Note: Descriptions are shown in the official language in which they were submitted.
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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
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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.
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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.
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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
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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.
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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
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AGAAAAATAAATATTTGCTTATAAATTAATAAATGAAGCCCTCAAAAATGTTCTAT
TAGATAATGTTAAGTACAGACATTTTTGTTATAAATACATAATATACAAAGAAATC
TATGTATAACATGATTAAAATGACCATAAGAACATAGATCCTAAACATGGCAAAT
ATTAGTGG GGTGGGGTTAGGGAAAG CGTTGTTTTfAACTTACACCTCTCTGTTA
GAGTTGGGAATGGGTTCAGGCGTAATTACAGGCACGACTGGGATCAGCTTGGA
CAAGTTCCCCCAGGCGGGCCAGAATTAGGATGTAGGGTCTAGGCCACCCCTGA
GAGGGGGTGAGGGCAAGAAAATGGCCCCAGAAGCCGGGCGCAGTGGCTCACG
CCTGTAATCCCAGCACTTTGCGGGGCCGAGGCGGGCACATCATGAGGTCAGGA
GATCGAGACCATTCTGGCCAACATAGTGAAACCCGGTCTCTACTAAAAATACAA
AAATTATCTGGGAGTGGTGGTGCGTGCCTGTAATCCCAGGTACTCGGGAGGCT
GAGGCAGGAGAATCACTTGAACCTGGGAGGCGGAGCTGGCAGTGAGCCGAGA
TCGCGCCACCGCACTCCAGCCTGGCGATAGAGAGAGACTCCATCCAAAAAAAA
GAAAGGAAGGGAGGGAGGGAGGAGGGAAGAAAGAAAGAAAACCGCCCCAGAG
AAGGACCCGAGCCAGAGCCTATTCTCTGAGCTCAGCGACTGC1TGAATCCCGC
TCCTGCCCCTCAGACCCAGCGCACCGGGTCCCTCCCCCGAGAGCAGCCAGGA
GGGACTGTGGGACCAGAATGTGCGGGGGCGCAGGAGCTGGGCACCGCCCGT
CCTTCGGAGGGAGGGTGGAGAGAGAGTGCAGTGGTGCCAATTGCTCTCGCTG
CGTCAGGGTTCCAGATAACCAGAACCGCAAATGCAGGCGGGGGTGTCCCAGAG
TCGGCTCCGCCTGCACCCCAGGGCGCTGGGGCCGGGCATGGGGCGGGGGGT
GATATAAGAGGACGGCCCAGCAGAGGGCTGAAGATTTTGGAGCCCAGCTGTGT
GCCAGCCCAAGTCGGAACTTGGATCACATCAGATCCTCTCGAGGTGAGAAGAG
GCTTCATCAAGGGTGCACCTGTAGGGGAGGGGGTGATGCTGGCTCCAAGCCTG
ACTCTGCTCTCGAGAGGTAGGGGCTGCAGCCTAGACTCCCGGTCCTGAGCAGT
GAGGGCCTGGAAGTCTGCAATTTG GGGCCTTTTAGGGAAAAACGAACTACAGA
GTCAGAAGTTTGGGTTCCACAGGGAAGGGCAAGATCGGAGCCTAGATTCCTGG
GTCTCTAGG GATCTGAAGAACAGGAATTTTGGGTCTGAGGGAGGAGGGGCTGG
GGTTCTGGACTCCTGGGTCTGAGGGAGGAGGGCCTGGGGGCCTGGACTCCTG
GGTCTGAGGGAGGAGGGGCTGGGGGTCTCGACTCCTGGGTCTGAGGGAGGAG
GGGCTGGGGGCCTGGACTCCTGGGTCTGAGGGAGGAGGGGCTGGGACCTGG
ACTCCTAGGTCTGAGGGAGGAGGAGCTGGGGCCTGGACTCCTGGGTCTGAGG
GAGGAGGGGCTGGGGCCTGGACTCCTGGGTCTGAGGGAGGATGGGCTGAGG
CCTAGACTCCTGGGTCTGAGGGAGGAGGGGCTGGGGCCTGGACTCCTGGGTC
TGAGGGAGGAGGGGCTGGAGCCTGGACTCCTGGGCCTGAGGGAGGAGGGAC
TGAGACCTGGACTCCTAGGTCTGAGGGAGGAGGGACTGGGACCTGGACTCCT
