CZ20002201A3 - Transgenic animal model for cartilage degenerative disease - Google Patents

Abstract

The present invention provides animal model systems for cartilage-degenerative disease, which comprise transgenic animals which can express recombinant matrix-degrading enzymes (MDEs), particularly matrix metalloproteinases (MMPs), in a temporally and spatially regulated manner. The invention also provides methods for producing phenotypic indicators of cartilage-degenerative disease in a mammal and methods for determining the potential of a composition to counteract cartilage-degenerative disease. The invention also provides isolated nucleic acids encoding proMMP polypeptides that exhibit constitutive enzymatic activity and isolated proMMP polypeptides.

Description

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The invention provides animal model systems for cartilage degenerative diseases that include transgenic animals that can express recombinant, matrix-degrading enzymes (MDEs), particularly matrix metalloproteinases (MMPs), in a temporary and spatially regulated manner. It also provides methods for inducing phenotypic indicators of cartilage degenerative diseases in animals and methods for determining the ability of agents to counteract cartilage degenerative diseases. It also provides isolated nucleic acids encoding proMMP polypeptides that exhibit constitutive enzymatic activity and isolated enzymatic proMMP peptides.

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Τίττη

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Transgenic animal model for cartilage degenerative disease.

Technical field

The present invention relates to transgenic mammals that temporarily and locally express recombinant matrix degrading enzymes. The invention further relates to model systems comprising such transgenic animals for the study of degenerative joint diseases, including systems for identifying therapeutic agents and treatment regimens.

BACKGROUND OF THE INVENTION

Cartilage degenerative diseases, including joint and plate diseases such as osteoarthritis, rheumatoid arthritis and osteochondrodysplasia, are widespread, especially in advanced age. Early symptoms common to these diseases include the progressive loss of proteoglycans in the joint (manifesting the loss of metachromasia); collagen degradation; fibrillation on the cartilage surface; and finally the loss of cartilage (which can radiologically act as a narrowing of the joint space).

One of the first targets that is affected by this disease is type II collagen, a major structural collagen found in articular cartilage. There is a balance between the production of type II collagen and catabolic enzymes that degrade type II collagen during normal cartilage and bone conversion. Disruption of this balance may result in pathological conditions such as degenerative joint diseases.

Enzymes that degrade components of the extracellular matrix include matrix metalloproteinases (MMPs), a family of enzymes requiring zinc for their activity, and aggrecanase (Table 1).

Table 1

enzyme degrading matrix substrates collagen fO C H P (Ό «- < 0> > N C O 1Z > 1 1 — i t? O Φ P O AT at. fibronectin laminin and ί 1 C P what O : other Γ. Metalloproteinases Collagenases MMP-1 (intestinal collagenases) I. T1, III, VII, X MMP-P (neutrophil lineagease) I, II, 111 MMP-1.3 (Coiagenase 3) I, II, III Piglets inase MMP-3 (atinase A) TV, in, VII, IX MMP-? (zelatinase P ‘ τ y y X in St romeo l.y ·: i ny MMP-3 (xtromeolysin-1) activates .MMP zymogens MMP-7 (mat r i 1 ys) i v ^M '' I '''* ^c: ' V · · ·; IV, v, 1X activates MM P zymogens ΜΪ-JF ·· — 11 ith: raeo 'yo i r. O! r y activate serpins Other M P -1Ϊ í me t 3 ί. o o i a s t d · MM ' pr? MMř-2 proMMP-13 MMP-I3 MMP-1-3 proMMP-2 MMP-1. 7 I. Ag rec. -ase

MMPs are synthesized in the joints by chondrocytes, which (in mature articular cartilage) are completely differentiated cells maintaining a specifically cartilaginous matrix phenotype. Overproduction of MMPs (due to endogenous MMP inhibitors) that occurs in joint diseases can lead to cartilage degradation. For example, type II collagen is a substrate for MMP-13 and MMP-1 (Knuper et al., J. Biol. Chem. 271: 1544, 1996) and both MMP-1 and MMP-13 proteins can be detected immunohistochemically in human osteoarthritic meshes. In some cases, MMP-13 and its cleavage products can be found in greater amounts than MMP-1 (Billinghurst et al.,

J. Clin. Inves. 99: 1534,1997). Thus, MMP-13 may play an important role in cartilage degradation associated with osteoarthritis and others. 4 4 4 4 · 4 · 4 · 4 · 4 4 · · · 4

4444 degenerative joint diseases (Mitchell et al., J. Clin. Inves. 97: 761, 1996).

Animal models for osteoarthritis-related syndromes have been described in guinea pigs (Watson et al., Arth. Rheum. 39: 1327, 1996) and in the STR / ORT of an inbred mouse strain (Das-Gupta et al., Int. J. Exp. Path.). 74: 627, 1993). In guinea pigs, spontaneous osteoarthritis develops for a long time (six months or more), and only some sublimions of SRT / ORT mice always develop degenerative joint disease. Thus, the length and / or variability of these models makes them less suitable for new drug development studies.

Other models of osteoarthritis include surgically induced joint destabilization, such as anterior cruciate ligament and / or partial menisectomy in rabbits and dogs that stimulate cartilage degradation (Hulth et al., Acta Orthop. Scand. 41: 522, 1970). Another model utilizes the injection of bacterial collagenase into the joint of an animal, thereby inducing biochemical ligament breakage (Van der Kraan et al., J. Exp. Pathol. 71:19, 1990). However, since (i) surgery or other surgery is necessary for each animal (ii) the animals are large and expensive and / or (iii) the course of the disease is not always the same, these models cannot easily be used for large studies, including drug development studies.

In principle, transgenic animal models can provide a reproducible animal model for degenerative joint disease. Previous attempts to create transgenic animals expressing MMPs, such as MMP-1 and stromeolysin, but did not lead to an observable phenotype of joint degeneration in these transgenic animals. This could be due to the lethality of embryos caused by the constitutive expression of these enzymes. Witty et al., Mol. Cell 6: 1287, 1995 produced transgenic animals that constitutively form MMP-1 and stromelysin in the mammary gland tissue, but these animals do not show symptoms of osteoarthritis. D'Armento et al., Cell 71: 955, 1992 introduced transgenic mice expressing human interstitial collagenase in the lung. Liu et al., J. Cell Biol. 130: 227, 1995 introduced transgenic animals overexpressing mutated type II collagen, resulting in connective tissue disorders but not osteoarthritis. None of these transgenic animal systems provide a useful animal model

for osteoarthritis (Khokha et al., Cancer and Metastasis Rev. 14:97, 1995; Shapiro, Natrix Biol. 15: 527, 1997).

Thus, there is a need in the art for an animal model system that mimics human degenerative joint disease, such as osteoarthritis, rheumatoid arthritis, and chondrodysplasia. Transgenic animals containing controllable heterologous genes whose expression would cause cartilage degeneration would be particularly useful as a reproducibly experimental control over time with the possibility to influence the expression of the transgene and thus to control the pathological syndrome itself. Such animals can be used to determine the level of transgene expression necessary to induce the disease and (and importantly) can be used to find new drugs and optimize treatment regimens. Of particular importance is that such transgenic animals can be used to further determine the role of matrix degrading enzymes in cartilage degradation and to (in in vivo testing) screen for compounds that alter these enzymes or compounds that inhibit the development of degenerative joint diseases.

Summary of the invention

The present invention provides transgenic non-human animals or their progeny whose somatic and germline cells contain (in a stably integrated form) one or more heterologous or recombinant genes encoding polypeptides forming enzymatically active matrix-degrading enzymes (MDE) ) especially MMP. MMPs used in the invention include MMP-1, MMP-2, MMP-3, MMP-7, MMP-8, MMP-9, MMP-10, MMP-11, MMP-12, MMP-13, MMP-14, MMP-15, MMP-16 and MMP-17, preferably one or more of MMP-1, MMP-3, MMP-8, and MMP-13, and more preferably either MMP-1 or MMP-13, or both. Also included are enzymatically active variants, fragments, and combinations of these polypeptides. Other enzyme degradation matrices, including aggrecanase, can also be used. MDEs can be derived from any species, preferably human.

In a preferred case, the recombinant MDE coding genes are selectively expressed in articular chondrocytes of transgenic animals. · ··

and expression results in pathological symptoms characteristic of joint degenerative diseases.

The invention provides transgenic animals or progeny thereof, wherein the somatic and germ cells comprise a stably integrated first recombinant gene encoding MDE or an enzymatically active derivative thereof, preferably a constitutively active variant of MMP-13 (designated MMP-13 *), comprising the sequence described in SEQ ID NO. : 4. Suitably, the first recombinant gene is under the control of the first regulatable promoter, and optimally the first regulatable promoter comprises a tet07 sequence, such as the promoter described in SEQ ID NO: 5. The transgenic animal may further comprise a second recombinant gene encoding a polypeptide regulating the first regulatable promoter, and is preferably a tTA polypeptide. In these cases, the second recombinant gene is under the control of a second controllable promoter, in particular one that contains sequences derived from a joint-specific promoter, preferably for collagen type

II. Such is, for example, the promoter designated in SEQ ID NO: 6. Thus, selective expression of the second recombinant gene in joint tissues results in regulated specific expression of recombinant MDE in the joint.

In another aspect, the invention provides isolated nucleic acids encoding enzymatically active variants of MMPs, particularly variants of human proMMP-13 and most preferably MMP-13 ®. The invention also encompasses recombinant cloning vectors comprising these nucleic acids; cells containing vectors; methods for producing MMP-13-derived polypeptides comprising growing the cells under conditions suitable for MMP-13 expression; and isolated polypeptides derived from MMP-13.

In another aspect, the invention provides methods for inducing phenotypic changes characteristic of degenerative cartilage disease in mammals, i.e., exposing the transgenic animals of the invention to conditions that result in the expression of MDE encoded by the transgenes. Preferably, the transgenic animals (comprising the first recombinant gene encoding MMP-13 * operably linked to the tet07 promoter and the second recombinant gene encoding tTAprotein operably linked to the type II colleague promoter) are maintained in the presence of a tetracycline or tetracycline analog. To induce MMP-13 * expression, the tetracycline or tetracycline analog is removed and MMP-13 * is selectively expressed in the joint tissue; the result is «· · · · · · ·

phenotypic changes characteristic of cartilage degenerative disease.

In another aspect, the invention provides methods for determining the possible effect of a composition against anti-degenerative cartilage diseases. These methods are based on administering known doses of the compositions to the transgenic animals of the invention either before or after the development of phenotypic traits typical of cartilage degenerative disease; monitoring the indicators for a predetermined time after administration of the mixture; and comparing the range of endpoints in the animals treated with the composition compared to the control transgenic animals not treated with the composition. Any difference in (i) the nature or extent of the phenotypic traits of cartilage degenerative diseases, (ii) the time required for manifestation of indicators, or (iii) the need for further improvement measures indicate the possibility that the composition counteracts cartilage degenerative diseases.

Brief description of the pictures

Figures 1A and 1B. (A) Schematic illustration of the human structure

MMP-13 (collagenase 3). The first window (corresponding to the extreme amino terminus) represents the pre-domain (signal peptide) leading to secretion of proMMP-13. The second window represents for the domain that participates in maintaining the latent state of the enzyme. See the conserved sequence (SEQ ID NO: 1) inside for domains that is important for maintaining latency of the enzyme. The third window represents a 170 amino acid catalytic domain that contains a conserved region (shown; SEQ ID NO: 2) important for catalytic activity. The fourth window represents the carboxy-terminus domain of 200 amino acids. (B) Scheme of the nucleic acid sequence (SEQ ID NO: 3) encoding a constitutively active variant of human proMMP-13, designated MMP-13 *, and the amino acid sequence of MMP-13 * (SEQ ID NO: 4). The residues that are mutated to the non-mutated type MMP-13 (shown by larger characters) are GTC nucleotides at position 299-301.

Figures 2Ά and 2B. The binary strategy used to obtain chondrocyte-specific, doxycycline (doxycycline, DOX) regulated expression of MMP-13 *. (A) The first diagram shows the rat promoter of type II collagen regulating the expression of the tetracycline repressor - VP16 - · · · · · · · · · · · · · · · · · · · · · · 99 activator fusion protein (TA) followed by splice site and polyadenylation signal from SV40 (this construct is called CPE - TA). (B) The second scheme shows the Tet07 promoter (Gossen and Bujard, Proc. Natl. Acad. Sci. USA, 89: 5547, 1992) controlling the expression of a constitutively active human MMP 13 protein, followed by a splicing and polyadenylation signal from SV40 (this construct is designated Tet07-MMP13 *). These two independent transgenic constructs were simultaneously introduced by microinjection into fertilized mouse embryos, resulting in a double transgene carrying both genes. In the presence of DOX, transgene expression is turned off; when DOX is removed, transgene expression is turned on. The arrow indicates the transcription start site, the asterisk indicates a constitutively active mutation in the MMP13 transgene.

Figures 3A and 3B. The type II collagen gene promoter targets expression to the joints of a test transgenic mouse expressing beta-galactosidase under the control of the rat type II collagen promoter. (A) Scheme of CPE-lacZ construct. The rat type II collagen promoter (first box) controls expression of the β-galactosidase (LacZ) gene (second box), followed by a β-globin splicing region and a polyadenylation signal (the last boxes joined by a splicing symbol). (B) Total staining for beta-galactosidase activity of E16 embryos. The transgenic embryo on the left shows color (arrows) as compared to the non-transgenic embryo. (C) Greater detail of elbow and forepaw in transgenic embryo; β-galactosidase staining (arrows) is seen. Figures 3B and 3C illustrate the invention more clearly when illustrated in color, but a black and white embodiment is sufficient to understand the invention.

