact gaattttccc ttgccgtaag tactagcgtg gctcagcaca gcgctgtact 6300 ggcactgtct tcatttaaaa tgtggatacc attcccatca tgcagtttta tgtattacat 6360 ttgatttcgt taagtactgc attgaagtat tgtgtattgc agttactgag attttgtgcc 6420 tgaagctgat gactcactca cctgaccctg gccctggtcc cggggaaaac actctttctc 6480 tccacctcct ctctgttccc tctttctggc cttttgtcat cccctctqtt tctgaacagt 6540 cttcccacat ctctctttgt gacataattt catttcattc ttttcctctt tgttttttct 6600 ctgtgttgag ctatcttgct ctc:cctccct tggtctctct: ccatgccctc ctctctgctc 6660 tctgtcttct ccctctttct cttgcttctc tctctctcct cccctccctc tctcctctcc 6720 ctgcccccct gctctctctt ttttcctctc tctctctctc ctctctgccc ctctcctctt 6780 tctctctctc ccccacttct ctgtctctct tca.tctctct ccctcatctc tccttgcccc 6840 ctccttttta ctgtctctct ctttctcttt cttctatctc tctcctct:cc ccgccgctcc 6900 cccatctctg tctttctttc tctctcttta ttctcctcct ctcttcccgt ctctctctcc 6960 tctccccacc cccaccccat ctctctcccc acaccttccc cccctttctc tttgtctctc 7020 tcttctacct ctttcttctc cacccccatc tct.ctctctc ttctcttccc acaccctccc 7080 catctccctc atctctttgt ctgtctctct tct:ccctcct tcttttccac ccccatctct 7140 ctgtctctct ctctccccat accctttccc tcttcctcat ctctctttgt ctctctctcc 7200 tttccctctt tcttctccac ctccaactct ctc:tgtctct ccacacccat cctccttgct 7260 cacatctgca ccttcagctg tcaggggatg tgggagagag agtgttaggg atagaggaga 7320 tgggagagag atgactgtcc tagagaatag ggt:gttcccc ttctcccctg gtgagggcca 7380 gtttcatgaa tgtgcaagct ctgcacggac acagagcccc acactcagaa gggtctcaaa 7440 cttagtctaa tgcattcctg ctgttgtctt gaaattctca ataatttttg aacaaagggc 7500 cctgcatttt cgttttgcac caagtcctgt aaattatgta actggtcttc accctggtct 7560 ccgagaccat cgtgtccccc tttcctgcgc cacagggcac gcatccaccc cttggagatg 7620 atgttccttc tcccactagc ttggagcagg gtccttaaca ttggaaaata aagagtgctc 7680 tgatcctgga agccccaccc cttctctgca att:ggtctca ttggccaagg gtcaaaccag 7740 tgtcttcaaa ggacctagtg tgtccctagc actagctttc ccattagtcc ccagagacaa 7800 tgagtctctt ctcattggct atggtggaag tccataatct gcaagacaaa gaccgataac 7860 tgaggaatgt atgagaatga gttgggcttt gat:ctgaagc caaagttaat ctccggctct 7920 attccctcta gggtgactca gggggaccgt ttgtgttcag aggtaccctg caaggtctgg 7980 tgtcctgggg aactttccct tgcggccaac ccaatgaccc aggagtctac actcaagtgt 8040 gcaagttcac caagtggata aatgacacca tgaaaaagca tcgctaacgc cacactgagt 8100 taattaactg tgtgcttcca acagaaaatg cacaggagtg aggacgccga tgacctatga 8160 agtcaaattt gactttacct ttcctcaaag atatatttaa acctcatgcc ctgttgataa 8220 accaatcaaa ttggtaaaga cctaaaacca aaacaaataa agaaacacaa aaccctcagt 8280 gctggagaag agtcagtgag accagcactc tcaaacactg gaactggacg ttcgtacagt 8340 ctttacggaa gacacttggt caacgtacac cgagaccctt attcaccacc tttgacccag 8400 taactctaat cttaggaaga acctactgaa acaaaaaaaa tccaaaatgt agaacaagac 8460 ttgaatttac catgatatta tttatcacag aaatgaagtg aaaccatcaa acatgttcca 8520 aaagtaccag atggcttaaa taatagtctg gcttggcaca acgatgtttt ttttctttga 8580 gacagagtct ctgttgcttg ggctgcaatg cagtgatgca atcttggctc actgcaacct 8640 ccgccccctg ggttcaagtg attctcgtgc ttcagcctcc caagtacctg ggactacagg 8700 tgtgcaccac cacaccaggc taattttttg tgcagtttta ctagagacag ggtttcacca 8760 tgttggccag cgtggtcttg aacgcctgac ctcagatgat ccacccacct tggcctccca 8820 aagtgctggg attacaggca tgagccacca ccgccactcc acaatgatat tacaaaccta 8880 ttaaaaatga tacttagaca gaattgtcag tattattcaa gaacatttag gctataggat 8940 gttaaatgac aaaaggaagg acaaaaatat atatgtatgt gaccctaccc ataaaaaatg 9000 aaatattcac agaatcagat ctgaaaacac atgtcccaga ctgcatactg gggtcgtcat 9060 gaggtgtctc cttccttctg tgtacttttc cttgaaggtg cacttttata acatgaaaaa 9120 taaaggtggg gaaaaaagtc tgaagatcta agattggaga gaggtgacct ttcaggaagg 9180 gagactagaa agaaatatgt gcctggtttt gagccctggt cctgccggcc ctgttccagg 9240 gcatatttcc atttcccaga tctcagtttt tcctgtctgt aaaatgggag agagaggaaa 9300 ggatggagag aggaagaagg aagggaggag ggaggagaga acaggccaac ttcatcagcg 9360 tgggaagggg tgtgaaagtg tttctgagca tctcacgagt gacaagtgag gagggaggct 9420 ggcggttttc agagggattg ggatgacagt agacaggaca caggggtccc acaggggtct 9480 gccagaagta agcaaacagt gccggaggaa gatggtggca cctgctcccc aagaagggag 9540 ggaaaggaac ctcgggaagc gggtaggatg agggaggagt cctctgtgac tcagagcctg 9600 gccacagccc cagccatcta acatcaaaga tcctctgtgt ggtcacacct cagacgctgc 9660 tgaccgagga gccactccag cccaggacac gccctcctac ctgttcttcc tgtttttctc 9720 ccagaattc 9729 (2) INFORMATION FOR SEQ ID NO: 4:

(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 23 (B) TYPE: amino acid (C) STRANDEDNESS: unknown (D) TOPOLOGY: unknown (vi) ORIGINAL SOURCE:
(A) ORGANISM: Bos taurus (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4:

Ala Gly Ile Pro Asn Ser Arg Thr Asn Ala Cys Asn Gly Asp Ser Gly Gly Pro Leu Met Cys Lys Gly (2) INFORMATION FOR SEQ ID NO: 5:
(U SEQUENCE CHARACTERISTICS:
(A) LENGTH: 23 (B) TYPE: amino acid (C) STRANDEDNESS: unknown (D) TOPOLOGY: unknown (vi) ORIGINAL SOURCE:
(A) ORGANISM: Sus scrofa (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 5:

Ala Gly Ile Pro Asn Ser Lys Thr Asn Ala Cys Asn Gly Asp Ser Gly Gly Pro Leu Val Cys Lys Gly (2) INFORMATION FOR SEQ ID NO: 6:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 23 (B) TYPE: amino acid (C) STRANDEDNESS: unknown (D) TOPOLOGY: unknown (vi) ORIGINAL SOURCE:
(A) ORGANISM: Homo sapiens (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 6:

Ala Gly Ile Pro Asp Ser Lys Lys Asn Ala Cys Asn Gly Asp Ser Gly Gly Pro Leu Val Cys Arg Gly (2) INFORMATION FOR SEQ ID NO: 7:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 23 (B) TYPE: amino acid (C) STRANDEDNESS: unknown (D) TOPOLOGY: unknown (vi) ORIGINAL SOURCE:
(A) ORGANISM: Rattus norvegicus (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 7:

Ala Gly Ile Pro Asp Ser Lys Thr Asn Thr Cys Asn Gly Asp Ser Gly Gly Pro Leu Val Cys Asn Asp (2) INFORMATION FOR SEQ ID NO: 8:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 23 (B) TYPE: amino acid (C) STRANDEDNESS: unknown (D) TOPOLOGY: unknown (vi) ORIGINAL SOURCE:
(A) ORGANISM: Mus musculus (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 8:

Ala Gly Ile Pro Asp Ser Lys Thr Asn Thr Cys Asn Gly Asp Ser Gly Gly Pro Leu Val Cys Asn Asp (2) INFORMATION FOR SEQ ID NO: 9:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 38 (B) TYPE: amino acid (C) STRANDEDNESS: unknown (D) TOPOLOGY: unknown (vi) ORIGINAL SOURCE:
(A) ORGANISM: Bos taurus (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 9:

Gln Glu Asp Gln Gly Asn Lys Ser Gly Glu Lys Ile Ile Asp Gly Val Pro Cys Pro Arg Gly Ser Gln Pro Trp Gln Val Ala Leu Leu Lys Gly Ser Gln Leu His Cys Gly (2) INFORMATION FOR SEQ ID NO: 10:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 37 (B) TYPE: amino acid (C) STRANDEDNESS: unknown (D) TOPOLOGY: unknown (vi) ORIGINAL SOURCE:
(A) ORGANISM: Sus scrofa (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 10:

Gln Glu Gly Gln Asp Lys Ser Gly Glu Lys Ile Ile Asp Gly Val Pro Cys Pro Gly Gly Ser Arg Pro Trp Gln Val Ala Leu Leu Lys Gly Asn Gln Leu His Cys Gly (2) INFORMATION FOR SEQ ID NO: 11:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 34 (B) TYPE: amino acid (C) STRANDEDNESS: unknown (D) TOPOLOGY: unknown (vi) ORIGINAL SOURCE:
(A) ORGANISM: Homo sapiens (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 11:

Glu Glu Ala Gln Gly Asp Lys Ile Ile Asp Gly Ala Pro Cys Ala Arg Gly Ser His Pro Trp Gln Val Ala Leu Leu Ser Gly Asn Gln Leu His Cys Gly (2) INFORMATION FOR SEQ ID NO: 12:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 31 (B) TYPE: amino acid (C) STRANDEDNESS: unknown (D) TOPOLOGY: unknown (vi) ORIGINAL SOURCE:
(A) ORGANISM: Rattus norvegicus (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 12:

Gln Gly Glu Arg Ile Ile Asp Gly Tyr Lys Cys Lys Glu Gly Ser His Pro Trp Gln Val Ala Leu Leu Lys Gly Asp Gln Leu His Cys Gly (2) INFORMATION FOR SEQ ID NO: 13:

(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 31 (B) TYPE: amino acid (C) STRANDEDNESS: unknown (D) TOPOLOGY: unknown (vi) ORIGINAL SOURCE:
(A) ORGANISM: Mus musculus (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 13:

Gln Gly Glu Arg Ile Ile Asp Gly Ile Lys Cys Lys Glu Gly Ser His Pro Trp Gln Val Ala Leu Leu Lys Gly Asn Gln Leu His Cys Gly (2) INFORMATION FOR SEQ ID NO: 14:

(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 7 (B) TYPE: amino acid (C) STRANDEDNESS: unknown (D) TOPOLOGY: unknown (vi) ORIGINAL SOURCE:
(A) ORGANISM: Artificial Sequence (ix) FEATURE:
(D) OTHER INFORMATION: Consensus sequence for cleavage site in C-terminal of SCCE.
(A) NAME/KEY: VARIANT
(B) LOCATION: 2 (D) OTHER INFORMATION: Asp = either aspartate (Asp) or glutamate (Glu).
(A) NAME/KEY: VARIANT
(B) LOCATION: 3 (D) OTHER INFORMATION: Lys = either lysine (Lys) or arginine (Arg).

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 14:
Gly Asp Lys Ile Ile Asp Gly (2) INFORMATION FOR SEQ ID NO: 15:

(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 8 (B) TYPE: amino acid (C) STRANDEDNESS: unknown (D) TOPOLOGY: unknown (vi) ORIGINAL SOURCE:
(A) ORGANISM: Artificial Sequence (ix) FEATURE:
(D) OTHER INFORMATION: consensus of the substrate specificity pouch.

(A) NAME/KEY: VARIANT
(B) LOCATION: 1 (D) OTHER INFORMATION: Thr = any amino acid residue.
(A) NAME/KEY: VARIANT
(B) LOCATION: 3 (D) OTHER INFORMATION: Ala = any amino acid residue.
(A) NAME/KEY: VARIANT
(B) LOCATION: 5 (D) OTHER INFORMATION: Asn = any amino acid residue.
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 15:

Thr Asn Ala Cys Asn Gly Asp Ser (2) INFORMATION FOR SEQ ID NO: 16:

(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH : 20 (B) TYPE: nucleic acid (C) STRANDEDNESS: unknown (D) TOPOLOGY: unknown (vi) ORIGINAL SOURCE:
(A) ORGANISM: Atificial sequence (ix) FEATURE:
(D) OTHER INFORMATION: PCR primer SYM3300.
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 16:

ggtggccctg ctcagtggca 20 (2) INFORMATION FOR SEQ ID NO: 17:

(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 22 (B) TYPE: nucleic acid (C) STRANDEDNESS: unknown (D) TOPOLOGY: unknown (vi) ORIGINAL SOURCE:
(A) ORGANISM: Artificial Sequence (ix) FEATURE:
(D) OTHER INFORMATION: PC&. primer SYM3301.
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 17:

caccatggat gacacagcct gg 22 (2) INFORMATION FOR SEQ ID NO: 18:

(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 (B) TYPE: nucleic acid (C) STRANDEDNESS: unknown (D) TOPOLOGY: unknown (vi) ORIGINAL SOURCE:
(A) ORGANISM: Artificial Sequence (ix) FEATURE:
(D) OTHER INFORMATION: PCR primer SYM3302.
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 18:

aataaagaaa cacaaaaccc 20 (2) INFORMATION FOR SEQ ID NO: 19:

(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 18 (B) TYPE: nucleic acid (C) STRANDEDNESS: unknown (D) TOPOLOGY: unknown (vi) ORIGINAL SOURCE:
(A) ORGANISM: Artificial Sequence (ix) FEATURE:
(D) OTHER INFORMATION: PCR primer SYM3418.
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 19:

tgtaatatca ttgtgggc 18 (2) INFORMATION FOR SEQ ID NO: 20:

(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 18 (B) TYPE: nucleic acid (C) STRANDEDNESS: unknown (D) TOPOLOGY: unknown (vi) ORIGINAL SOURCE:

(A) ORGANISM: Artificial Sequence (ix) FEATURE:
(D) OTHER INFORMATION: PCR. primer SYM4118.
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 20:

ggatgtgaag ctcatctc 18 (2) INFORMATION FOR SEQ ID NO: 21:

(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 18 (B) TYPE: nucleic acid (C) STRANDEDNESS: unknown (D) TOPOLOGY: unknown (vi) ORIGINAL SOURCE:
(A) ORGANISM: Artificial Sequence (ix) FEATURE:
(D) OTHER INFORMATION: PCR primer SYM4121.
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 21:

tggagtcggg gatgccag 18 (2) INFORMATION FOR SEQ ID NO: 22:

(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 25 (B) TYPE: nucleic acid (C) STRANDEDNESS: unknown (D) TOPOLOGY: unknown (vi) ORIGINAL SOURCE:
(A) ORGANISM: Artificial Sequence (ix) FEATURE:
(D) OTHER INFORMATION: PCR primer SYM4720.
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 22:

gggagggtgg agagagagtg cagtg 25 (2) INFORMATION FOR SEQ ID NO: 23:

(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 (B) TYPE: nucleic acid (C) STRANDEDNESS: unknown (D) TOPOLOGY: unknown (vi) ORIGINAL SOURCE:
(A) ORGANISM: Artificial Sequence (ix) FEATURE:
(D) OTHER INFORMATION: PCR. primer SYM4899.
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 23:

agtctaggct gcagccccta c 21 (2) INFORMATION FOR SEQ ID NO: 24:

(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 22 (B) TYPE: nucleic acid (C) STRANDEDNESS: unknown (D) TOPOLOGY: unknown (vi) ORIGINAL SOURCE:
(A) ORGANISM: Artificial Sequence (ix) FEATURE:
(D) OTHER INFORMATION: PCR primer hEXON1.
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 24:

ctggagtgat ctgatgtgat cc 22 (2) INFORMATION FOR SEQ ID NO: 25:

(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 24 (B) TYPE: nucleic acid (C) STRANDEDNESS: unknown (D) TOPOLOGY: unknown (vi) ORIGINAL SOURCE:
(A) ORGANISM: Artificial Sequence (ix) FEATURE:
(D) OTHER INFORMATION: PCR primer mEXON1.
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 25:

ctgggagtga cttggcgtgg ctct 24 (2) INFORMATION FOR SEQ ID NO: 26:

(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 23 (B) TYPE: nucleic acid (C) STRANDEDNESS: unknown (D) TOPOLOGY: unknown (vi) ORIGINAL SOURCE:
(A) ORGANISM: Artificial Sequence (ix) FEATURE:
(D) OTHER INFORMATION: PCR primer specific for human SCCE
IE2.

(xi) SEQUENCE DESCRIPTION: SEQ ID NC): 26:

gctctcccat tagtccccag aga 23 (2) INFORMATION FOR SEQ ID NO: 27:

(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 24 (B) TYPE: nucleic acid (C) STRANDEDNESS: unknown (D) TOPOLOGY: unknown (vi) ORIGINAL SOURCE:
(A) ORGANISM: Artificial Sequence (ix) FEATURE:
(D) OTHER INFORMATION: PCR primer specific, for human SCCE
MJ2.

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 27:

ccacttggtg aacttgcaca cttg 24 (2) INFORMATION FOR SEQ ID NO: 28:

(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 18 (B) TYPE: nucleic acid (C) STRANDEDNESS: unknown (D) TOPOLOGY: unknown (vi) ORIGINAL SOURCE:
(A) ORGANISM: Artificial Sequence (ix) FEATURE:
(D) OTHER INFORMATION: forward primer covering the position 427 - 444 of the human SCCE cDNA sequence.

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 28:

gcgaaccccc tggaacaa 18 (2) INFORMATION FOR SEQ ID NO: 29:

(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 (B) TYPE: nucleic acid (C) STRANDEDNESS: unknown (D) TOPOLOGY: unknown (vi) ORIGINAL SOURCE:
(A) ORGANISM: Artificial Sequence (ix) FEATURE:
(D) OTHER INFORMATION: reverse primer covering the position 490 - 510 of the human cDNA sequence in exon five.
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 29:

acatccacgc acatgaggtc a 21 (2) INFORMATION FOR SEQ ID NO: 30:

(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 29 (B) TYPE: nucleic acid (C) STRANDEDNESS: unknown (D) TOPOLOGY: unknown (vi) ORIGINAL SOURCE:
(A) ORGANISM: Artificial Sequence (ix) FEATURE:
(D) OTHER INFORMATION: The real time amplification probe covering the position 445 - 473 of the human cDNA sequence in exon four.

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 30:

cctgtactgt ctccggctgg ggcactacc 29 (2) INFORMATION FOR SEQ ID NO: 31:

(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 25 (B) TYPE: nucleic acid (C) STRANDEDNESS: unknown (D) TOPOLOGY: unknown (vi) ORIGINAL SOURCE:
(A) ORGANISM: Artificial Sequence (ix) FEATURE:
(D) OTHER INFORMATION: PCR primer mS3.
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 31:

caaggagaaa ggattataga tggct 25 (2) INFORMATION FOR SEQ ID NO: 32:

(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 (B) TYPE: nucleic acid (C) STRANDEDNESS: unknown (D) TOPOLOGY: unknown (vi) ORIGINAL SOURCE:
(A) ORGANISM: Artificial Sequence (ix) FEATURE:
(D) OTHER INFORMATION: PCk primer 698.
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 32:

aaggctccgc acccatggca g 21 (2) INFORMATION FOR SEQ ID NO: 33:

(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 26 (B) TYPE: nucleic acid (C) STRANDEDNESS: unknown (D) TOPOLOGY: unknown (vi) ORIGINAL SOURCE:
(A) ORGANISM: Artificial Sequence (ix) FEATURE:
(D) OTHER INFORMATION: PCR primer 696.
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 33:

tgcaatggtg actcaggggg gccctt 26 (2) INFORMATION FOR SEQ ID NO: 34:

(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 23 (B) TYPE: nucleic acid (C) STRANDEDNESS: unknown (D) TOPOLOGY: unknown (vi) ORIGINAL SOURCE:
(A) ORGANISM: Artificial Sequence (ix) FEATURE:
(D) OTHER INFORMATION: PCR primer H2.
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 34:

gacccaggcg tctacactca agt 23 (2) INFORMATION FOR SEQ ID NO: 35:

(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 25 (B) TYPE: nucleic acid (C) STRANDEDNESS: unknown (D) TOPOLOGY: unknown (vi) ORIGINAL SOURCE:
(A) ORGANISM: Artificial Sequence (ix) FEATURE:
(D) OTHER INFORMATION: PCR. primer mS4.
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 35:

gagaccatga aaacccatcg ctaac 25 (2) INFORMATION FOR SEQ ID NO: 36:

(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 26 (B) TYPE: nucleic acid (C) STRANDEDNESS: unknown (D) TOPOLOGY: unknown (vi) ORIGINAL SOURCE:
(A) ORGANISM: Artificial Sequence (ix) FEATURE:
(D) OTHER INFORMATION: PCR primer KO 0905.
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 36:

tgactttctt cacactggac gacagc 26 (2) INFORMATION FOR SEQ ID NO: 37:

(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 26 (B) TYPE: nucleic acid (C) STRANDEDNESS: unknown (D) TOPOLOGY: unknown (vi) ORIGINAL SOURCE:
(A) ORGANISM: Artificial Sequence (ix) FEATURE:
(D) OTHER INFORMATION: PCR primer GR 0905.
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 37:

cttcacactg gctgatagcc tggccg 26 (2) INFORMATION FOR SEQ ID NO: 38:

(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 28 (B) TYPE: nucleic acid (C) STRANDEDNESS: unknown (D) TOPOLOGY: unknown (vi) ORIGINAL SOURCE:
(A) ORGANISM: Artificial Sequence (ix) FEATURE:
(D) OTHER INFORMATION: PCR. primer Ngr.
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 38:

cagggtggcg gaatgacctc atggccct 28 (2) INFORMATION FOR SEQ ID NO: 39:

(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 28 (B) TYPE: nucleic acid (C) STRANDEDNESS: unknown (D) TOPOLOGY: unknown (vi) ORIGINAL SOURCE:
(A) ORGANISM: Artificial Sequence (ix) FEATURE:
(D) OTHER INFORMATION: PCR primer RA 1016.
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 39:

ctactccaca aggacccatg tcaatgac 28 (2) INFORMATION FOR SEQ ID NO: 40:

(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 28 (B) TYPE: nucleic acid (C) STRANDEDNESS: unknown (D) TOPOLOGY: unknown (vi) ORIGINAL SOURCE:
(A) ORGANISM: Artificial Sequence (ix) FEATURE:
(D) OTHER INFORMATION: PCR primer nRA 1016.
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 40:

gctgtgtgct ggcattcccg actctaag 28 (2) INFORMATION FOR SEQ ID NO: 41:

(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 30 (B) TYPE: nucleic acid (C) STRANDEDNESS: unknown (D) TOPOLOGY: unknown (vi) ORIGINAL SOURCE:
(A) ORGANISM: Artificial Sequence (ix) FEATURE:
(D) OTHER INFORMATION: SMART II oligonucleotide.
(xi) SEQUENCE DESCRIPTION: SEQ II) NO: 41:

aagcagtggt aacaacgcag agtacgcggg 30 (2) INFORMATION FOR SEQ ID NO: 42:

(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 27 (B) TYPE: nucleic acid (C) STRANDEDNESS: unknown (D) TOPOLOGY: unknown (vi) ORIGINAL SOURCE:
(A) ORGANISM: Artificial Sequence (ix) FEATURE:
(D) OTHER INFORMATION: 5"-RACE cDNA synthesis primer.
(A) NAME/KEY: variation (B) LOCATION: 27 (D) OTHER INFORMATION: n == a or g or c or t (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 42:

tttttttttt tttttttttt tttttvn 27 (2) INFORMATION FOR SEQ ID NO: 43:

(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 45 (B) TYPE: nucleic acid (C) STRANDEDNESS: unknown (D) TOPOLOGY: unknown (vi) ORIGINAL SOURCE:
(A) ORGANISM: Artificial Sequence (ix) FEATURE:
(D) OTHER INFORMATION: Long universal primer.
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 43:

ctaatacgac tcactatagg gcaagcagtg gtaacaacgc agagt 45 (2) INFORMATION FOR SEQ ID NO: 44:

(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 23 (B) TYPE: nucleic acid (C) STRANDEDNESS: unknown (D) TOPOLOGY: unknown (vi) ORIGINAL SOURCE:
(A) ORGANISM: Artificial Sequence (ix) FEATURE:
(D) OTHER INFORMATION: Short universal primer.
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 44:

ctaatacgac tcactatagg gcc 23 (2) INFORMATION FOR SEQ ID NO: 45:

(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 23 (B) TYPE: nucleic acid (C) STRANDEDNESS: unknown (D) TOPOLOGY: unknown (vi) ORIGINAL SOURCE:
(A) ORGANISM: Artificial Sequence (ix) FEATURE:
(D) OTHER INFORMATION: Nested universal primer.
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 45:

aagcagtggt aacaacgcag agt 23 (2) INFORMATION FOR SEQ ID NO: 46:

(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 243 (B) TYPE: amino acid (C) STRANDEDNESS: unknown (D) TOPOLOGY: unknown (vi) ORIGINAL SOURCE:
(A) ORGANISM: Artificial Sequence (ix) FEATURE:
(D) OTHER INFORMATION: Deducecd amino acid sequence from the C-terminal part of SCCE from cow.