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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
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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
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TTACAAGGACTTACTGGAAAATTCCATGCTGTGCGCTGGCATCCCCGACTCCAA
GAAAAACGCCTGCAATGTGAGACCCTCCCCCCCAATTCCTCCCCAGTCCTGGG
TACCCTGTCTGCATGCCCCAGGGACAGAGCTTGACCCAAGTGACTGGGTACCA
AGCCCGGCCTTGCCCTCCCCCCAGGCCTGGCCTCCTCAGCTTTTTCCACCTCA
5 TTCTCTGCCTAGGTCAGGGGTGGGAGTTTACTTAGGGGCCGATGTGGCCCTGG
GGATGGGACAGAGAGTTTAATAGGGGTGAGAAAGTGGGGGTGGGACCAGGGA
AGGAGACTGAGGTGCTGGCCTCAGGCCCAAACCCTAAGGGGGCACCAAAAACC
TCAGTGATTGAGATAAATCATAATGCAATATTTAAAAATAAAAATAAAAACTCATG
CAGAAGTCCATGATGGACAAAATGTCACATTTTAAATAAAGAGCAGGTGGATCTT
10 ACTGAATTTTCCCTTGCCGTAAGTACTAGCGTGGCTCAGCACAGCGCTGTACTG
GCACTGTCTTCATTTAAAATGTGGATACCATGCCCATCATGCAGTTTTATGTATT
ACATTTGATTTCGTTAAGTACTGCATTGAAGTATTGTGTATTGCAGTTACTGAGAT
TTTGTGCCTGAAGCTGATGACTCACTCACCTGACCCTGGCCCTGGTCCCGGGG
AAAACACTCTTTCTCTCCACCTCCTCTCTGTTCCCTCTTTCTGGCCTTTTGTCATC
CCCTCTGTTTCTGAACAGTCTTCCCACATCTCTCTTTGTGACATAATTTCATTTCA
TTCTTTTCCTCTTTGTTTTTTCTCTGTGTTGAGCTAGCTTGCTCTCCCTCCCTTGT
TCTCTCTCCATGCCCTCCTCTCTGCTCTCTGTCTTCTCCCTCTTTCTCTTGCTTCT
CTCTCTCTCCTCCCCTCCCTCTCTCCTCTCCCTGCCCCCCTGCTCTCTCTTTTIT
CCTCTCTCTCTGTCTCCTCTCTGGCCCTCTCCTCTTTCTCTCTCTCCCCCACTTC
TCTGTCTCTCTTCATCTCTCTCCCTCATCTCTCCTTGCCCCCTCCTTTTTACTGTC
TCTCTCTTTCTCTTTCTTCTATCTCTCTCCTCTCCCCGCCGCTCCCCCATCTCTG
TCTTTCTTTCTCTCTCTTTATTCTCCTCCTCTCTTCCAGTCTCTCTCTCCTCTCCC
CACCCCCACCCCATCTCTCTCCCCACACCTTCCCCCCCTTTCTCTTTGTCTCTCT
CTTCTACCTCTTTCTTCTCCACCCCCATCTCTCTCTCTCTTCTCTTCCCACACCCT
CCCCATCTCCCTCATCTCTTTGTCTGTCTCTCTTCTCCCTCCTTCTTTTCCACCC
CCATCTCTCTGTCTCTCTCTCTCCCCATACCCTTTCCCTCTTCCTCATCTCTCTTT
GTCTCTCTCTCCTTTCCCTCTTTCTTCTCCACCTCCAACTCTCTCTGTCTCTCCA
CACCCATCCTCCTTGCTCACATCTGCACCTTCAGCTGTCAGGGGATGTGGGATG
GTGAGTGTTAGGGATAGAGGAGATGGGAGAGAGATGACTGTCCTAGAGAATAG
GGTGTTCCCCTTCTCCCCTGGTGAGGGCCAGTTTCATGAATGTGCAAGCTCTGC
ACGGACACAGAGCCCCACACTCAGAAGGGTCTCAAACTTAGTCTAATGCATTCC
TGCTGTTGTCTTGAAATTCTCAATAATTTTTGAACAAAGGGCCCTGCATTTTCGTT
TTGCACCAAGTCCTGTAAATTATGTAACTGGTCTTCACCCTGGTCTCCGAGACC
ATCGTGTCCCCCTTTCCTGCGCCACAGGGCACGCATCCACCCCTTGGAGATGA
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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
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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
CA 02332655 2002-03-08
13
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
CA 02332655 2002-03-08
14
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
CA 02332655 2002-03-08
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
CA 02332655 2010-03-31
16
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.