Figures 4A and 4B. Expression profile of TA and MMP13 RNA performed by RT-PCR. (A) Amplification of TA cDNA from total RNA. (B)

Amplification of MMP13 * cDNA from total RNA. Lane 1: 0X164 / Hae III molecular weight markers; lane 2: PCR amplification of transgenic (lane 6) genomic DNA; lane 3: PCR amplification of non-transgenic genomic DNA; lane 4: non-transgenic (DOX) wild type mouse; lane 5: non-transgenic mouse without DOX; lanes 6-7: transgenic mouse (about 4 months) with DOX; lanes 8-9: transgenic mouse (about 4 months) from birth without DOX. Arrows 1a and 1b show a 648 bp MMP13 * specific fragment and a 859 bp specific MMP13 * fragment. For each reaction, c-fos primers were used as internal control; the truncated mRNA was 187 bp (arrow 3) and the uncut mRNA was 303 bp (arrow 2). Similar fragments were not detectable in the corresponding RNA-containing lanes for PCR where no reverse transcription was performed (data not shown).

Figures 5A and 5B. Photographs illustrating the immunohistochemical localization of collagen II type cleavage products in the growth plate and articular cartilage of the transgenic mouse expressing the transgene of Figure 2. The mesh was stained with an antibody specific for collagen II type cleavage products. (A) Mesh obtained from doxycycline-treated mice to prevent MMP-13 * expression. (B) Mesh obtained from doxycycline-free mice (allowing MMP-13 * expression) for 30 days at 3 months of age. Figures 5A and 5B illustrate the invention more clearly when colored, but a black and white embodiment is sufficient to understand the invention.

Figures 6A, 6B and 6C. A color photograph documenting staining of the articular cartilage and patella growth plate in a transgenic mouse with two Safranin O transgenes. (A) Mesh derived from doxycycline-treated mice. (B) Mouse-derived mesh 7 days after doxycycline was stopped. (C) Mice derived from the mouse 14 days after doxycycline was stopped. Figures 6A-C illustrate the invention more clearly when colored, but a black and white embodiment is sufficient to understand the invention.

Figures 7A, 7B, 7C and 7D. Longitudinal cross-section through the rear knee joints (A and B) and synovium (C and D). (A and C) Age matched litter control and (B and D) line 6 without DOX. Abbreviations: L, lesions; AG, articular cartilage; AG, angiogenesis; IH, infiltration hyperplasia; BM, bone marrow; and PL, patella ligament. Localization of the femur and patella are indicated in the figure. Figures 7A-D illustrate the invention more clearly when colored, but a black and white embodiment is sufficient to understand the invention.

Detailed description of the invention.

The present invention is based on the discovery that the regulated expression of matrix-degrading enzymes in the cartilage of transgenic mice induces characteristic phenotypic changes associated with matrix degenerative joint and intervertebral disc diseases.

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Animal models of the invention provide novel model systems for disease-degenerating matrix syndromes that can be used to characterize in detail the pathologies of human joints and intervertebral discs, as well as to research new drugs and optimize treatment regimens.

A transgenic animal of the invention is an animal with cells that contain a transgene that has been introduced into either the animal or the ancestors of the animal in a prenatal (germ) state. For example, a transgenic animal can be made by introducing a gene of interest into a male pronucleus of a fertilized oocyte, e.g., microinjection; then, the oocyte is allowed to develop in a pseudogravid female foster animal. A given gene may contain appropriate promoter sequences, intron sequences, and polyadenylation signal sequences. Methods for preparing transgenic animals are disclosed, for example, in U.S. Pat. Nos. 4,736,866 and 4,870,009 and in Hogan et al., A Laboratory Manual, Cold Spring Harbor Laboratory, 1986. An animal found to be transgenic can be used to breed other animals carrying the transgene. A transgenic animal carrying one transgene can mate with another transgenic animal that carries another transgene to give rise to a "double transgenic animal that owns both transgenes. Alternatively, both transgenes can be introduced together by microinjection, thereby also forming a "double transgenic animal." There may also be animals with more than two transgenes. In addition, heterozygous transgenic animals can be mated, i.e. animals with only one copy of the transgene, with a second heterozygous animal with the same transgene to obtain homozygous animals with two copies of the transgene.

The present invention encompasses transgenic animals, preferably mammals, that express from a recombinant MDE gene, in particular MMPs and most preferably MMPs with collagenase activity. MDEs for use in the invention include MMP and aggrecanase without limitation. Useful MMPs include, but are not limited to, collagenases designated MMP-1, MMP-8, and MMP-13; stromelysins designated MMP-3, MMP-10 and MMP-11; gelatinases designated MMP-2 and MMP-9; MMP-12 labeled metalloelastases; and membrane MMPs designated MMP-14, MMP-15, MMP-16 and MMP-17 (Matrisian, BioEssays, 14: 455, 1992). As used herein, the term matrix degrading activity refers to the proteolytic degradation of matrix components, including, for example, collagen, particularly collagen type II, and especially the triple helical form of collagen

type II (triple helical form of type II collagen). Any polypeptide having an activity-degrading matrix can be used in the practice of the invention, including enzymatically active fragments of the aforementioned enzymes. Preferably, the enzymatic activity of MMP-13 is expressed. The enzymatic activity of MMP-13 used herein relates to the proteolytic degradation of type II collagen. Thus, any MMP-13 polypeptide or fragment or derivative thereof with MMP-13 enzymatic activity may be used. The enzymes may be derived from any animal species, including, for example, humans, mice, rats, rabbits, pigs, cows or non-human primates, or combinations thereof. Preferably, MMP-13 or a derivative thereof is of human origin.

Normally, MMPs are synthesized as precursors (i.e., zymogens or proenzymes) whose enzymatic activity is latent; proteolytic removal for secretion regions results in an enzymatically active protein. In a preferred embodiment of the invention, it is possible to circumvent the need for proteolytic processing using enzyme or proenzyme variants that are enzymatically active even in the non-cleaved state. Such variants can be produced by conventional local or random mutagenesis techniques associated with collagenase enzyme activity analysis (see below). In this way, modifications (including, for example, insertions, deletions, or substitutions) can be introduced into the proenzyme sequence, particularly at or near the pro-region cleavage site, thereby obtaining constitutively active polypeptides that do not require proteolytic processing for activation. Alternatively, the entire pro-region may be removed. In addition, recombinant genes can be used in which a sequence encoding a natural signal peptide is replaced by a heterologous sequence that functions as a signal peptide, i.e., elicits secretion. The use of genes encoding any such modified MMP polypeptides is contemplated by the invention.

It is preferred that constitutively active MMP-13 be used in the practice of the invention. It is particularly preferred that the MMP-13 variant comprises a sequence comprising a mutation in the sequence encoding the PRCGVPDV region, SEQ ID NO: 7, particularly the Pro 'substitution ^; ' ^:! to Val; the sequence of this polypeptide is shown in SEQ ID NO: 4 and this polypeptide is designated MMP-13 *. Alternatively, the constitutively active variant of MMP-13 comprises a Val ' ^iB substitution for Gly.

The transgenic animals of the invention preferably exhibit MMP activity in a controlled manner. Regulated expression means temporary and / or spatial control. Temporary control refers to the ability to suppress the expression of MMP activity by a predetermined time in the development of transgenic animals for which MMP expression can be activated and maintained at that level for as long as desired. It is good if MMP expression is suppressed during embryonic development and activated in adult animals. Spatial control means the ability to selectively express MMPs in certain tissues. It is preferred that the MMP activity is expressed in joint tissues and in particular in joint chondrocytes.

Temporary control of MMP expression is achieved by using one or more polypeptides comprising a transcriptional repressor, transcriptional activator, or transcription enhancer, or a combination thereof, in conjunction with a transcriptional repressor / activator responsive promoter operably linked to an MMP coding sequence. In certain instances, transient control of MMP expression is achieved by (i) expressing (in transgenic animals) a repressor polypeptide operably linked to a polypeptide that directly or indirectly activates transcription in eukaryotic cells to form a repressor-activator fusion polypeptide; and (ii) coupled using a target promoter operably linked to an MMP coding sequence whose transcriptional activity responds to a repressoractivator fusion polypeptide. Typically, the nucleotide sequences encoding the repressor polypeptide are ligated in reading frame to sequences encoding the transcriptional activator polypeptide to form a chimeric gene encoding the fusion protein.

Useful repressor polypeptides include all polypeptides comprising sequences derived from bacterial repressors, including, e.g., tetracycline repressors, LacR repressors, KRAB domains, and lambda repressors (cro and cl), and others, as well as eukaryotic repressors, including those involved in amino acid or sugar synthesis. . Useful polypeptide direct transcriptional activators include herpes simplex virus 16 protein (VP 16); yeast GAL 14; yeast STATE; steroid receptors such as the progesterone and estrogen receptor; and constitutive activators such as c-fos, c-jun and SP-1. Alternatively, the repressor polypeptide may be linked to a polypeptide that indirectly activates transcription by recruiting the transcriptional activator fcfc for interaction with the repressor fusion protein. -activator; such indirect activator proteins include the TATA Box Binding Protein (TBP) and basic transcription factors, including basic transcription factor D.

According to the invention, each repressoractivator fusion protein is used in conjunction with a target promoter that responds to a particular fusion protein and which regulates the transcription of the MDE coding sequence. A promoter typically comprises at least one operator sequence responsive to the repressor portion of a repressoractivator fusion polypeptide that is operably linked to at least one (to at least a minimal promoter) that affects transcription in eukaryotic cells. Examples of suitable repressor-sensitive operator sequences are those derived from the tetracycline resistance operon encoded by Tn10 in E. coli, the lamda repressor operon, and the yeast GAL repressor operon. Examples of suitable eukaryotic promoters from which minimal promoters can be derived are, for example, the cytomegalovirus (CMV) IE promoter, the PtK-1 promoter (thimidine kinase), the HSP (heat shock protein) promoter, and any eukaryotic promoter comprising a TATA box. Minimal promoter sequences can be derived from these promoters by (i) creating deletion mutants using conventional methods and (ii) testing the ability of the resulting sequence to activate transcription in a cell line. U.S. Pat. Patent No. No. 5,650,298 discloses a repressor-activator fusion protein comprising sequences derived from a tetracycline repressor fused to VP16 sequences, designated as tTA and a tTA-susceptible promoter, designated tet07, which comprises a Tn10-derived sequence linked to a portion of the CMV IE promoter.

Alternatively, the transient control may be achieved by (i) expressing a heterologous or recombinant polypeptide (or polypeptides) of a transcriptional activator in a transgenic animal, and (ii) associated using a target promoter operably linked to a MMP coding sequence whose transcriptional activity responds to a heterologous or recombinant transcriptional activator. Useful transcriptional activators include a modified ecdysone receptor in which the VP16 transcriptional domain (linked to the amino-terminal glucocorticoid receptor transactivating domain) is fused to the ligand-binding domain and the carboxy-12 ecdyzone receptor sequence.

* * Konce · konce ♦ «konce konce konce konce konce konce ((((((((((((((((No No No No No No No (No et al. USA 93: 3346,1996); a chimeric protein designated pGL-VP, comprising VP16 activator sequences, GAL4 activating sequences, and a mutated human progesterone receptor ligand binding domain (Wang et al., Proc. Natl. Acad. Sci. USA 91: 8180, 1994; Wang et al., Gene Therapy 4: 432

1997); and chimeric proteins containing transcriptional activators fused to an estrogen (or other steroid) binding domain (Mattioni et al., Meth Cell Biol. 43: 335, 1994). The ecdysone receptor system utilizes the retinoid X receptor (RXR) to form heterodimers with a chimeric receptor and responds to ecdozyn muristerone (an analogue of ecdozyne) or dexamethasone (dexamethasone). The pGL-VP system reacts to mifepristone (mifepristone) (RU486). Chimeric receptors containing the estrogen-binding domain respond to hydroxytamoxifen (an estrogen analog).

Spatial control of MDE expression is achieved using transcriptional promoters that target transcription selectively into the joint tissues. The term specific expression in joints is used herein to mean expression greater in joints than in other cells; typically, the expression level in the non-articular tissues is less than 10 ^; expression levels in the joints. Preferably, the expression is undetectable in the non-articular tissues. Useful promoter sequences that induce joint-specific expression of a sequence to which they are operably linked include sequences derived from the type II collagen promoter. It will be appreciated that the joint-specific promoter of the invention may comprise one or more copies of certain sequences or sub-sequences, and these sequences may be in a direct or opposite orientation relative to each other and relative to a sequence whose expression is controlled by the promoter.

Coordinated spatial and temporal control of MDE expression is preferably achieved by (i) placing the expression of a repressor-activator fusion polypeptide or transcriptional activator polypeptide under joint-specific promoter control (ii) placing the expression of MDE or an MDE derivative under control of a promoter corresponding to a repressor-activator fusion polypeptide; a transcriptional activator polypeptide; and (iii) such that transgenic animals are during fetal development and early in birth under conditions where expression of MDE is suppressed.

»« ·· ·· ·

The method by which transgenic animals are maintained during fetal development and soon after birth under conditions that do not express MDE depends on which particular transgenes are expressed. When a repressor-activator fusion polypeptide is used, repression is accomplished by administering to the animal a substance that binds to the repressor-activator fusion protein resulting in repression of the transcription of the target MDE gene. In animals containing a transgene encoding a repressor-activator fusion polypeptide comprising tet repressor sequences, repression is accomplished by administering tetracycline or a tetracycline analogue in food or drinking water to the mother and to the offspring after birth. Tetracycline or a tetracycline analog can also be administered by surgically implanting subcutaneously pellets that release these substances over time (Innovative Research of America, Inc., Sarasota FL). In this case, binding of the tetracycline or tetracycline analog to the repressor-activator fusion protein prevents binding of the fusion protein to the analog promoter and activation of transcription. Tetracycline analogues are compounds closely related to tetracycline, which bind to the tet repressor with a Ka of at least 10 ^'J M, or preferably with an affinity of ^10? M '* or greater. Effective tetracycline analogs include doxycycline (doxycycline), anhydrytrotetracycline (anhdryrotetracycline), chlortetracycline (chlortetracycline), epioxytetracycline (epioxytetracycline) and others. The doses employed are such as to substantially reduce MMP expression. Typically, the tetracycline or tetracycline analog is administered in drinking water for animals at a dose of about 1 mg / ml. When MMP is to be expressed, tetracycline or an analogue thereof is discontinued.