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 46:

Met Thr Thr Pro Leu Val Ile Leu Leu Leu Thr Phe Ala Leu Gly Ser Val Ala Gln Glu Asp Gln Gly Asn Lys Ser Gly Glu Lys Ile Ile Asp Gly Val Pro Cys Pro Arg Gly Ser Gln Pro Trp Gln Val Ala Leu Leu Lys Gly Ser Gln Leu His Cys Gly Gly Val Leu Leu Asn Glu Gln Trp Val Leu Thr Ala Ala His Cys Met Asn Glu Tyr Asn Val His Met Gly Ser Val Arg Leu Val Gly Gly Gln Lys Ile Lys Ala Thr Arg Ser Phe Arg His Pro Gly Tyr Ser Thr Gln Thr His Ala Asn Asp Leu Met Leu Val Lys Leu Asn Gly Arg Ala Lys Leu Ser Ser Ser Val Lys Lys Val Asn Leu Pro Ser His Cys Asp Pro Pro Gly Thr Met Cys Thr Val Ser Gly Trp Gly Thr Thr Thr Ser Pro Asp Val Thr Phe Pro Gly Gln Leu Met Cys Thr Asp Val Lys Leu Ile Ser Pro Gin Asp Cys Arg Lys Val Tyr Lys Asp Leu Leu Gly Asp Ser Met Leu Cys Ala Gly Ile Pro Asn Ser Arg Thr Asn Ala Cys Asn Gly Asp Ser Gly Gly Pro Leu Met Cys Lys Giy Thr Leu Gln Gly Val Val Ser Trp Gly Ser Phe Pro Cys Gly Gln Pro Asn Asp Pro Gly Val Tyr Thr Gln Val Cys Lys Tyr Val Asn Trp Ile Lys (2) INFORMATION FOR SEQ ID NO: 47:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 249 (B) TYPE: amino acid (C) STRANDEDNESS: unknown (D) TOPOLOGY: unknown (vi) ORIGINAL SOURCE:
(A) ORGANISM: Artificial Sequence (ix) FEATURE:
(D) OTHER INFORMATION: Deducecd amino acid sequence from the C-terminal part of SCCE from pig.

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 47:

Met Ala Arg Pro Leu Leu Pro Pro Arg Leu Ile Leu Leu Leu Ser Leu Ala Leu Gly Ser Ala Ala Gln Glu Giy Gln Asp Lys Ser Gly Glu Lys Ile Ile Asp Gly Val Pro Cys Pro Gly Gly Ser Arg Pro Trp Gln Val Ala Leu Leu Lys Gly Asn Gln Leu His Cys Gly Gly Val Leu Val Asn Gln Gin Trp Val Leu Thr Ala Ala His Cys Met Met Asn Asp Tyr Asn Val His Leu Gly Ser Asp Arg Leu Asp Asp Arg Lys Gly Gln Lys Ile Arg Ala Met Arg Ser Phe Arg His Pro Gly Tyr Ser Thr Gln Thr His Val Asn Asp Leu Met Leu Val Lys Leu Ser Arg Pro Ala Arg Leu Ser Ala Ser Val Lys Lys Val Asn Leu Pro Ser Arg Cys Glu Pro Pro Gly Thr Thr Cys Thr Val Ser Gly Trp Gly Thr Thr Thr Ser Pro Asp Val Thr Phe Pro Ala Asp Leu Met Cys Thr Asp Val Lys Leu Ile Ser Ser Gln Asp Cys Lys Lys Val Tyr Lys Asp Leu Leu Gly Ser Ser Met Leu Cys Ala Gly Ile Pro Asn Ser Lys Thr Asn Ala Cys Asn Gly Asp Ser Gly Gly Pro Leu Val Cys Lys Gly Thr Leu Gln Gly Leu Val Ser Trp Gly Thr Phe Pro Cys Gly Gln Pro Asn Asp Pro Gly Val Tyr Thr Gln Val Cys Lys Tyr Ile Asp Trp Ile Asn (2) INFORMATION FOR SEQ ID NO: 48:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 253 (B) TYPE: amino acid (C) STRANDEDNESS: unknown (D) TOPOLOGY: unknown (vi) ORIGINAL SOURCE:
(A) ORGANISM: Artificial Sequence (ix) FEATURE:
(D) OTHER INFORMATION: Deducecd amino acid sequence from the C-terminal part of SCCE from homo.

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 48:

Met Ala Arg Ser Leu Leu Leu Pro Leu Gln Ile Leu Leu Leu Ser Leu Ala Leu Glu Thr Ala Gly Glu Glu Ala Gln Gly Asp Lys Ile Ile Asp Gly Ala Pro Cys Ala Arg Gly Ser His Pro Trp Gln Val Ala Leu Leu Ser Gly Asn Gln Leu His Cys Gly Gly Val Leu Val Asn Glu Arg Trp Val Leu Thr Ala Ala His Cys Lys Met Asn Glu Tyr Thr Val His Leu Gly Ser Asp Thr Leu Gly Asp Arg Arg Ala Gln Arg Ile Lys Ala Ser Lys Ser Phe Arg His Pro Gly Tyr Ser Thr Gln Thr His Val Asn Asp Leu Met Leu Val Lys Leu Asn Ser Gln Ala Arg Leu Ser Ser Met Val Lys Lys Val Arg Leu Pro Ser Arg Cys Glu Pro Pro Gly Thr Thr Cys Thr Val Ser Gly Trp Gly Thr Thr Thr Ser Pro Asp Val Thr Phe Pro Ser Asp Leu Met Cys Val Asp Val Lys Leu lie Ser Pro Gln Asp Cys Thr Lys Val Tyr Lys Asp Leu Leu Glu Asn Ser Met Leu Cys Ala Gly Ile Pro Asp Ser Lys Lys Asn Ala Cys Asn Gly Asp Ser Gly Gly Pro Leu Val Cys Arg Gly Thr Leu Gln Gly Leu Val Ser Trp Gly Thr Phe Pro Cys Gly Gln Pro Asn Asp Pro Gly Val Tyr Thr Gln Val Cys Lys Phe Thr Lys Trp Ile Asn Asp Thr Met Lys Lys His Arg (2) INFORMATION FOR SEQ ID NO: 49:

(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 226 (B) TYPE: amino acid (C) STRANDEDNESS: unknown (D) TOPOLOGY: unknown (vi) ORIGINAL SOURCE:
(A) ORGANISM: Artificial Sequence (ix) FEATURE:
(D) OTHER INFORMATION: Deducecd amino acid sequence from the C-terminal part of SCCE from rat.

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 49:

Met Gly Val Trp Leu Leu Ser Leu Leu Thr Val Leu Leu Ser Leu Ala Leu Glu Thr Ala Gly Gin Gly Glu Arg Ile Ile Asp Gly Tyr Lys Cys Lys Glu Gly Ser His Pro Trp Gln Val Ala Leu Leu Lys Gly Asp Gln Leu His Cys Gly Gly Val Leu Val Gly Glu Ser Trp Val Leu Thr Ala Ala His Cys Lys Met Gly Gln Tyr Thr Val His Leu Gly Ser Asp Lys Ile Glu Asp Gln Ser Ala Gln Arg Ile Lys Ala Ser Arg Ser Phe Arg His Pro Gly Tyr Ser Thr Arg Thr His Val Asn Asp Ile Met Leu Val Lys Met Asp Lys Pro Val Lys Met Ser Asp Lys Val Gln Lys Val Lys Leu Pro Asp His Cys Glu Pro Pro Gly Thr Leu Cys Thr Val Ser Gly Trp Gly Thr Thr Thr Ser Pro Asp Val Thr Phe Pro Ser Asp Leu Met Cys Ser Asp Val Lys Leu Ile Ser Ser Gln Glu Cys Lys Lys Val Tyr Lys Asp Leu Leu Gly Lys Thr. Met Leu Cys Ala Gly Ile Pro Asp Ser Lys Thr Asn Thr Cys Asn Gly Asp Ser Gly Gly Pro Leu Val Cys Asn Asp Thr Leu Gln Gly Leu Val Ser Trp Gly Thr Tyr Pro Cys Gly Gln Pro Asn (2) INFORMATION FOR SEQ ID NO: 50:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 249 (B) TYPE: amino acid (C) STRANDEDNESS: unknown (D) TOPOLOGY: unknown (vi) ORIGINAL SOURCE:
(A) ORGANISM: Artificial Sequence (ix) FEATURE:
(D) OTHER INFORMATION: Deducecd amino acid sequence from the C-terminal part of SCCE from mouse.

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 50:

Met Gly Val Trp Leu Leu Ser Leu Ile Thr Val Leu Leu Ser Leu Ala Leu Glu Thr Ala Gly Gln Gly Glu Arg Ile Ile Asp Gly Ile Lys Cys Lys Glu Gly Ser His Pro Trp Gln Val Ala Leu Leu Lys Gly Asn Gln Leu His Cys Gly Gly Val Leu Val Asp Lys Tyr Trp Val Leu Thr Ala Ala His Cys Lys Met Gly Gln Tyr Gln Val Gln Leu Gly Ser Asp Lys Ile Gly Asp Gln Ser Ala Gln Lys Ile Lys Ala Thr Lys Ser Phe Arg His Pro Gly Tyr Ser Thr Lys Thr His Val Asn Asp Ile Met Leu Val Arg Leu Asp Glu Pro Val Lys Met Ser Ser Lys Val Glu Ala Val Gln Leu Pro Glu His Cys Glu Pro Pro Gly Thr Ser Cys Thr Val Ser Gly Trp Gly Thr Thr Thr Ser Pro Asp Val Thr Phe Pro Ser Asp Leu Met Cys Ser Asp Val Lys Leu Ile Ser Ser Arg Glu Cys Lys Lys Val Tyr Lys Asp Leu Leu Gly Lys Thr Met Leu Cys Ala Gly Ile Pro Asp Ser 6 ;9 Lys Thr Asn Thr Cys Asn Gly Asp Ser Gly Gly Pro Leu Val Cys Asn Asp Thr Leu Gln Gly Leu Ala Ser Arg Gly Thr Tyr Pro Cys Gly Gln Pro Asn Asp Pro Gly Val Tyr Thr Gln Val Cys Lys Tyr Lys Arg Trp Val Met Glu Thr Met Lys Thr His Arg

Claims (21)