CA 02332655 2010-03-31
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
CA 02332655 2002-03-08
17
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
CA 02332655 2002-03-08
18
antibodies, contra-staining with hematoxylin. A and C: #1010 transgenic mice,
5
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.
CA 02332655 2002-03-08
19
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.
CA 02332655 2002-03-08
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).
5
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.
CA 02332655 2002-03-08
21
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):
X3-
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.
CA 02332655 2002-03-08
22
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
CA 02332655 2002-03-08
23
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
CA 02332655 2002-03-08
24
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
CA 02332655 2002-03-08
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.
5
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
CA 02332655 2008-10-22
26
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.
CA 02332655 2008-10-22
27
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
CA 02332655 2008-10-22
28
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
CA 02332655 2002-03-08
29
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.
CA 02332655 2008-10-22
EXAMPLE 2
Generation and gross phenotypic characterization of of scce transgenic mice
with the
hscce gene under control of the SV40e promoter
5
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
CA 02332655 2002-03-08
31
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
CA 02332655 2002-03-08
32
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
CA 02332655 2002-03-08
33
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
CA 02332655 2002-03-08
34
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.
CA 02332655 2002-03-08
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.
EXAMPLE 4
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.
CA 02332655 2002-03-08
36
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
CA 02332655 2002-03-08
37
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.
CA 02332655 2002-03-08
38
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
CA 02332655 2002-03-08
39
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.
EXAMPLE 6
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.
CA 02332655 2008-10-22
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)
CA 02332655 2008-10-22
41
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
CA 02332655 2002-03-08
42
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,
56-63
(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).
CA 02332655 2002-03-08
43
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.
CA 02332655 2002-10-31
44
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
1 5
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
15 20 25
cag ggt gac aag att att gat ggc gcc cca tgt gca aga ggc tcc cac 147
CA 02332655 2002-10-31
Gln Gly Asp Lys Ile Ile Asp Gly Ala Pro Cys Ala. Arg Gly Ser His
30 35 40
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
45 50 55
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
60 65 70
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
90 95 100 105
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
125 130 135
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