In other cases, repression is accomplished by not providing the animal with a substance that is necessary for the activity of the transcriptional activator polypeptide. For example, if the transcriptional activator is a modified ecdysone receptor, the animals are free of ecdysone or an ecdysone analog during fetal and early postnatal development. Ecdysone analogs are compounds closely related to ecdysone that bind to a modified ecdysone receptor with a Ka of at least 10''M ‘. Useful ecdysone analogs include, for example, muristerone A (muristerone A). When MDE expression is desired, animals are administered, e.g., ecdysone or muristerone A by intraperitoneal injection at doses between about 10 mg and about 20 mg / animal. Similarly, when pGL-VP is used, activation is achieved by administration of mefiprestone (mefiprestone).

In a preferred case, a transgenic animal is produced whose somatic and germ cells contain two recombinant genes in a stable integrated form: (i) the first recombinant gene comprises a sequence encoding MMP-13 *, wherein the sequence is operably linked to the tetO7 promoter; and (ii) the second recombinant gene encodes a tTA protein operably linked to a type II collagen promoter. In this case, the animals are maintained during fetal and early postnatal development in the presence of tetracycline or a tetracycline analog, thereby repressing the gene. Later, the tetracycline or tetracycline analog is removed and the joint tissues selectively express the enzymatic activity of MMP-13.

Animal models for cartilage degenerative diseases

The present invention provides animal model systems that reproducibly exhibit phenotypic changes characteristic of cartilage degenerative diseases, such as articular or plate diseases. These diseases include, but are not limited to, osteoarthritis, rheumatoid arthritis, chondrodysplasia, and degenerative diseases of the intervertebral discs. The model systems of the invention exhibit one or more phenotypic indicators common to these diseases, including, for example, loss of proteoglycan (detected, for example, by loss of Safranin 0 staining) and cleavage of type II collagen in the affected tissues. The systems include the transgenic animals described above, in which recombinant or heterologous MDE, particularly MMPs, are expressed in cartilage at a specified time during the life of the transgenic animals. The timing of the appearance of cartilage degeneration indicators is determined by the activation of MDE expression and by monitoring the effect on cartilage (see below). Preferably, one or more MDEs are expressed after birth, preferably when the animals have reached maturity.

Transgene expression is typically monitored by extracting mRNA from various tissues and by one of the following: (i) reverse transcription with polymerase chain reaction (RT-PCR) using primers homologous to the transgenes; (ii) RNase protection

..... · · '♦ ♦ · * - - - * · · · · · · protection protection protection protection protection protection protection protection protection protection protection protection protection protection protection protection protection protection protection protection protection protection protection protection protection protection protection) and (iii) Northern blot analysis. In situ hybridization can also be performed.

The physiological effects of MDE expression on articular cartilage are monitored in test animals after sacrifice, staining of paraffin decalcified cartilage (i) with hematoxylin and eosin (using conventional techniques), followed by double staining with (ii) Safranin 0 and fast green (Peter et al. (J. Exp. Pathol. 71:19, 1990). Alternatively, frozen sections can be stained with antibodies that are specific for type II collagen-derived cleavage fragments (Billinghurst et al., J. Clin. Invest. 99: 1534, 1997). Typically, at least seven days expression of the MMP transgene (s) induces a detectable proteoglycan loss and changes in growth plate morphology (see, eg, Example 5 below).

Within the scope of this invention there are animal models in which the expression of MDE, in particular MMP, and in particular of enzymatically active forms of MMP-13, results in the loss of proteoglycan and / or cleavage of type II collagen.

Other phenotypic indicators of cartilage degenerative diseases that can be monitored in the transgenic animals of the invention include, for example, an overall observation of changes in joint function and histological evidence of (i) fibrilation and articular cartilage loss and (ii) osteophyte formation.

The transgenic animals of the invention are a useful model for pathological condition syndromes that exhibit impaired cartilage composition, morphology, and / or function, including osteoarthritis; rheumatoid arthritis; intervertebral disc degenerative diseases; chondriodysplasia including, but not limited to, Kneist dysplasia, achondrogenesis, and hypophosphatasia; and proteoglycan-induced disorders such as occur in brachymorphic (brachymorphic) animals (Halí et al., Cartilage: Molecular Aspects, CRC Press, 1991, pp. 201-203).

The transgenic animals of the invention may undergo further interventions to influence the cartilage degeneration indicators and / or complement the phenotype of diseased animals with other physiological effects, such as those associated with a particular disease. For example, transgenic animals can be treated with inflammatory agents to increase collagen degradation and / or induce proteoglycan loss (see, eg, Example 6 below). In addition, the pro

The recurrence of symptoms of a particular disease or syndrome may use the timing and extent of MDE induction, with or without further intervention.

Methods for evaluation of substances that affect cartilage degenerative diseases.

The present invention encompasses methods for discovering and evaluating the efficacy of medicaments and treatments against degenerative cartilage diseases, particularly joint degenerative diseases. In one embodiment, transgenic animals are maintained under conditions where expression of one or more MDEs results in one or more phenotypic markers of cartilage degenerative disease. Once symptoms have occurred, substances can be evaluated to counteract degenerative cartilage disease. They are administered at known doses to animals that have experienced symptoms;

monitoring phenotypic indicators for a predetermined time after administration of test substances; and comparing the range of phenotypic indicators in the treated animals with the control animals. Control animals include transgenic animals of the same sex and age bred under the same conditions (ie, expressing transgenes) but not treated with the test substance. Any statistically significant variations in the extent or nature of the phenotypic indicators indicate the possible action of the test substance against degenerative cartilage disease. Phenotypic indicators of cartilage degenerative disease include proteoglycan loss, joint space narrowing, collagen degradation and cartilage degradation.

The ability of a substance to act against cartilage degenerative diseases, particularly joint degenerative diseases, is assessed by applying it to a transgenic animal at a known dose prior to and / or concurrently with inducing MDE expression in the transgenic animal; monitoring phenotypic indicators of cartilage degenerative disease for a specified period after drug administration and MDE induction; and comparing the range of phenotypic indicators and / or disease in the animal to which the test substance was administered and the control animal to which the test substance was not administered. Any statistically significant variation in the extent or nature of phenotypic phenotypes.

indicators and / or diseases indicate the possible action of the test substance against degenerative cartilage disease.

Another indication of the ability of a substance to counteract degenerative cartilage disease is its ability to cause any reduction in the extent or duration of other treatments, including, eg, the dose and time of administration of another therapeutic agent used to alleviate the symptoms of the disease.

Compounds that can be tested for their ability to counteract degenerative cartilage disease can be found, for example, among natural source sources, fermentation sources (including plants and microorganisms), combination sources, compound sources, synthetic compounds and compounds resulting from targeted design and synthesis. Sources of synthetic compounds are commercially available, for example, from Maybridge Chemical Co. (Trevillet, Cornwall, UK); Comgenex (Princeton, NJ); Brandon Associates (Merrimack, NH); and Microsource (New Milford, CT). Sparse chemical sources are available from Aldrich Chemical Company, Inc. (Milwaukee, WI). Sources of natural substances in the form of extracts from bacteria, fungi, plants and animals are available, for example, from Pan Laboratories (Bothell, WA) or MycoSearch (NC), or can be easily prepared. In addition, libraries of natural or synthetically prepared substances can be easily modified by conventional chemical, physical and biochemical methods (Blondelle et al., TibTech 14:60, 1996)

Transgenic animals

The term transgenic animals is used herein to refer to animals in which one or more heterologous and / or recombinant genes have been introduced. The transgenes may be from a different species, or from the same species as a transgenic animal, but do not naturally occur in the animal in the form and / or at the chromosomal locus as the transgene. Transgenes may contain foreign DNA sequences, i.e., sequences not normally found in the genome of the host animal. Instead of (or in addition to) these transgenes, they may contain endogenous DNA sequences that have been rearranged or mutated in vitro to affect normal in vivo gene expression, or to affect or eliminate the biological activity of an endogenous gene product encoded by the gene.

• ·

• · ······· · ··· · · · · ···

The invention also relates to DNA fragments which are introduced into a pre-existing gene in order, for example, to alter expression patterns or to provide means for regulating gene expression (Watson et al., "Introduction to Foreign Genes Into Mice." In Recombinant DNA, 2 ^nd Ed. , WH Freeman & Co., New York, 1992, pp. 255-272; Gordon, JW,

Intl. Roar. Cytol. 115: 171 (1989); Jaenisch, Science 240: 1468,1989; Rossant, Neuron 2: 323 (1990).

The transgenic, non-human animals of the invention are obtained by introducing transgenes into the germ cell of non-human animals. Target embryonic cells at different stages of development are used to introduce the transgene of the invention. Depending on the state of development of the target embryonic cell (s), different methods are used. Such methods include, for example, zygote microinjection, virus integration, embryonic stem cell transformation as described below, and other methods.

A preferred method for introducing a transgene into an animal genome is by microinjection of a zygote. As a target cell for microinjection of the transgenic DNA sequence, a zygote, which is a fertilized egg that has not undergone prononuclear fusion or subsequent cell division, is preferred. The mouse male pronucleus reaches a size of about 20 microns in diameter, a size sufficient to reproducibly inject 1-2 picolitre solutions with transgenic DNA. The use of a zygote for transgene delivery has the advantage that in most cases the transgenic DNA sequence will be incorporated into the genome of the host animal before the first cell division (Brinster et al., Proc. Natl. Acad. Sci. USA 82: 4438, 1985). As a result, all cells of the founder animals stably carry the incorporated transgene at a particular genetic locus, referred to as the transgenic allele. The transgenic allele shows Mendelian inheritance; half of the progeny from the cross between the transgenic animal and the non-transgenic animal will inherit the transgenic allele in accordance with Mendelian randomization rules.

2. Virus integration may also be used to introduce a transgene of the invention into an animal. Developing embryos are sampled in vitro to the blastocyte developmental stage. Blastomers can be infected with the corresponding retroviruses (Jaenich, Proc. Natl. Acad. Sci. USA 73: 1260). Blastomer infection can be increased by enzymatic removal of the pellucid zone. Transgenes are introduced by viral vectors that typically carry a replication defect but which remain capable of integrating a virus-linked DNA sequence (including a transgenic DNA sequence linked to such a viral sequence) into the host animal genome. Transfection is readily and efficiently achieved by culturing the blastomeres in the monolayer of cells producing the transgene containing the viral vector. Infection can also be achieved by using cells at a later developmental stage, for example blastocel. In any case, most of the founder animals will be obtained by integrating the mosaic virus for the transgenic allele; that is, the transgene is incorporated into only a portion of all cells of the founder transgenic animal. In addition, multiple integration of the virus into a single founder animal can occur, thereby creating more transgenic alleles that segregate in future generations of offspring. The introduction of the transgene into germ cells by this method is possible, but is likely to occur at a lower frequency. However, once the transgene is introduced into the germ cell by this method, it is possible to obtain progeny where the transgenic allele will be present in all animal cells, i.e. both somatic and germ cells.

3. Embryonic stem (ES) cells can also serve as target cells for introduction of the transgene of the invention into animals. ES cells are obtained from pre-implantation embryos that are cultured in vitro (Evans et al., Nature 292: 154, 1981). ES cells that have been transformed with a transgene can be combined with an animal blastocyte; The ES cells then populate the embryo and contribute to the formation of the germ cell of the resulting animal (which is a chimera, ie consisting of cells derived from two or more animals). Again, once the transgene is introduced into the germ cells by this method, we can obtain offspring in which the transgenic allele is present in all animal cells, i.e. both somatic and germ cells.

Although the initial introduction of the transgene is a Larkark (non-Mendelian) event, the transgenes of the invention can be stably integrated into the germ cells and transferred to the progeny of the transgenic animal as a Mendelian loci. Other transgenic techniques give rise to mosaic transgenic animals in which some cells carry transgenes and others do not. U • · · ·

The mosaic transgenic animals where the germ cells do not carry the transgene do not transfer the transgene to the offspring. However, mosaic transgenic animals can be used to demonstrate the phenotype associated with transgenes.

In the practice of the invention, the animals of the maintenance line are crossed with animals of a genetic origin where expression of the transgene induces symptoms of cartilage degenerative disease. Progeny with inherited transgenes of the invention are distinguished from other offspring in the litter who have not inherited the transgene by analyzing the genetic material from the progeny for the presence of nucleic acid sequences from the transgene of the invention. For example, biological fluids containing polypeptides exclusively encoded by transgenes of the invention can be analyzed by immunoassays for the presence of these polypeptides. A simpler and more reliable way of identifying progeny is to obtain a tissue sample from the "edge of the animal, such as the tail," and analyze the sample for the presence of nucleic acid sequences corresponding to a portion or portions of the DNA sequence of the transgene of the invention. The presence of such nucleic acid sequences can be performed, for example, by hybridization ("Southern") analysis with DNA sequences corresponding to certain portions of the transgene, analysis of the PCR reaction product using the DNA sequence from the sample as substrate, oligonucleotides derived from the DNA sequence of the transgene, and the like.

Nucleic acids, vectors, expression systems and polypeptides.