1. A transgenic mammal cell, having integrated within its genome a nucleotide sequence (SCCE-construct) comprising a heterologous nucleotide sequence of SEQ ID NO:1 coding for a human stratum corneum chymotryptic enzyme (SCCE), operably linked to a SV40 early promoter that drives expression of said heterologous nucleotide sequence in skin, wherein a transgenic mammal comprising the transgenic mammal cell exhibits epidermal hyperplasia and hyperkeratosis and a mild cellular inflammatory reaction of the skin.
2. A transgenic mammal cell according to claim 1 wherein said operably linked SV40 early promoter drives expression of scce in epidermis.
3. A transgenic mammal cell according to claim 1, wherein the nucleotide sequence codes for the human SCCE corresponding to amino acid no. 23 through no. 253 of the amino acid sequence shown in SEQ ID NO. 2.
4. A transgenic mammal cell according to claim 1, wherein the nucleotide sequence codes for the human SCCE corresponding to amino acid no. 30 through no. 253 of the amino acid sequence shown in SEQ ID NO. 2.
5. A transgenic mammal cell according to claim 1, wherein the nucleotide sequence codes for the human SCCE shown in SEQ ID NO. 2.
6. A transgenic mammal cell according to any one of claims 1 to 5, wherein the mammal is a mouse, rat or rabbit.
7. A transgenic mammal cell according to claim 6, wherein the mammal is a mouse.
8. A method for making a transgenic mammal comprising the transgenic mammal cell according to any one of claims 1 to 7, the method comprising (a) inserting said heterologous sequence downstream from the SV40 early promoter, (b) constructing and amplifying said heterologous nucleotide sequence, (c) introducing said heterologous nucleotide sequence into a cell from a mammal, where said cell is selected from the group consisting of a mammal ovum, a mammal embryonic cell, and a mammal embryonic stem cell, (d) using said cell or the progeny of said cell to create a number of putative transgenic mammals, and (e) selecting said mammal having said heterologous nucleotide sequence integrated within its genome.
9. A method for making a transgenic mammal according to claim 8 wherein said operably linked promoter drives expression of scce in epidermis.
10. A method according to claim 8 comprising introducing the SCCE-construct into an ovum or embryo of the mammal.
11. A method according to claim 8 comprising microinjecting the SCCE-construct into embryonal stem cells of the mammal.
12. A method according to claim 8, wherein the mammal is a mouse and the SCCE-construct is microinjected into C57BU6JxCBA-f2 mouse ova or embryos.
13. A method according to claim 8, wherein the mammal is a mouse and the SCCE-construct is introduced into C57BL/6JxCBA-f2 mouse ova or embryos and breeding the resulting mice with C57BL/6JxCBA or with C57BL/6J to obtain transgenic progeny and stable mouse lines.
14. A method of identifying a compound or composition effective for the prevention or treatment of epidermal hyperkeratosis, acanthosis, epidermal inflammation, dermal inflammation and pruritus, the method comprising (a) administering a compound or composition to a transgenic mammal comprising the transgenic mammal cell according to any one of claims 1 to 7, (b) evaluating the appearance of the skin and/or the behavior of a mammal treated according to step (a), and (c) comparing the appearance of the skin and/or the behavior of a treated mammal with an untreated control mammal, (d) identifying the compound or composition as being effective for the prevention or treatment of epidermal hyperkeratosis, acanthosis, epidermal inflammation, dermal inflammation and pruritus.
15. A method according to claim 14 of identifying a compound or composition effective for the prevention or treatment of epidermal hyperkeratosis.
16. The method of claim 8 in which said introduction is by electroporation, transfection, microinjection or viral infection.
17. The method of claim 8 in which said introduction is by microinjection.
18. The method of claim 17 in which the microinjection is into a mouse ovum.
19. Use of a transgenic mammal comprising the transgenic mammal cell according to any one of claims 1 to 7, as a model for the study of disease with the aim of improving treatment, relieve or ameliorate a pathogenic skin condition, for development or testing of a cosmetic or a pharmaceutical formulation or for the development of a diagnostic method.
20. The use according to claim 19 of the transgenic mammal as a model for a skin disease.
21. The use according to claim 19 of the transgenic mammal as a model for skin cancer.
CA2332655A 2001-02-09 2001-02-09 Scce modified transgenic mammals and their use as models of human diseases Expired - Fee Related CA2332655C (en)

Priority Applications (9)

Application Number Priority Date Filing Date Title
CA2332655A CA2332655C (en) 2001-02-09 2001-02-09 Scce modified transgenic mammals and their use as models of human diseases
PT02720389T PT1367886E (en) 2001-02-09 2002-02-08 Scce transgenic mice and their use as models of human disease
AT02720389T ATE515940T1 (en) 2001-02-09 2002-02-08 SCCE TRANSGENIC MICE AND THEIR USE AS A MODEL FOR HUMAN DISEASES
AU2002251429A AU2002251429B2 (en) 2001-02-09 2002-02-08 SCCE modified transgenic mammals and their use as models of human disease
EP02720389A EP1367886B1 (en) 2001-02-09 2002-02-08 Scce transgenic mice and their use as models of human disease
JP2002562148A JP4262479B2 (en) 2001-02-09 2002-02-08 SCCE modified transgenic mammals and their use as models of human disease
DK02720389.2T DK1367886T3 (en) 2001-02-09 2002-02-08 SCCE transgenic mice and their use as models for human diseases
PCT/IB2002/001300 WO2002062135A2 (en) 2001-02-09 2002-02-08 Scce modified transgenic mammals and their use as models of human disease
CY20111100962T CY1111935T1 (en) 2001-02-09 2011-10-07 SCCE DIAGNOSTIC MICE AND USE THEMSELVES IN HUMAN DISEASE

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