140 145 150
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
155 160 165
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
170 175 180 185
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
190 195 200
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
205 210 215
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
220 225 230
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
235 240 245
aaa aag cat cgc taa cgccacactg agttaattaa ctgtgtgctt ccaacagaaa 826
Lys Lys His Arg
250
atgcacagga gtgaggacgc cgatgaccta tgaagtcaaa tttgacttta cctttcctca 886
aagatatatt taaacctcat gccctgttga taaaccaatc aaattggtaa agacctaaaa 946
CA 02332655 2002-10-31
4E
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
1 5 10 15
Ala Leu Glu Thr Ala Gly Glu Glu Ala Gin Gly Asp Lys Ile Ile Asp
20 25 30
Gly Ala Pro Cys Ala Arg Gly Ser His Pro Trp Gln Val Ala Leu Leu
35 40 45
Ser Gly Asn Gln Leu His Cys Gly Gly Val Leu Val Asn Glu Arg Trp
50 55 60
Val Leu Thr Ala Ala His Cys Lys Met Asn Glu Tyr Thr Val His Leu
65 70 75 80
Gly Ser Asp Thr Leu Gly Asp Arg Arg Ala Gln Arg Ile Lys Ala Ser
85 90 95
Lys Ser Phe Arg His Pro Gly Tyr Ser Thr Gln Thr His Val Asn Asp
100 105 110
Leu Met Leu Val Lys Leu Asn Ser Gln Ala Arg Leu Ser Ser Met Val
115 120 125
Lys Lys Val Arg Leu Pro Ser Arg Cys Glu Pro Pro Gly Thr Thr Cys
130 135 140
Thr Val Ser Gly Trp Gly Thr Thr Thr Ser Pro Asp Val Thr Phe Pro
145 150 155 160
Ser Asp Leu Met Cys Val Asp Val Lys Leu Ile Ser Pro Gln Asp Cys
165 170 175
Thr Lys Val Tyr Lys Asp Leu Leu Glu Asn Ser Met Leu Cys Ala Gly
180 185 190
Ile Pro Asp Ser Lys Lys Asn Ala Cys Asn Gly Asp Ser Gly Gly Pro
195 200 205
Leu Val Cys Arg Gly Thr Leu Gln Gly Leu Val Ser Trp Gly Thr Phe
210 215 220
Pro Cys Gly Gln Pro Asn Asp Pro Gly Val Tyr Thr Gln Val Cys Lys
225 230 235 240
Phe Thr Lys Trp Ile Asn Asp Thr Met Lys Lys His Arg
245 250
(2) INFORMATION FOR SEQ ID NO: 3:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 9729
(B) TYPE: nucleic acid
(C) STRANDEDNESS: unknown
(D) TOPOLOGY: unknown
CA 02332655 2002-10-31
47
(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
CA 02332655 2002-10-31
48
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
CA 02332655 2002-10-31
49
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
CA 02332655 2002-10-31
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
1 5 10 15
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
1 5 10 15
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
1 5 10 15
CA 02332655 2002-10-31
51
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
1 5 10 15
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
1 5 10 15
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
1 5 10 15
Pro Cys Pro Arg Gly Ser Gln Pro Trp Gln Val Ala Leu Leu Lys Gly
CA 02332655 2002-10-31
52
20 25 30
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
1 5 10 15
Cys Pro Gly Gly Ser Arg Pro Trp Gln Val Ala Leu Leu Lys Gly Asn
20 25 30
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
1 5 10 15
Gly Ser His Pro Trp Gln Val Ala Leu Leu Ser Gly Asn Gln Leu His
20 25 30
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
CA 02332655 2002-10-31
53
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 12:
Gln Gly Glu Arg Ile Ile Asp Gly Tyr Lys Cys Lys Glu Gly Ser His
1 5 10 15
Pro Trp Gln Val Ala Leu Leu Lys Gly Asp Gln Leu His Cys Gly
20 25 30
(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
1 5 10 15