The present invention encompasses isolated nucleic acids encoding MDE, particularly MMPs and enzymatically active fragments derived therefrom, as well as constitutively active variants of MMPs and enzymatically active fragments derived therefrom. The invention also encompasses the above-mentioned nucleic acid additions; nucleic acid containing vectors; cells containing vectors; and isolated polypeptides encoded by nucleic acids.

Many techniques common in molecular biology, microbiology, recombinant DNA, and protein biochemistry are used in the practice of the present invention, such as those explained, for example, in Sambrook et al., 1989, Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spriong Harbor Laboratory Press. Cold Spring Harbor, New York; DNA Cloning: A Practical Approach, Volume and II

99 ·· ··

1985 (D. N. Glover ed.); Oligonucleotide Synthesis, 1984, (M. L. Gait ed.); Transcription and Translation, 1984 (Hames &amp; Higgins eds.);

A Practical Guide to Molecular Cloning; series, Methods in Enzymology (Academic Press, Inc); and Protein Purification: Principles and Practice, Second Edition (Springer-Verlag, N.Y.), etc.

As used herein, the term "nucleic acid or" polynucleotides refers to purines and pyrimidines comprising polymers of any length, either polyribonucleotides or polydeoxyribonucleotides or mixtures of polyribo-polydeoxyribo nucleotides. This includes single- and double-stranded molecules, e.g. DNA-DNA, DNA-RNA and RNA-RNA hybrids as well as protein nucleid acids (PNAs) produced by conjugation of bases to the amino acid backbone. Also included are nucleic acids containing modified bases.

A "coding sequence" or "protein coding sequence" is a polynucleotide sequence capable of transcription into mRNA and / or capable of translation into a polypeptide. The boundaries flanking the coding sequence are typically given by a translation start codon at the 5'-end and a translation stop codon at the 3'-end.

As used herein, the term "nucleic acid complications" refers to an antisense (antisense) sequence that participates in Watsnon-Crick pairing with the original sequence.

As used herein, the term "isolated nucleic acid or polypeptide" refers to components that are obtained from their normal site of occurrence (for example, their natural occurrence, if naturally occurring). The isolated nucleic acid or polypeptide typically comprises less than 50% (preferably less than 75%, and most preferably less than 90%) of the cellular components with which they were originally associated.

A nucleic or polynucleotide sequence that is "derived from a designated sequence" refers to a sequence that corresponds to a region of said sequence. For nucleic acid sequences, this includes sequences that are homologous or complementary to a given sequence, as well as "sequence conserved variants" and "functionally conserved variants." For polypeptide sequences, this refers to &quot; functionally conserved variants. Sequence conserved variants are those in which a change in one or more nucleotides in a given codon does not affect the amino acid encoded by that position. Functionally conserved variants are those in which a given amino acid residue in a polypeptide has been altered without affecting the overall conformation and function of the natural polypeptide. For example, the amino acid may be replaced with another having the same physicochemical properties (such as acidic, basic, hydrophobic and the like). Functionally conserved variants also include any polypeptides having the ability to elicit antibodies specific to the polypeptide.

Nucleic acids, including any of the sequences disclosed herein, or variants thereof, can be prepared by conventional methods. For example, DNA can be synthesized chemically, e.g., using a solid surface phosphoramidite method (Matteucci et al., 1981, J. Am. Chem. Socc 103: 3185) or the method described in Yoo et al., 1989, J. Biol. Chem. 764: 17708, or using another generally known method. This can also be accomplished by sequentially joining a series of oligonucleotide cassettes containing pairs of synthetic oligonucleotides.

Due to the degeneracy of the genetic code, a given amino acid sequence, or a portion thereof, can be encoded by many different nucleotides. Thus, codons can be selected for optimal expression in porocaryotic or eukaryotic systems. Such degenerate variants are also included in the invention.

Nucleic acids can also be modified by methods known to those skilled in the art. Such modifications may include, for example, methylation, capping, substitution of one or more naturally occurring nucleotides with their analogs, internucleotide modifications such as modifications with uncharged bonds (e.g., methyl phosphonates, phosphotriesters, phosphoramidates, carbamates, etc.) and charged bonds ( eg phosphorothioates (phosphorothioates), phosphorodithioates (phosphorodithioates, etc.). The nucleic acids may comprise one or more covalently attached moieties such as proteins (e.g. nucleases, toxins, antibodies, signal peptides, poly-L-lysine, etc.), intercalators (e.g. acridine, psoralen, etc.), chelators (e.g. metals, radioactive metals, iron, oxidizing metals, etc.) and alkylators. The term "nucleic acid" also includes PNA. The nucleic acid may be modified by the formation of a methyl or ethyl phosphotriester or an alkyl.

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4 by phosphoramidate bond. The nucleic acid sequences of the present invention may also be modified by a label capable of producing a detectable signal, either directly or indirectly. The labels may be, for example, radioisotopes, fluorescent molecules, biotin, and the like.

Polypeptides of the invention can be expressed using a variety of known vectors such as pUC plasmid, pET plasmids (Novagen, Inc, Madison, WI) or pRSET or pREP plasmids (Invitrogen, San Diego, CA) and many suitable host cells, using methods disclosed or cited herein, or known to those skilled in the art. The particular vector / host selection is not critical to the practice of the invention. Recombinant cloning vectors will often contain one or more replication systems for cloning or expression; one or more markers for selection in the host, for example antibiotic resistance; and one or more expression cassettes. The inserted coding sequences can be synthesized by standard methods, isolated from natural materials, prepared as hybrids, and the like. Ligation of coding sequences to transcriptional regulatory elements and / or other amino acid coding sequences can be performed by known methods. Suitable host cells can be transformed / transfected / infected by any suitable method including electroporation, CaCL-mediated absorption, fungal infection, microinjection, microprojectiles or other established methods.

Suitable host cells include bacteria, archebacteria, fungi, yeast, plant and animal cells (particularly mammalian cells). In particular, E. coli, S. aureus, B. subtilis, Saccharomyces cerevisiae, Saccharomyces carlsbergensis, Schizosaccharomyces pombi, SF9 cells, C129 cells, 293 cells, Neurospora, CHO, COS, HeLa cells and immortalized mammalian myeloid and lymphoid cell lines are used. Preferred replication systems include M13, ColE1, SV40, baculovirus, lambda, adenovirus, cytomegalovirus and others. A large number of regulatory regions initiating transcription and termination have been isolated that are effective in transcription and translation of heterologous proteins in various hosts. Examples of these fields, isolation methods, methods of manipulation, etc. are known (Ausubel et al., Current Protocols in Molecular Biology, John Willey, 1977). Under the conditions · · · · * ♦ · · * * *

Suitable for expression, the host cells can be used as a source of recombinantly obtained peptides and polypeptides.

The MDEs of the present invention, including functionally conserved variants, can be isolated from natural or heterologous organisms or cells (including, for example, bacteria, fungi, insects, plant or mammalian cells) into which protein coding sequences have been introduced and subsequently expressed. These polypeptides can also be produced in non-cellular protein synthesis systems, which can subsequently be enriched in microsomal membranes to achieve glycosylation and signal peptide processing of preprocollagenases. In addition, the polypeptides may be chemically synthesized by commercially available automated techniques, including, for example, exclusive solid phase synthesis, partial solid phase methods, fragment condensation, or classical synthesis with solutions.

Methods for purifying the polypeptide are well known to those skilled in the art and include, for example, prepathic disk gel electrophoresis, isoelectric focusing, HPLC, reverse phase HPLC, gel filtration, ion exchange and partition chromatography, and contercurrent distribution. For some purposes, it is preferable to produce the polypeptide in a recombinant system, where the protein comprises an additional tag, which facilitates purification. Such a sequence may be, for example, a polyhistidine sequence. The polypeptide may then be purified from coarse host cell lysates by chromatography on a suitable solid-phase matrix, or antibodies prepared against these peptides may be used as the purifying agent. Other purification methods are also useful.

The construction and analysis of variants of MMPs and derivatives that exhibit enzymatic activity and preferably constitutive enzymatic activity can be achieved by routine application of conventional methods. First, the nucleic acid encoding the MMP is modified by either local or random mutagenesis, or is used in the design as a single segment of the fusion gene. It is good that the process results in a modification either contained within the pro-region coding sequence or near the cleavage site for the region; it involves complete removal of pro-areas. Alternatively, fusion coding sequences may be prepared

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4449 9 44 99 44 444 proteins either between enzymatically active MMP domains and other polypeptides, or between different MMPs. The modified nucleic acid is then used to program the synthesis of the MMP variant, either in cell-free systems or in intact cells (including permeabilized cells), or in a transgenic animal. Preferably, either cell-free systems or cell systems are used to express MMP variants or derivatives. The extent of cleavage of the pro-region is assessed by metabolic labeling and distribution of MMP product by SDS-PAGE. Finally, the enzymatic activity of MMPs is measured using conventional assays, such as quantification of cleavage of natural substrates or model peptides, as described, for example, in Weingarten et al., Biochem. 24: 6730 (1985); Woessner et. Al, J. Biol. Chem., 263: 16918 and Knight et al., FEBS Letts., 296: 263, 1992. In this case, a large number of MMP variants and their derivatives, including, for example, functionally conserved MMP variants, can be routinely prepared and tested for MMP enzymatic activity. -13*.

Description of preferred forms

The following examples are intended as some demonstrations of the present invention.

Example 1: Construction of a gene encoding modified, constitutively active ProMMP-13

The following experiments were performed to create a gene encoding MMP-13-derived procollagenase, which is enzymatically active in the absence of pro-region cleavage. The sequence of this variant for MMP-13, designated MMP13 *, is shown in SEQ ID NO: 4.

Local mutagenesis to modify the MMP-13 cDNA

Cleavage of plasmid pNot3A (Freije et al., J. Biol. Chem.

269: 16766 (1994); GENBANK accession number X75308) enzymes XbaI a

HindIII and purification of the resulting fragment of about 1515 bp yielded a cDNA fragment encoding proMMP. This fragment was cloned into the Tet-resistant / Amp-sensitive pAlter plasmid (Promega,

Madison, WI), which was digested with XbaI and HindIII.

Local mutagenesis was performed using the Altered Sites II Kit in vitro Mutagenesis System (Promega, Madison, Wi). Briefly, phagemid single-stranded DNA was purified from cultures containing helper phage R408 (Promega). In addition to ampicillin - not tetracycline (Amp repair - Tet knock-out) conversion oligo (Promega), a phosphorylated oligonucleotide with sequence was added to single-stranded DNA

5'-AAGCCAAGATGCGGGGTTGTCGATGTGGGTGAATACAAT-3 ', SEQ ID NO: 8; then the mutated chain was synthesized. The reaction mixture was then used to transform E. coli strain ES1001 mutS (no repair) and the culture was grown in selective medium with ampicillin. DNA was transformed from the isolated clones to transform JM109 cells and further grown on ampicillin LB dishes (120 µg / ml).

The above procedure achieved the substitution of proline for valine at position 99. The modified MMP was designated as MMP13 * (SEQ ID NO: 4).

A similar local metagenesis technique was also used to replace valine with glycine at position 98. A nucleotide with the sequence GAAAAAGCCAAGATGCGGGGGGTCCTGATGTGGGTGAATAC-5 ', SEQ ID NO: 9 was used similarly as above. This procedure replaced valine with glycine at amino acid position 98.

After confirming the above mutations by direct sequencing, cDNA encoding MMP13 * was released from the pAlter-MMP13 * vector using ExoRI and HindIII.

Example 2: Determination of MMP-13 enzymatic activity

CDNA material and methods encoding both mutant forms of MMP-13 and unmutated MMP-13 were subcloned into the BS (SK-) vector (Stratagene) containing the CMV promoter (XhoI - EcoRI) and SV40 splice poly (A) n (XbaI - Ncol) ). Duplicate cultures of Hela cells (10 cm dishes) were transfected with 50 µg of these plasmids by CaPO 3 precipitation method (Promega). After 5 hours, cells were exposed to 1 minute glycerol shock; a solution containing the same amount of 2x HBS + 303 glycerol was used. This

the procedure is described in the Profection Mammalian technical manual

Transfection Systems (Promega).

24 hours after transfection, culture medium (D-MEM with 10% fetal bovine serum) was replaced with serum-free D-MEM with 10 µM CGS-27023A, an MMP inhibitor (Ciba-Geigy). It is believed that if no MMP inhibitor is added, the MMP produced by the culture is degraded; therefore, the addition of an MMP inhibitor to the culture medium results in a detectable MMP13 band.

48 hours after the addition of serum-free medium with the MMP inhibitor, 10 ml of the supernatant was collected and concentrated approximately 200-fold (Centriprep-30 and Centricon-10 concentrators, Amicon). Then an equal amount of 2x Tris-glycine SDS running buffer was added to each sample. The samples were then loaded onto a 4-16% stained beta casein zymogram SDS polyacrylamide gel (Novex). After electrophoresis, the gels were renatured for 30 minutes at room temperature in renaturation buffer (Novex) and incubated overnight at 37 ° C in zymogram development buffer (Novex).

Results

Normally, MMPs are synthesized as precursors (i.e., procollagenases or zymogens) whose enzymatic activity is hidden. Proteolytic removal of the proregion upon secretion produces the active protein. The need for proteolytic processing can be overcome by the use of a procollagenase variant, which is active even without cleavage. The constitutively active variant of MMP13 used as a transgene comprises proline-to-valine substitution in the sequence encoding the PRCGVPDV region (SEQ ID NO: 7). This region is highly conserved among MMPs and important for controlling enzyme activity.

The activity of the altered MMP13 protein was determined using a casein zymogram (data not shown). HeLa cells secrete three MMP species. One moves like the 92kDa MMP9 enzyme, also referred to as gelatinase B (gelatinase B). Cleavage of the MMP prodomain during enzyme activation results in a loss of about 10 kDa. The molecular weight of the second band corresponds to a modified form of gelatinase B. Both stromelysin-1 (MMP3) and stromelysin-2 (MMP10) have 54 kDa and one or both may correspond to the fourth band.