Pro Trp Gln Val Ala Leu Leu Lys Gly Asn Gln Leu His Cys Gly
20 25 30
(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
1 5
(2) INFORMATION FOR SEQ ID NO: 15:
CA 02332655 2002-10-31
54
(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
1 5
(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
CA 02332655 2002-10-31
(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:
CA 02332655 2002-10-31
56
(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
CA 02332655 2002-10-31
57
(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
CA 02332655 2002-10-31
58
(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
CA 02332655 2002-10-31
59
(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
CA 02332655 2002-10-31
(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
CA 02332655 2002-10-31
61
(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
CA 02332655 2002-10-31
62
(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
CA 02332655 2002-10-31
63
(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
CA 02332655 2002-10-31
64
(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
1 5 10 15
Val Ala Gln Glu Asp Gln Gly Asn Lys Ser Gly Glu Lys Ile Ile Asp
20 25 30
Gly Val Pro Cys Pro Arg Gly Ser Gln Pro Trp Gln Val Ala Leu Leu
35 40 45
CA 02332655 2002-10-31
Lys Gly Ser Gln Leu His Cys Gly Gly Val Leu Leu Asn Glu Gln Trp
50 55 60
Val Leu Thr Ala Ala His Cys Met Asn Glu Tyr Asn Val His Met Gly
65 70 75 80
Ser Val Arg Leu Val Gly Gly Gln Lys Ile Lys Ala Thr Arg Ser Phe
85 90 95
Arg His Pro Gly Tyr Ser Thr Gln Thr His Ala Asn Asp Leu Met Leu
100 105 110
Val Lys Leu Asn Gly Arg Ala Lys Leu Ser Ser Ser Val Lys Lys Val
115 120 125
Asn Leu Pro Ser His Cys Asp Pro Pro Gly Thr Met Cys Thr Val Ser
130 135 140
Gly Trp Gly Thr Thr Thr Ser Pro Asp Val Thr Phe Pro Gly Gln Leu
145 150 155 160
Met Cys Thr Asp Val Lys Leu Ile Ser Pro Gin Asp Cys Arg Lys Val
165 170 175
Tyr Lys Asp Leu Leu Gly Asp Ser Met Leu Cys Ala Gly Ile Pro Asn
180 185 190
Ser Arg Thr Asn Ala Cys Asn Gly Asp Ser Gly Gly Pro Leu Met Cys
195 200 205
Lys Giy Thr Leu Gln Gly Val Val Ser Trp Gly Ser Phe Pro Cys Gly
210 215 220
Gln Pro Asn Asp Pro Gly Val Tyr Thr Gln Val Cys Lys Tyr Val Asn
225 230 235 240
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
1 5 10 15
Ala Leu Gly Ser Ala Ala Gln Glu Giy Gln Asp Lys Ser Gly Glu Lys
20 25 30
Ile Ile Asp Gly Val Pro Cys Pro Gly Gly Ser Arg Pro Trp Gln Val
35 40 45
Ala Leu Leu Lys Gly Asn Gln Leu His Cys Gly Gly Val Leu Val Asn
50 55 60
Gln Gin Trp Val Leu Thr Ala Ala His Cys Met Met Asn Asp Tyr Asn
65 70 75 80
CA 02332655 2002-10-31
66
Val His Leu Gly Ser Asp Arg Leu Asp Asp Arg Lys Gly Gln Lys Ile
85 90 95
Arg Ala Met Arg Ser Phe Arg His Pro Gly Tyr Ser Thr Gln Thr His
100 105 110
Val Asn Asp Leu Met Leu Val Lys Leu Ser Arg Pro Ala Arg Leu Ser
115 120 125
Ala Ser Val Lys Lys Val Asn Leu Pro Ser Arg Cys Glu Pro Pro Gly
130 13S 140
Thr Thr Cys Thr Val Ser Gly Trp Gly Thr Thr Thr Ser Pro Asp Val
145 150 155 160
Thr Phe Pro Ala Asp Leu Met Cys Thr Asp Val Lys Leu Ile Ser Ser
165 170 175
Gln Asp Cys Lys Lys Val Tyr Lys Asp Leu Leu Gly Ser Ser Met Leu
180 185 190
Cys Ala Gly Ile Pro Asn Ser Lys Thr Asn Ala Cys Asn Gly Asp Ser
195 200 205
Gly Gly Pro Leu Val Cys Lys Gly Thr Leu Gln Gly Leu Val Ser Trp
210 215 220
Gly Thr Phe Pro Cys Gly Gln Pro Asn Asp Pro Gly Val Tyr Thr Gln
225 230 235 240
Val Cys Lys Tyr Ile Asp Trp Ile Asn
245
(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
1 5 10 15
Ala Leu Glu Thr Ala Gly Glu Glu Ala Gln Gly Asp Lys Ile Ile Asp
20 25 30
Gly Ala Pro Cys Ala Arg Gly Ser His Pro Trp Gln Val Ala Leu Leu
35 40 45
Ser Gly Asn Gln Leu His Cys Gly Gly Val Leu Val Asn Glu Arg Trp
50 55 60
Val Leu Thr Ala Ala His Cys Lys Met Asn Glu Tyr Thr Val His Leu
65 70 75 80
Gly Ser Asp Thr Leu Gly Asp Arg Arg Ala Gln Arg Ile Lys Ala Ser
85 90 95
Lys Ser Phe Arg His Pro Gly Tyr Ser Thr Gln Thr His Val Asn Asp
100 105 110
CA 02332655 2002-10-31
67
Leu Met Leu Val Lys Leu Asn Ser Gln Ala Arg Leu Ser Ser Met Val
115 120 125
Lys Lys Val Arg Leu Pro Ser Arg Cys Glu Pro Pro Gly Thr Thr Cys
130 135 140
Thr Val Ser Gly Trp Gly Thr Thr Thr Ser Pro Asp Val Thr Phe Pro
145 150 155 160
Ser Asp Leu Met Cys Val Asp Val Lys Leu lie Ser Pro Gln Asp Cys
165 170 175
Thr Lys Val Tyr Lys Asp Leu Leu Glu Asn Ser Met Leu Cys Ala Gly
180 185 190
Ile Pro Asp Ser Lys Lys Asn Ala Cys Asn Gly Asp Ser Gly Gly Pro
195 200 205
Leu Val Cys Arg Gly Thr Leu Gln Gly Leu Val Ser Trp Gly Thr Phe
210 215 220
Pro Cys Gly Gln Pro Asn Asp Pro Gly Val Tyr Thr Gln Val Cys Lys
225 230 235 240
Phe Thr Lys Trp Ile Asn Asp Thr Met Lys Lys His Arg
245 250
(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
1 5 10 15
Leu Glu Thr Ala Gly Gin Gly Glu Arg Ile Ile Asp Gly Tyr Lys Cys
20 25 30
Lys Glu Gly Ser His Pro Trp Gln Val Ala Leu Leu Lys Gly Asp Gln
35 40 45
Leu His Cys Gly Gly Val Leu Val Gly Glu Ser Trp Val Leu Thr Ala
50 55 60
Ala His Cys Lys Met Gly Gln Tyr Thr Val His Leu Gly Ser Asp Lys
65 70 75 80
Ile Glu Asp Gln Ser Ala Gln Arg Ile Lys Ala Ser Arg Ser Phe Arg
85 90 95
His Pro Gly Tyr Ser Thr Arg Thr His Val Asn Asp Ile Met Leu Val
100 105 110
Lys Met Asp Lys Pro Val Lys Met Ser Asp Lys Val Gln Lys Val Lys
115 120 125
Leu Pro Asp His Cys Glu Pro Pro Gly Thr Leu Cys Thr Val Ser Gly
130 135 140
CA 02332655 2002-10-31
68
Trp Gly Thr Thr Thr Ser Pro Asp Val Thr Phe Pro Ser Asp Leu Met
145 150 155 160
Cys Ser Asp Val Lys Leu Ile Ser Ser Gln Glu Cys Lys Lys Val Tyr
165 170 175
Lys Asp Leu Leu Gly Lys Thr. Met Leu Cys Ala Gly Ile Pro Asp Ser
180 185 190
Lys Thr Asn Thr Cys Asn Gly Asp Ser Gly Gly Pro Leu Val Cys Asn
195 200 205
Asp Thr Leu Gln Gly Leu Val Ser Trp Gly Thr Tyr Pro Cys Gly Gln
210 215 220
Pro Asn
225
(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
1 5 10 15
Leu Glu Thr Ala Gly Gln Gly Glu Arg Ile Ile Asp Gly Ile Lys Cys
20 25 30
Lys Glu Gly Ser His Pro Trp Gln Val Ala Leu Leu Lys Gly Asn Gln
35 40 45
Leu His Cys Gly Gly Val Leu Val Asp Lys Tyr Trp Val Leu Thr Ala
50 55 60
Ala His Cys Lys Met Gly Gln Tyr Gln Val Gln Leu Gly Ser Asp Lys
65 70 75 80
Ile Gly Asp Gln Ser Ala Gln Lys Ile Lys Ala Thr Lys Ser Phe Arg
85 90 95
His Pro Gly Tyr Ser Thr Lys Thr His Val Asn Asp Ile Met Leu Val
100 105 110
Arg Leu Asp Glu Pro Val Lys Met Ser Ser Lys Val Glu Ala Val Gln
115 120 125
Leu Pro Glu His Cys Glu Pro Pro Gly Thr Ser Cys Thr Val Ser Gly
130 135 140
Trp Gly Thr Thr Thr Ser Pro Asp Val Thr Phe Pro Ser Asp Leu Met
145 150 155 160
Cys Ser Asp Val Lys Leu Ile Ser Ser Arg Glu Cys Lys Lys Val Tyr
165 170 175
Lys Asp Leu Leu Gly Lys Thr Met Leu Cys Ala Gly Ile Pro Asp Ser
180 185 190
CA 02332655 2002-10-31
6 ;9
Lys Thr Asn Thr Cys Asn Gly Asp Ser Gly Gly Pro Leu Val Cys Asn
195 200 205
Asp Thr Leu Gln Gly Leu Ala Ser Arg Gly Thr Tyr Pro Cys Gly Gln
210 215 220
Pro Asn Asp Pro Gly Val Tyr Thr Gln Val Cys Lys Tyr Lys Arg Trp
225 230 235 240
Val Met Glu Thr Met Lys Thr His Arg
245