• 9

Pro-MMP13 should be about 60 kDa; is seen in lane 1 containing uncleaved recombinant MMP13. HeLa cells were transfected with constructs expressing both parental and mutant forms of MMP13. However, only the -60 kDa proMMP13 form was detected in cells expressing both constructs, suggesting that autoproteolysis of the proenzyme to the final form does not appear to occur in the exogenous system. These results indicate that the substitution of proline for valine does not affect the natural activity of MMP13 or its substrate specificity.

This method allows rapid screening of variants of MMP13 that retain MMP13 activity.

Example 3: Construction of transgenic vectors

Construction of MMP-13 * attached to tet07

In a next step, the cDNA encoding MMP-13 * was operably linked to the tet07 promoter-derived transcriptional regulatory sequence as follows:

The BS (SK-) vector (Stratagene) was digested with KpnI and NotI. A synthetic duplex oligonucleotide with the following sequence was digested with KpnI and NotI and ligated into the vector:

5 '-GGTACCACTAGTAAGCTTAGATCTCATATGGTCGACCCCGGGGAATATCTCTGCAGGGATCC TCTAGAAGTACTCCATGGGTATACATCGATGCGGCCGC-3', SEQ ID NO: 10.

The BS (SK-) vector modified by the above procedure was digested with XbaI and NcoI. A vector (745 bp) containing the SV40 splice site and the polyadenylation signal, which was obtained by digesting pcDNAI / Amp (Invitrogen, Carlsbad, CA) with XbaI and NcoI, was ligated into the vector.

The resulting vector was linearized by digestion with XhoI and EcoRI, and then a 460 bp XhoI-EcoRI fragment containing the tetO7 promoter region from pUHD 10-3 was ligated (Gossen et al., Proc. Natl. Acad. Sci. USA 89: 5547, 1992). This vector was subsequently digested with SpeI, the ends blunt-ended with Klenow polymerase and further digested with EcoRI. pAlter-MMP13 * was digested with HindIII, blunted with Klenow polymerase, and digested with EcoRI; to obtain a fragment encoding MMP-13 *. The MMP13 * EcoRI fragment was further cloned into an EcoRI digested vector (see above).

• ftftftft ftft ftft ftft ftft ftftftft ftftftftft ftft ftftftft ftftft ftftftftftft ftftftftftftftftftftftftftftftftftftftftftftftftftftftftftftftftftftftftftftftftftftftftftftftftftftftftftftftftftftftftftftftftftftftftftftftftftftftftftftftftftftftftftftftftftftftftftftftftftftftftftftftftftftftftftftftftftftft

The 2792 bp long transgene, SEQ ID NO: 11 (FIG. 2B), was released by XhoI and NotI and purified in a CsCl gradient prior to microinjection into fertilized eggs.

Construction of type II collagen promoter - tTa gene

The gene encoding the tTa repressor-activator fusion protein was operably linked to a joint-specific promoter (type II collagen).

To the modified BS (SK-) vector containing the SV40 junction site and polyadenylation signal described above in Example 1 and digested with NdeI and SmaI, an 1897 bp fragment containing the promoter and enhancer for collagen II was added. This fragment was obtained by digesting the plasmid PBSAH1 with HindIII, alignment with Klenow polymerase, and digesting with NdeI.

The plasmid was then digested with EcoRI and BamHI and ligated to the 1025 bp fragment encoding the tetracycline / VP16 repressor-activator fusion protein. This was released from EcoRI and BamHI from the plasmid pUHG15-1 (Gossen et al., Supra). The plasmid was linearized by digestion with BglII, dephosphorylated with calf intestinal phosphatase, and ligated to the 1554 BamHI enhancer fragment obtained from plasmid PBSAH1.

Finally, with the enzyme KpnI and NotI, a transgene, SEQ ID NO: 12 (Fig. 2A) was released from the 5276 bp vector, purified by gel, and dialyzed by means of CsCl centrifugation against the buffer used for microinjection (5 mM Tris-HCl pH 7.4). and 0.1 mM EDTA pH 8.0) and used for microinjection.

Gene promoter of type II collagen - β galactosidase

A reporter gene suitable for assessing expression caused by the type II collagen promoter in individual tissues was operably linked to the type II collagen promoter.

A 4179 bp BamHI-BglII fragment containing. The β-galactosidase gene fused to the β-globin linker sequence and polyadenylation signal was excised from the plasmid pUGH16-3 (Gossen et al., supra) and cloned into the BamHI site of unmodified BS (SK-) (Stratagene). This plasmid was digested with EcoRI and HindIII and ftftft ftft ftft ftft ftft ftft ftft ftft ftft ftft ftftftft ftft ftft ftft ftft ftft ftft ftft ftftft was ligated to a 655 bp HindIII-EcoRI fragment with the type II collagen promoter sequence cleaved from the plasmid described above. The plasmid was then digested with EcoRI and ligated to the 2807 bp EcoRI fragment which was excised from the collagen type II promoter described above. Restriction enzyme mapping was used to verify the orientation of each insert. The 7664 bp long transgene sequence (SEQ ID NO: 13, Fig. 3A) was released with HindIII and NotI enzymes, gel purified and CsCl centrifuged, dialyzed against the buffer used for microinjection (5 mM Tris-HCl pH 7.4 and 0). , 1 mM EDTA pH 8.0) and used for microinjection of mouse embryos.

Results

Giant. 2A and 2B show a schematic representation of synthetic genes designed to achieve regulated expression of MMP13 in chondryocytes. Inducible expression of the transgene was achieved using a tetracycline-regulatable expression system (Gossen et al., Supra; Furth et al., Proc. Natl. Acad. Sci. USA, 91: 93029306, 1994). In the first construct (FIG. 2A), the expression of the tetracycline driven VP16 transactivator fusion protein is under the control of the type II collagen promoter. This construct directs expression of the VP16 fusion protein into chondryocytes. In the second construct (FIG. 2B), cDNA expression encoding a modified version of the MMP13 protein (MMP13 *) is under the control of the VP16 fusion protein. In the presence of doxycycline, a tetracycline analog supplied in drinking water, the VP16 fusion protein does not bind to the Tet07 promoter of the synthetic MMP13 * gene and the gene is inactive. Upon removal of doxycycline, the transactivator stimulates transcription of human MMP13 * cDNA and production of the modified protein.

Both transgenic constructs, shown in Figures 2A and 2B, were tested in primary bovine chondryocytes and germline mouse fibroplasts in addition to validating MMP13 * activity prior to microinjection. The results showed the ability of the type II collagen promoter to induce expression of a second construct with either Tet07-luciferase or TetO7-MMP13 * (data not shown).

Figure 3A is a schematic illustration of the transgene used to assess the tissue-specific regulation induced by the collagen promoter.

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4444 4 44 44 44 Type II. The nucleic acid construct comprises: (i) a sequence derived from a type II collagen promoter; (ii) a sequence encoding bacterial β-galactosidase (Lac Z); and (iii) a sequence comprising a β-globin splice and polyadenylation splice signal.

Example 4: Production and characterization of transgenic mice expressing tetracycline-regulated LacZ or MMP-13 * in joint tissues.

The following experiments were performed to obtain transgenic mice expressing the MMP-13 * gene or the LacZ (β-galactosidase) reporter gene.

Material and methods:

Preparation and testing of transgenic mice. To generate MMP-13 * expressing mice under the control of tetracycline, an approximately 2784 bp XhoI-NotI tetO7-MMP13 * DNA fragment and a KpnI-NotI CPE-tTA DNA fragment were microinjected together in equimolar amounts in fertilized mouse embryos. 5265 bp (FIG. 2A). To generate mice expressing the reporter gene, a 7641 bp HindIII-NotI LacZ fragment was injected into (FVB / N) fertilized eggs (Fig. 3A), as described (Hogan et al., Manipulation the Mouse Embryo, Cold Spring Harbor Laboratories, 1966). . (The size of the restriction fragments is smaller than the transgenes described in Example 3 because the endonuclease cleavage sites are within the transgenes).

Founders animals were first identified by PCR as follows. The transgene encoding tTA was identified using primers corresponding to the tTA sequence (5'-CGAGGGCCTGCTCGATCTCC-3 ', SEQ ID NO: 14) and primers corresponding to the non-transcribed 3' end (5 '-GGCATTCCACCACTGCTCCC-3', SEQ IDNO: 15). The resulting PCR product was 584 bp in size. The transgene encoding MMP13 * was identified using primers corresponding to the sequences encoding MMP13 * (5'-GAGCACCCTTCTCATGACCTC-3 ', SEQ ID NO: 16) and the non-transcribed 3' end. The resulting PCR product was 731 bp in size.

The transgen encoding lacZ was identified using primers corresponding to the signal located in the core of the β-galactosidase gene (5'-GTTGGTGTAGATGGGCGCATCG-3 ', SEQ ID NO: 17) and (5'-GCGGGGTCTCAGGTTACAGCC-3', NO: 18). The resulting PCR product was 673 bp in size.

To verify the PCR results, Southern blot analysis of the lower end of the DNA digested with BamHI / NcoI or PvuII / NcoI and hybridized to the 3 'untranslated end under high stringency conditions was performed. The number of copies of the transgenic DNA that had been integrated into the genome was determined by comparing the relative intensity of the hybridization signal from the transgenic mice to the signal obtained using control DNA containing 10 and 100 genomic equivalents of similar DNA that was injected.

The copy number was determined using Taqman quantitative PCR according to the manufacturer's instructions (Perkin Elmer). Transgenic lines were obtained by mating founder animals with non-mutated FVB / N mice, and subsequent generations were identified by PCR.

All mice were given doxycycline (Sigma Chemical Co., St. Louis MO) prepared as a stock solution at 100 mg / ml in 501 ethanol and diluted to a final concentration of 1 mg / ml in acidic drinking water that changed daily (Schultze et al. al., Nature Biotech. 14: 499, 1996).

Staining of whole embryo for β-galactosidase (lacZ) activity. Non-mutated females mated with transgenic males with the CPE-lacZ construct. On the 16th embryonic day (E16), the females were sacrificed and the embryos were stained for β-galactosidase activity as described by Hogan et al., 1996 (supra). Prior to fixation, tails were removed from E16 embryos and analyzed for transgene transfer by PCR.

Expression analysis by RT-PCR. Transgenic expression was determined by RT-PCR. Total RNA was isolated after tissue homogenization in Trizol (Life Technologies). First chain synthesis was performed using the Superscript preamplification kit from Gibco / BRL. Briefly, 5 µg of total RNA was treated with Dnase I for 15 minutes at room temperature; followed by inactivation by adding 2 µl of 25 mM EDTA and heating to 65 ° C / 15 minutes. 0.5 µg of oligo dT was then added to the RNA and reverse transcription was performed according to the manufacturer's instructions. PCR analysis of cDNA was performed using the following primer sets (5'-3 'sequence): tet activator:

CGCCCAGAAGCTAGGTGTAGAG (SEQ ID NO: 19) and CGGCCATATCCAGAGCGCCG (SEQ ID NO: 20); MMP13 *: GCCCTCTGGCCTGCTGGCTCATG (SEQ ID NO: 21) and CAGGAGAGTCTTGCCTGTATCCTC (SEQ ID NO: 22). The resulting PCR products are 859 and 648 bp large. To verify the mRNA integrity and reverse transcription efficiency, each PCR reaction also contained a c-fos primer set: 5'-AGGAGGGAGCTGACAGATACACTCC-3 '(SEQ ID NO: 23) and 5'-AGGCCACAGACATCTCCTCTGG-3' (SEQ ID NO: 24). PCR analysis was performed with cDNA using Taq-gold polymerase (Perkin Elmer) - 10 minutes 95 ° C and then 35 cycles - 60 seconds 96 ° C, 90 seconds 67 ° C, 60 seconds 72 ° C. The final synthesis was performed at 72 ° C for 12 minutes. Reaction products were analyzed on a 2% agarose gel and visualized by ethidium bromide staining.

RT-PCR of germline fibroblasts. Tet07-lacZ females mated with transgenic males with both (CPE-tTA and TetO7-MMP13 *) transgenes. Females were sacrificed on day E15 and fibroblasts were prepared from embryos (Graham et al., Virology, 52: 456, 1973; Lopata et al., Nucl. Acids Res., 12: 5707, 1984). Fibroblasts were grown in DMME medium (Gibco / BRL) with 10 3 FBS (fetal bovine serum). Fibroblasts were further transfected with the desired calcium expression plasmid by the phosphate precipitation method. At 48 post transfection, total RNA was obtained by the Trizol method (Gibco / BRL). First strand cDNA synthesis was performed using the Superscript preamplification system (Gibco / BRL). MMP13 * expression was identified by PCR using primers specific for human MMP13 (GCCCTCTGGCCTGCTGGCTCATG) (SEQ ID NO: 21) and (CAGGAGAGTCTTGCCTGTATCCTC) (SEQ ID NO: 22). The resulting PCR product was 648 bp in size.

Immunohistochemistry. Expression of MMP13 * in double transgenic lines was further analyzed by immunohistochemistry using antibodies specific for MMP-13 derived collagen type II cleavage fragments. For this purpose, the joints were fixed for 60 minutes in 45 paraformaldehyde in PBS at neutral pH and room temperature. They were then washed twice in PBS and incubated overnight in 0.1M Tris-HCl, pH 7.4 and partially decalcified in 0.2M EDTA at neutral pH. Samples were transferred to TOC. media and Hacker / Bright cryostat were cut 6 mm

9 tt · t · • * • «t t t t t t t t t t t t t

Frozen slices. Sections were stained with antibodies specific to the epitope present in the degraded product of type II collagen, namely the TC ^A degradation product, which is also the designated 3/4 piece. Billinghurst et al., J. Clin. Invest. 99: 1534,1997.

Results

Tetracycline and its analogs are known inhibitors of MMP activity. We compared serum DOX levels by adding 1 mg / ml to drinking water and in vitro IC ₅₀ . In the MCA fluorescence assay, IC 50 = 59.1 µM, while serum levels showed 2.64 µM using the zone inhibition assay. These data indicate that the amount of DOX in serum is 22.4 times lower than the amount at which 50-MMP activity can be inhibited. Therefore, significant inhibition of DOX is unlikely.

Figure 3B is a photograph of whole mount staining for β-galactosidase activity 16 embryonic day in the transgenic mice expressing the transgene shown in Figure 3A. The blue color (arrows) is evident in the joints throughout the body of the transgenic animals, while it is not in the non-transgenic offspring. observable any coloring. Specifically, the joints including the ankles, knees, hips, finger, wrist, elbows, shoulder joints and vertebrae showed transgene expression. In addition to the articular cartilage, the cartilage that has not yet been ossified to the bone at a given stage of development, ie. some facial, skull and rib bones. Figure 3C shows an enlargement of the elbow and paw. These data confirm the expression capabilities of the type II collagen promoter and are useful for determining those tissues (joints) that will express the MMP13 * transgene.

The constructs shown in Figures 2A and 2B were both co-injected into fertilized mouse embryos. Of the 112 newly born mice, 7 carried both transgenes, but only four derived lines were capable of reproduction. Transgenes were identified by PCR and verified by Southern blot analysis using a transgene-specific probe (data not shown). The copy number of each of the four transgenes was further determined by Taqman quantitative PCR. (data fc fc fc fc fc fc fcfc fcfc fcfc fcfc fcfc fcfc fcfc not listed). Briefly, the copy number of transgenes ranged from 1 to 32 for tet activator and from 1 to 20 for MMP13 *. Specifically, lane 6 contained about 8 copies of tet activator and about 3 copies of MMP13 transgenes.

The expression of TA and MMP13 * transgenes was first evaluated in the knees of the hind limb joint in 4 month old mice (line 6). To check the efficiency of each reaction, c-fos endogenous cDNA was amplified. Figure 4 shows the amplification of a 890 bp long fragment obtained using TA-specific primers. RT-PCR showed that the TA transgene is expressed in transgenic mice both with and without DOX but. was not expressed in non-transgenic controls (lanes 4-5). Constitutive expression of TA is expected to be driven by a constitutively active promoter. In addition, the expression of TA is limited to the joints and has not been demonstrated by RT-PCR in other tissues, including brain, heart, liver, kidney, spleen or skeletal muscle (data not shown).

Figure 4B shows the amplification of the 645 bp long fragment obtained with primers specific for MMP13 *. Note that the primers for MMP13 * are specific for human MMP13 and do not react with their endogenous mouse homologue, collagenase-1. RT-PCR showed that MMP13 * expression did not occur in non-transgenic controls (lanes 4-5). Lanes 6-7 show that MMP13 * transgenes are expressed in DOX-treated mice. Removal of DOX from drinking water resulted in a significant increase in expression (lanes 8-9), according to our estimates, about 5-10x. After electrophoresis, the PCR fragments were then transferred to a nylon membrane and hybridized with TA or MMP13 * specific probes to verify the identity of the PCR products (data not shown).

Fibroblasts from several transgenic lines. (eg, lines 8 and 42) were able to express MMP13 * as evidenced by the presence of a PCR product of predicted length. No MMP13 * RT-PCR band was detected in cells transformed with the carrier alone. These results indicate that in these mice the MMP13 * transgene is integrated into the transcriptionally active region of chromatin.

As early as three days after stopping doxycycline, immunologically cleavable MMP-13 products could be detected. After 30 days without doxycycline, a significant increase in staining was observed with: / * * * · · · ««!!!!

»· ·" · »* <

·· «·

• * •··

Growth plates and articular cartilage (Figure 5), but the results differed between mouse lines (see Table 1 below).

Table 1

Imunohist ^ chemistry Fi animals days without DOX HMMP13 Ab antibodies for cleavage fragments type II collagen loss of color Network 0 line 8 wt - - invisible line 8 10 d - - invisible 2 d + + invisible 7 d + + e + rn í r n á 14 d + + · + + + + small wt - - invisible lilii e 6 30 d nezniká line 8 30 d + L i + 'ř + n - / »Lk lazy e -12 30 d •and - invisible

Example 5: Phenotype of MMP13 * transgenic mice.

Materials and methods

To study the effect of MMP13 * activity on cartilage in adult transgenic animals, mice were stopped receiving doxycycline over an increasing period of time after which they were sacrificed. Paraffin-formaldehyde-fixed sections of decalcified cartilage were cut and stained with (i) hematoxylin and eosin (H&E) and (ii) Safranin O followed by fast green (American Histo Labs, Gaithhersburg MD). Staining techniques for articular cartilage are described (Peter et al., J. Exp. Pathol. 71:19, 1990).

Results

Control transgenic animals without MMP13 * expression retained a significant amount of Safranin O staining in the articular cartilage and patella growth plate (Fig. 6A). In contrast, transgenic line 8 animals exhibited a significant loss of Safranin O staining in their joints after the doxycycline was stopped. After seven days, a slight reduction in Safranin 0 staining in the patella cartilage (Fig. 6B) was observed, which increased and a somewhat greater staining loss was observed in 14 days. articular cartilage and growth discs (Fig. 6C). Significant loss of staining with Safranin O was also observed in other joints, including cartilage of inflammation and femur, wrist, and ankle, indicating a decreased proteoglycan concentration in these areas compared to control.

To record all changes in articular cartilage induced by transgene expression, Line 6 mice were maintained with or without DOX for 114 days; slices were prepared from her joints and stained with H&E. In comparison to age-matched controls, transgenic animals without DOX developed a pathological finding resembling osteoarthritis. Figure ΊΑ shows that no control lesions or other osteoarthritic pathology occurred in the control animal, while transgenic animals showed lesion formation on their articular cartilage (Figure 7B). Specifically, the H&E sections show significant cartilage loss, focal disturbance, disturbance that extends to the bones, and inflamed synovium. Inside the synovium is evidence of fibroid necrosis, metaplasia, and synovial cell hyperplasia. In addition to these symptoms of osteoarthritis, some changes characteristic of rheumatoid arthritis are observed. These changes include angiogenesis - observable blood cell, monocyte and macrophage cell infiltration. Figures 7C and 7D show synovium at a higher resolution.

Discussion

We succeeded in creating transgenic mice expressing constitutively active human MMP13 protein in articular cartilage of adult animals. We showed that a unique combination of technologies, ie the tetracycline-regulated gene expression system and chondryocyte-specific expression of a constitutively active MMP protein, allowed us to develop a transgenic model that produces lesions and other osteoarthritic pathological changes.

Using a controllable / inducible system, we avoided deleterious effects on embryos, although we achieved significant expression of hMMP13 * in adult mice. Lac Z staining shown in Fig. 3 * ·

• · i i · ··. ·. ·. It shows that collagen II expression occurs during development around E16, and type II collagen is involved in skeletal formation. Type II collagen together with type I collagen are known as substrates for proteolytic cleavage by MMP13. We therefore assumed that unregulated expression of MMP13 during embryogenesis would be lethal.

We have established four transgenic lines containing both transgenes. In the two lines that expressed hMMP13 *, the expression level in one was very low, but the other expressed a significant amount of hMMP13 *. A line that expressed only minor amounts of transgene showed histological evidence of proteoglycan loss. However, no lesions were observed after six months without DOX. Lane 6, which expresses significantly greater amounts of hMMP13 *, shows osteoarthritis pathology including lesion formation, cartilage degeneration and synovium inflammation after four months without DOX. In addition, expression of hMMP13 * in line 6 can be driven, i.e., initiated or stopped by the investigator. These results further confirm that the phenotype is dependent on expression of hMMP13 * and is not the result of integration at a particular site in chromatin.

As with Line 6, osteoarthritis patients also experience joint destruction / injury, lesions, fibroid necrosis, metaplasia, synovial cell hyperplasia, and synovium inflammation (in the absence of T-cells). Not all pathological conditions observed in transgenic mice resemble osteoarthritis. For example, angiogenesis and infiltration of monocytes and macrogages are pathological conditions observed during the inflammatory process associated with rheumatoid arthritis. Note the absence of neutrophils in the synovial fluid. The pathway of neutrophils to the site of inflammation is a hallmark of the pathology of rheumatoid arthritis.

The data presented in this work provide direct evidence that MMP13 is a critical player in osteoarthritis. Moreover, the transgenic animals described in this work provide an animal model for testing the efficacy of therapeutics. Compounds that alter MMP13 activity or inhibit the progression of osteoarthritis can be monitored for the formation of lesions and other pathological osteoarthritic (OA) changes at different times during disease progression. The ability to induce and stop expression of hMMP13 * and thus control the formation / timing of lesion formation will be an advantage in determining the potency of a compound.

··· • 0 • · ·

* • 000 *

This osteoarthritically-like (OA-like) transgenic model can also be used to answer the expanding list of biological questions. In addition to MMP13, type II collagen also cleaves interstitial collagenase (MMP1) and neutrophil collagenase (MMP8). Thus, transgenic mice constitutively expressing active MMP1 or MMP8 can be expected to have a similar phenotype of their articular cartilage.

Example 6: Extension of the Development of Symptoms of Joint Diseases in MMP-13 Transgenic Mice

The following treatments are made to highlight the symptoms of joint degeneration exhibited by the transgenic animals of the invention.

In the group of transgenic mice, transgene expression was induced at the age of 4-12 months. Two to six weeks after induction, mice are given an intraperitoneal inflammatory agent, such as lipopolysaccharides (10-100 µg), zamosan (1-10 mg), staphylococcal enterotoxin B superantigen (1-100 µg), or TGF-β (1-10 µg) ) or some other. Alternatively, the animals may be injected into the joint with an inflammatory or function-influencing agent, such as lipopolysaccharide (1-100 ng), zymosan (50-250 µg), papain (10-100 µg), TGF-β (0.01-1 µg) ), bone morphogenic protein - 2 (2-1000 ng) IL-1 (1-100 ng). TGF-α (10-200 ng), IGF (0.01-1 µg) or FGF (0.01-1 µg) or some others. Transgenic mice of the same age and sex bred to avoid transgene expression were treated in the same way and served as a control.

The development of symptoms of degenerative joint diseases is monitored by overall observation of joint swelling and functions and histological evaluation of joints at selected time series after exposure to the inflammatory agent.

The agents will induce an acute inflammatory response and / or short-term loss of proteoglycan for less than one week. Acute inflammatory response and / or temporary cartilage changes will further regulate gene expression in chondryocytes, increase expression of transgenes, and increase the level of MMP-13 produced.

All application patents, articles, publications and test methods mentioned above are hereby incorporated by reference as ceiek.

<φ · ♦ φ ·

In light of the above descriptions, many variations of the present invention will be offered to those skilled in the art. Such obvious variations are fully consistent with the appended claims.

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SEQUENCE LISTING

<110> Neuhold, Lisa Killar, Loran <120> TRANSGENIC ANIMAL MODEL FOR DEGENERATIVE DISEASES OF CARTILAGE <130> AHP-97285PCT <140> TBA <141>

<150> 60 / 060,312 <151> 1997-12-19 <150> 08 / 994.609 <151> 1997-12-19 <160> 24 <170> FastSEQ for Windows Version 3.0 <210> 1 <211> 7 <212 > PRT <212> Homo sapiens <400> 1

For Arg Cys Gly Xaa For Asp 1 5 <21O> 2 <211> 12 <212> PRT <213> Homo sapiens <400> 2

His Glu Xaa Gly His Xaa Xaa Xaa Xaa Xaa His Ser 1 5 10 <210> 3 <211> 1521 <212> DNA <213> Homo sapiens <400> 3 caagatgcat ccaggggccc tggccgcccc accaccaccc cacaaacccc gcgggaaccc cgaaggagaa cgcagcaagc cccacgaccg agaggccccg agaaacgcag cccccccccg gctcagaggt gactggcaaa cttgacgata acaccccaga cgccacgaaa aagccaagat gcggggccgc cgacgcgggc gaacacaacg cccccccccg aacccccaaa cggcccaaaa cgaatccaac ccacagaacc gcgaaccaca cccccgacac gacccacccc gaagccgaaa aggcacccaa aaaagccccc aaagcccggc ccgacgcaac ccccccgaac cccaccagac cccacgacgg caccgccgac accacgaccc cccccggaac caaggagcac ggcgaccccc acccacccga cgggcccccc ggcccgccgg cccacgcccc cccccccggg ccaaaccacg gaggagacgc ccaccccgac gacgacgaaa cccggacaag cagccccaaa ggccacaacc cgccccccgt cgccgcgcac gagcccggcc accccccagg ccccgaccac cccaaggacc ccggagcacc cacgcccccc acccacaccc acaccggcaa aagccacccc acgccccccg acgacgacgc

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Met His For Gly Wall Leu. Ala Ala Phe Leu Phe Leu Ser ¹ Trp ¹ Thr: His 1 5 10 15 Dec Cys Arg Ala Leu For Leu For Ser Gly Gly Asp Glu Asp Asp Leu Ser 20 May 25 30 Glu Glu Asp Leu Gin Phe Ala Glu Arg Tyr Leu Arg Ser Tyr Tyr His 35 40 45 For Thr Asn Leu Ala Gly Ile Leu Lys Glu Asn Ala Ala Ser Ser Met 50 55 60 Thr Glu Arg Leu Arg Glu Met Gin Ser Phe Phe Gly Leu Glu Wall Thr 65 70 75 80 Gly Lys Leu Asp Asp Asn Thr Leu Asp Wall Met Lys Lys For Arg Cys 85 90 95 Gly Wall Wall Asp Wall Gly Glu Tyr Asn Wall Phe For Arg Thr Leu Lys 100 ALIGN! 105 110 Trp Ser Lys Met Asn Leu Thr Tyr Arg Ile Wall Asn Tyr Thr For Asp 115 120 125 Met Thr His Ser Glu Wall Glu Lys Ala Phe Lys Lys Ala Phe Lys Wall 130 135 140 Trp Ser Asp Wall Thr For Leu Asn Phe Thr Arg Leu His Asp Gly Ile 145 150 155 160 Ala Asp Ile Met Ile Ser Phe Gly Ile Lys Glu His Gly Asp Phe Tyr 165 170 175 For Phe Asp Gly For Ser Gly Leu Leu Ala His Ala Phe For For Gly 180 185 190 For Asn Tyr Gly Gly Asp Ala His Phe Asp Asp Asp Glu Thr Trp Thr 195 200 205 Ser Ser Ser Lys Gly Tyr Asn Leu Phe Leu Wall Ala Ala His Glu Phe 210 215 220 Gly His Ser Leu Gly Leu Asp His Ser Lys Asp For Gly Ala Leu Met 225 230 235 240 Phe For Ile Tyr Thr Tyr Thr Gly Lys Ser His Phe Met Leu For Asp 245 250 255 Asp Asp Wall Gin Gly Ile Gin Ser Leu Tyr Gly For Gly Asp Glu Asp 260 265 270 For Asn For Lys His For Lys Thr For Asp Lys Cys Asp For Ser Leu 275 280 285 Ser Leu Asp Ala Ile Thr Ser Leu Arg Gly Glu Thr Met Ile Phe Lys 290 295 300 Asp Arg Phe Phe Trp Arg Leu His For Gin Gin Wall Asp Ala . Glu . Leu 305 310 315 320 Phe Leu Thr Lys Ser Phe Trp For Glu . Leu . For i Asr i Arg 'Ile : ASp i Ala

325 330 • • · «· • • • ··· • 0 « * • ♦ · • • · * »» * · ♦ · »» »· ·· »· · • ♦ · ·· 335 and 0 • • · • 4 Ala Tyr Glu His For Ser His Asp Leu Ile Phe Ile Phe Arg Gly Arg 340 345 350 Lys Phe Trp Ala Leu Asn Gly Tyr Asp Ile Leu Glu Gly Tyr For Lys 355 360 365 Lys Ile Ser Glu Leu Gly Leu For Lys Glu Wall Lys Lys Ile Ser Ala 370 375 380 Ala Val His Phe Glu Asp Thr Gly Lys Thr Leu Leu Phe Ser Gly Asn 385 390 395 400 Gin Val Trp Arg Tyr Asp Asp Thr Asn His Ile Met Asp Lys Asp Tyr 405 410 415 For Arg Leu Ile Asp Phe Pro Gly Ile Gly Asp Lys Wall Asp 420 425 430 Ala Val Tyr Glu Lys Asn Gly Tyr Ile Tyr Phe Phe Asn Gly For Ile 435 440 445 Gin Phe Glu Tyr Ser Ile Trp Ser Asn Arg Ile Wall Arg Wall Met For 450 455 460 Ala Asn Ser Ile Leu Trp Cys

465 470 <210> 5 <211> 470 <212> Artificial Sequence <220>

<223> Tet promoter cloned into cytomegaloviral promoter, tet 07 promoter <400> 5 ctcgagttta ccactcccta tcagtgatag agaaaagtga aagtcgagtt taccactccc tatcagtgat agagaaaagt gaaagtcgag tttaccactc cctatcagtg atagagaaaa gtgaaagtcg agtttaccac tccctatcag tgatagagaa aagtgaaagt cgagtttacc actccctatc agtgatagag aaaagtgaaa gtcgagttta ccactcccta tcagtgatag agaaaagtga aagtcgagtt taccactccc tatcagtgat agagaaaagt gaaagtcgag ctcggtaccc gggtcgagta ggcgtgtacg gtgggaggcc tatataagca gagctcgttt agtgaaccgt cagatcgcct ggagacgcca tccacgctgt tttgacctcc atagaagaca ccgggaccga tccagcctcc gcggccccga attagcttga tatcgaattc <210> 6 <211> 34791 <211>

<223> Sprague Dawley rat type II collagen promoter.

<400> 6 ggtaccacta gtaagcttag atccactgtc tgggattaca tcaggacaac cgaagcctgg aaagtgtatt aggtagagca ttttcttcca cgtgtttggg cacgtttccg acagctagga ttccagctct gtctttgtat gttacagact gtaaatcaat cgcaggtgaa actgtttgga cagtaggtgg ggatcaaaga ccctccgccc gtgagactct aggcgctttc ccctgccacc agcctgtctc cagagatgct ctggaaggag gcgggcccgg gcggtctttc tgctctttag cgtggcggac gcggcggcgg gggcagggct ggagcagaga gcgctgcagt gatagaactt tctgaccccg ctgcgcaggg cggcagggtg gcagggtggc agggtggcga gctaagccag agccgaacgc tggagctctg ggaggaacat cgaaggtttg tatgtggtct gagatcggcc tgactatatt tttttgtcct aaatttgcaa gcacacaccc acaaagctgc ggtcttgacc ggtattcttt atagagcgca atggagtgag ctgagtgtct aaacgatttc cctaattcat ctgatagcag aggcgctctc ctaattggcg aagagctgcc tcatgtccgc aactttttgg cagagtgaat tccacagctt tgtgtgtgtg tgtggggggg ggtgtaaggg gtgtctaaaa ctttcggtct cctactattc tgtatctcga ccggttggtt ttacaccccg gctcatctca

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ccaacgcaaa accaggaacg gaaactcccc aagcgccgca agggaccggc gtcccccgcg agaggcaagg ccccccccgc aagcaacaca atgccgcacg cctttccgcg ttacactgtt ctgtctgatg cacccggccg aagccagcca caaaatgtcc gacccaaaac ataggccggg cagcacacct gctccaaagt cgggggttcc ttcgagacaa aagttcccta acccccttgt ggggtaaggt ggggaagacg tggcggcgga ccggagccgc cgctgctccc aagagccgaa ctcggttgtc gtccccctct cgcctaccta cgcgagcctc ctggaaaact ccccccgccc cgcctttcca ttctaacccc agagggggca cgatcaggcc cttggcaggc cagccccgcg cgaccgggag ccgggtctcc cccggacccg cacccccact tcagaaacct ggctgcgaag ggttcgggag agcgatggct gagagctgtg cccagtgggt gcccgcctgc gttccccgaa gcggtaacga ccacaattag ggctcctttc gagggctgtg gatctcatat agctagcttg cccgggctaa cagctttcta aagatgcaag gcatagaaat ctgaggtggt atgggggtag aggagttact gaggaccatc tatggaagga cggggttggg tgtgtgtgtg caactaggaa ctccagactc tccaggctaa ttagacaagt tgcagctcct gtgtaggaat gatttttttc caggcgggag gtcccccaga cctaaactcc gccttgagtc agcagaactc gtgtggcagt actgggcaca gtgggccctg gggggccggg catataaccg tgcctcctcc ctccgcgctc cccctatgga agagcccacg accccgaccc gggagacccc tccagatggg aaccgggctc ccccccgacc cgccacctcc agaggcagcc ggggaaccgg ggtggcgccg tcgggcagcc tccggagccc ggcgcacccc cgcaagctag gatccgacag gcagaggcag aacggcaccc caagcctcac gcggcacgcc ccccccccaa gcctaaacca ccacccccaa gaccccggga gggagcaagg cgcgcgtgtg actctggcgc tccggccagg gggcacccac gtctccaatc ttagtccctt acaccagacc atagccagtt ttcaccgaca tttaggattc ccatccccac taaagctcca cgaggaaagg cccaagtggg tcgggggcgg gcccggcctg gggcggcccg gagcccctgc tcctgctcct tgccgccccg

1L cccaaggacc cccctcccct acatccgcat agtcctcctt ctgaaaccct tgtatgcgct ccccgacccc cgggctccgg ttctaatcgg atcgggcggc gcttcctccg atttaatcct catccacaaa cagtacaatc ctcgcgatcc ccatggtcca aaggatctag tacataaaga acttgaagtt gggtaattct ggacttccag ggccctaatt agaaaaggtg acagagcaag aaggcaagtg tgtgtgtgtg tttccccccc gcctcagagt tcccccggag cggctgcgtg ccctggcctg ctctcttagc ggatggggga ggtaccccgc tgggcagctt cttcctttct tgcttatgcc ggccggaggc ggcgaccgga gaagctgggc ggcaggctcc cggcttgggc tgggagaaga agagcctcct ccgagctccg • · • »t» · · · • · · ··· * ♦♦ cgacgcgggg ccacccttcc cccccagccc tgcgaggctt gcccgtattc cgagaaaagc cctcccccac ccccgcgcgc ccagccacaa caggatgcta accgcaccta actccttacc gagtcagcca cgctctgatg gtaaaaatgt aagaagactt agccaaaggc acactctctc ctttgtttcc ccccaccccc cggcaacaca tccaaggttc tattcgggga ggccgagccc aggctggagg tgtgtgtgtc cctcacaaaa ggtcaacagt attcctgaac cggattttgc ccccttacac cacacacacc tgggttaggg aaaggagctg ccatcagctt cccgttactt tctgcaaaca cccccctctc ggccgtctcg tcaccaaagg ggggg cgggg tggtttgcca cgcagagcgc gcatgagggc cccgcaagct gaaggcggac acgagtttgg cagcctccaa gtttgcgttg atttaaactg cccattcatg aatatatccc agcggcgacg agaatcactt tctgtgtagc ggcgatctgg ctacgaatgt gcagctctca ccgcatagtt gtgagagtca cggcactgca aaagactcga cttttccagc atcctgagaa caacattccc gaaatcccac cctttgctta cccatcctca ccggcagttt cccagggata tcggggatgg ctgagcccag ccctggccag ctgggccagg tgcggtgtcc ctccacacag tccagtcccc aggctgggct gaaggcaggt atactttggc cgtcctccct accccctccc gcctgtggtt gtgccccgcc ggcgactggc tcccaggtta gcccttggag cgccgggctg gcggtagaga ggggaattc

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3479 <210> 7 <211> 8 <212> PRT <213> Horr. Sapiens <400> Pro Arg Cys

Gly Val Pro Asp Val 5 <210> 8 <211> 39 <212> DNA <213> Artificial Sequence <220>

· «♦ ···

<223> MMP13 * Mutagenic Oligonucleotide <400> 8 aagccaagac gcggggttgt cgatgtgggt gaatacaat <210> 9 <211> 40 <212> DNA <213> Artificial Sequence <220>

<223> MMP13 * Mutagenic Oligonucleotide <400> 9 gaaaaagcca agatgcgggg gtcctgatgt gggtgaatac 40 <210> 10 <211> 98 <212> DNA <213> Artificial Sequence <220>

<223> BS (EN-) vector <400> 10 ggtaccacta gtaagcttag atctcatatg gtcgaccccg gggaattcct gcagggatcc tctagaagta ccccatgggt atacatcgat gcggccgc <210> 11 <211> 2792 <212> DNA <213> Artificí

<223> tet-07 / MMP-13 * transgene <400> 11 ctcgagttta ccactcccta tcagtgatag agaaaagtga aagtcgagtt taccactccc 60 tatcagtgat agagaaaagt gaaagtcgag tttaccactc cctatcagtg atagagaaaa 120 gtgaaagtcg agtttaccac tccctatcag tgatagagaa aagtgaaagt cgagtttacc 180 actccctatc agtgatagag aaaagtgaaa gtcgagttta ccactcccta tcagtgatag 240 agaaaagtga aagtcgagtt taccactccc tatcagtgat agagaaaagt gaaagtcgag 300 ctcggtaccc gggtcgagta ggcgtgtacg gtgggaggcc tatataagca gagctcgttt 360 agtgaaccgt cagatcgcct ggagacgcca tccacgctgt tttgacctcc atagaagaca 420 ccgggaccga tccagcctcc gcggccccga attagcttga tatcgaattc gagctcggta 480 cccggggatc ctctagacaa gatgcatcca ggggtcctgg ctgccttcct cttcttgagc 540 tggactcatt gtcgggccct gccccttccc agtggtggtg atgaagatga tttgtctgag 600 gaagacctcc agtttgcaga gcgctacctg agatcatact accatcctac aaatctcgcg 660 ggaatcctga aggagaatgc agcaagctcc atgactgaga ggctccgaga aatgcagtcc 720 ttcttcggct tagaggtgac tggcaaactt gacgataaca ccttagatgt catgaaaaag 780 ccaagatgcg gggttgtcga tgtgggtgaa tacaatgttt tccctcga c tcttaaatgg 840 tccaaaatga atttaaccta cagaattgtg aattacaccc ctgatatgac tcattctgaa 900 gtcgaaaagg cattcaaaaa agccttcaaa gtttggtccg atgtaactcc tctgaatttt 960 accagacctc acgatggcat tgctgacatc atgatctctt ttggaattaa ggagcatggc 1020 gacctctacc catttgatgg gccctctggc ctgctggctc atgcttttcc tcctgggcca 1080 aatcatggag gagatgccca ttttgatgat gacgaaacct ggacaagtag ttccaaaggc 1140 cacaactcgt ttcttgttgc cgcgcatgag ttcggccact ccttaggtct tgaccactcc 1200 aaggaccctg gagcactcat gtttcctatc tacacctaca ccggcaaaag ccactttatg 1260 cttcctgatg acgatgtaca agggatccag tctctctatg gtccaggaga tgaagacccc 1320 aaccccaaac acccaaaaac gccagacaaa tgtgaccctt ccttatccct tgatgccatt 1380 accagtcccc gaggatcat aatcat

ftft · ftft · ftft ftftft ftftft ftft ftftft ftftftft ftftftft

ft ft •

ft cagcaggccg atgcggagct gtttttaacg aaatcactcc ggccagaact ccccaaccgt actgacgctg catatgagca cccttctcat gacctcatct tcatcttcag aggtagaaaa ttttgggctc ttaatggtta tgacattctg gaaggttatc ccaaaaaaac atctgaactg ggtcttccaa aagaagttaa gaagataagt gcagctgttc actttgagga tacaggcaag actctcccgt tctcaggaaa ccaggtctgg agatatgatg atactaacca tattatggat aaagactatc cgagactaat agaagaagac ttcccaggaa ttggtgataa agtagatgct gtctatgaga aaaatggtta tatctatttt ttcaacggac ccatacagtt tgaatacagc atctggagta accgtattgt tcgcgtcatg ccagcaaatt ccattttgtg gtgttaagtg tctttttaaa aattgttatt taaatcccga agagcatttg gggtaatact tccagaagtg cggggtaggg gaagaagagc tatcaggaga aagccccagt tccagagggc cctattctat agtgtcacct aaacgccaga ggatctttgt gaaggaacct tacttctgtg gtgtgacata attggacaaa ctacctacag agatttaaag ctctaaggta aatataaaat ttttaagtgt ataatgtgtt aaactactga ttctaattgt ttgtgtattt tagattccaa cctatggaac cgacgaacgg gagcagcggt ggaatgcctt taatgaggaa aacctgtttt gctcagaaga aatgccatct agtgatgatg aggctactgc cgactcccaa cattctactc ctccaaaaaa gaagaga AAG gtagaagacc ccaaggactt tccctcagaa tcgctaagtt ttttgagtca tgccgtgttt agtaatagaa ctcttgcttg ctttgctatt tacaccacaa aggaaaaagc tgcactgcta tacaagaaaa ttatggaaaa atatttgacg tatagtgcct tgactagaga tcataatcag ccataccaca tttgtagagg ttttacttgc tttaaaaaac ctcccacacc tccccctgaa cctgaaacat aaaatgaatg caattgttgt tgttaacctg tttattgcag ctcataatgg ttacaaataa agcaatagca tcacaaattt cacaaataaa gcattttttt cactgcattc tagttgtggt ttgtccaaac tcaccaacgt atcttatcat gtctggatca tcccgccacg ggtatacatc gacgcggccg cc <210> 12 <211> 5276 <212> DNA <213> Artificial Sequenee <220>

<223> tetracycline / VP16 repressor-activator fusion Linked to and type II collagen promoter and enhancer, CPE-tTA transgene <400> 12 ggtaccacta gtaagcttag atccactgtc tgggattata tcaggacaac cgaagcctgg aaagtgtatt aggtagagca ttttcttcca cgtgtttggg cacgtttccg acagctagga ttccagctct gtctttgtat gttacagact gtaaatcaat cgcaggtgaa actgtttgga cagtaggcgg ggatcaaaga ccctccgccc gtgagactct aggcgctttc ccctgccacc agcctgtctc cagagatgct ctggaaggag gcgggcccgg gcggtctttc tgctctttag cgtggcggac gcggcggcgg gggcagggct ggageagaga gcgctgcagt gatagaactt tctgaccccg ctgcgcaggg cggcagggtg gcagggtggc agggtggcga gctaagccag agccgaacgc tggagctctg ggaggaacat cgaagtgttt gtatgtggtc tgagatcggc ctgactatat ttttttgtcc taaatttgca agcacacacc cacaaagctg cggtcttgac cggtattctt tatagagcgc aatggagtga gctgagtgtc taaacgattt ccctaattca tetgatagea gaggcgctct cctaattggc gaagagctgc ctcatgtccg caactttttg gcagagtgaa ttccacagct ttgtgtgtgt gtgtgggggg gggtgtaagg ggtgtctaaa actttcggtc tcccactatt ctgtatctcg accggttggt tttacacccc ggctcatctc atcaacgcaa acacccccac tctcctatgg acccaaggac ctgacgtggg ggaaggtgga cattaggaac gtcagaaacc tagagtccac gctcctcctc tccatctttc cacgagtttg ggaaacttct tggctgcgaa gactttgacc cacatctgca tttctcagcc ccagcttcca aaagtgccgc aggttcggga ggggagacct cagtcctcct ttgcgaggct tgtttgcgtt gagggattgg cagcgatggc ttccagatgg gctgaaaccc tgcccgtatt tatttaaact ggttcctcgt ggagagctgt gaaccgggcc ctgtatgcgc tcgagaaaag ccccattcat gagaggcaag gcccagcggg tccccccgac tccccgaccc ccctctccca caatacatcc cccctccctg tgcccgcctg ccgccacctc ccgggctccg gccccgcgcg cagcggcgac gaagcaacac agttccccga aagaggtagc tttttaattg gccagccaca aagaatcacc tatgccgcac ggcggtaacg aggggaaccg gatcgggcgg ccaggatgct atctgtgtag cccttctcgt gccacaatta gggcggcgct ggcttcctcc gaccgcacct aggcgatctg gttacaccgc tggctccttt cctgggcagc cacttaatcc tactttttac tctacgaacg cccgcctgac ggagggctgt gcccggagcc ccatccacaa agagtcagcc agcagctccc

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• · *

«* * ·« ··· a · · aacgtacccc accacgcccg gatcatcccg ccacgggtat acatcgatgc ggccgc <210> 13 <211> 7664 <212> DNA <213> Artificial Sequence <220>

<223> beta-galactosidase (lacZ) gene linked the beta-globin splice sequence and polyadenylation signal, and linked to type II collagen promoter sequence <400> 13 ggtaccacta gtaagcttag atccactgtc tgggattata tcaggacaac cgaagcctgg aaagtgtatt aggtagagca ttttcttcca cgtgtttggg cacgtttccg acagctagga ttccagctct gtctttgtat gttacagact gtaaatcaat cgcaggtgaa actgtttgga cagtaggtgg ggatcaaaga ccctccgccc gtgagactct aggcgctttc ccctgccacc agcctgtctc cagagacgct ctggaaggag gcgggcccgg gcggtctttc tgctctttag cgtggcggac gcggcggcgg gggcagggcc ggagcagaga gcgctgcagt gatagaactt tctgaccccg ctgcgcaggg cggcagggtg gcagggtggc agggtggcga gctaagccag agccgaacgc tggagctctg ggaggaacac cgaagtgttt gcacgtggcc tgagatcggc ctgactatat ttttttgtcc taaatttgca agcacacacc cacaaagctg cggccttgac cggtattctt tatagagcgc aatggagtga gctgagtgtc taaacgattt ccctaattca tctgatagca gaggcgctct cctaattggc gaagagctgc ctcatgtccg caactttttg gcagagtgaa ttccacagct ttgtgtgtgt gtgtgggggg gggtgtaagg ggtgtctaaa actttcggtc tcctactatt ctgtatctcg accggttggt t ttacacccc ggctcatctc atcaacgcaa acacccccac tctcctatgg acccaaggac ctgacgtggg ggaaggtgga cattaggaat gtcagaaacc tagagtccac gctcctcctc tccatctttc cacgagtttg ggaaacttct tggctgcgaa gactttgacc cacatctgca tttctcagcc ccagcttcca aaagtgctgc aggttcggga ggggagacct cagtcctcct ttgtgaggct tgtttgcgtt gagggattgg cagcgatggc ttccagatgg gctgaaaccc tgcccgtatt tatttaaacc ggccccccgc ggagagctgt gaatcgggct ctgtatgcgc tcgagaaaag ccccacccac gagaggcaag gcccagtggg tccccccgac tccccgaccc ccctctccca caacacaccc cccctccccg tgcccgcctg ccgccacctc ccgggctccg gccccgcgcg cagcggcgac gaagcaacac agttccccga aagaggcagc cccctaaccg gccagccaca aagaaccacc tatgccgcac ggcggtaacg aggggaaccg gatcgggcgg ccaggatgct atctgtgtag cccttttcgt gccacaatta gggcggcgcc ggcttcctcc gaccgcacct aggcgatctg gttacaccgt tggctccttt cttgggcagt catttaatcc tactttttac tctacgaatg tctgtctgat ggagggctgt gtccggagcc ccatccacaa agagtcagcc agcagctctc acacccggct ggatctcata tggtgcactc ccagcacaac ctgctctgat gccgcacagc caagccagcc aagctagctt gcgcaagcta gcttgcgatc cgtaaaaatg tgcgagagcc acaaaatgtc ttccgggcca agatccgaca gccatggtcc aaagaagact tcggcactgc agactcaaaa ccagctttct agcagaggca gaaggatcta gagccaaagg caaagacttg aataggctgg gaagatgcaa gaacggcacc ttacataaag aacactctct ccttttccag ccagcacact tgcatagaaa ttaagtttta cacttgaagt tctttgtttc catcctgaga agccccaaag tctgaggtgg cgcggcacgc tgggtaatcc tccccacccc ccaacattcc ccgggggttc cacgggggca gcttctccca aggacttcca gcggcaacac agaaatccca cttcgagaca aaggagttac cgcccaaacc aggccctaat ttccaaggtt ccctttgctc aaagttccct agaggaccat ctcacttcta aagaaaaggt gtattcgggg acccatcctc aacccccccg ttatggaagg agacttcggg aacagagcaa gggctgagcc cccggcagcc cggggcaagg ccggggccgg ggggagcaag gaaggcaagt gaggctggag gcccagggac aggggaagac gtgtgtgcgt gcgcgcgcgc gcgcgcgcgc gtgtgtgtgt ctcggggatg gcggcggcgg acaactagga aactctggcg ctttctcctc ccctcacaaa actgagtcca gctggagccg cctccagact ctctggccag ggcctcagag tggtcaacag tccctggcca gcgttgcccc ccccaggcca agggcaccca ctcccctgga gattcctgaa cctgggccag gaagagccga accagacaag cgcccccaac ccggctgcgt gcggattttg ttgcggtgtc ccccggccgc ctgcagttcc tttagccccc cccccggccc gccccttaca cccccacaca ggcccccccc tgtgtaggaa tacaccagac cctctcctag ccacacacac ctccagtccc

9

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caagccagcc i acaaaatgcc t agactcaaaa <.

aataggctgg <

ccagcacact i agctccaaag 1 ctgggggttc <

ctccgagaca i aaagttcccc i aacctccttg 1 tggggtaagg i aggggaagat <

gtggtggcgg and gccggagccg gcgttgctct gaagagccga ccccggttgt ggtccccctc ccgtctacct ttgcgagcct tccggaaaac ctcccccgcc tcgcctttcc cttctaaccc tagagggggc ccgatcaggc ccttggcagg acagccccgc gcgaccggga gccgggtctc acccggaccc atatgtctag tcggaatcga cattgtattg cagataggca gtaataacgc atttaggtac tatgccaaca ttaccttagg cacctactac aaggtgcaga aacttaaatg ctaccatcga cggctccgcg cgaccgatgt acgccgacgc cgggatttac ttgagcagat gatcctctag aacctcacct aggtaaatat tattttagat aggaaaacct ctcaacattc cagaattgct ctatttacac tgatgtatag cttgctttaa gttgttgtta aatttcacaa ggatcccata aagctagctt ttccgggcta ccagctttct gaagatgcaa tgcatagaaa tctgaggtgg catgggggta aaggagttac agaggaccat ttatggaagg ttggggttgg gtgtgtgtgt acaaccagga cctccagact ctccaggcta attagacaag ctgcagttcc tgtgtaggaa agattttttt ccaggtggga tgtcccccag ccctaaactc agccttgagt cagcagaacc agtgtggcag cactgggcac tgtgggctct ggggggctgg gcatataacc ctgcctcctc gctccgtgct attagataaa aggcttaaca gcatgtaaaa ccatactcac taaaagtttt acggcctaca aggtttttca ttgcgtattg tgatagtatg gccagccttc tgaaagtg gg gggcctgctc cctgtccttt cagcctgggg gctagacgat: cccccacgac gtttaccgat i agggccctat ctgtggtgtg. aaaattctca: tccaacctat: gttttgctca: tactcctcca aagtctcctg: cacaaaggaa j tgccttgacc i aaaacccccc i acttgtttat i ataaagcatc cggtgcaccc gcgcaagcca agatccgaca agcagaggca gaatggcatt tcaagtttta tgtggtatgc ctcttcta cg

agacttcggg

Sgggagcaag gtgtgtgtgt aaccctggcg ctctggccag agggcaccca tgtctccaat tttagtccct tacaccagac catagctagt gttcaccgac atttaggatt cccaccccca ctaaagctcc ccgaggaaag ccccaagtgg atcgggggcg ggtccggccc ggggcggccc ggagcctccg cccctgctcc ctgccgcctc agtaaagtga acccgcaaac aataagcggg tcttgccctt agatgtgctt gaaaaacagc ctagagaatg gaagatcaag ccgccattat tcattcggcc tccgcgtaca gatctcccgg ccccccgcgg gacgagctcc ttcgacctgg tccgcccccc gccctcggaa tctatagtgt acataattgg agtgtataac ggaaccgatg gaagaaacgc aaaaagaaga agccatgctg aaagccgcac agagatcata acacctcccc cgcagcttac ccccccaccg

4 »· #« ·· • 9 · • 4 · 3 «4 ·· 4 4 4 4 4 4 45 4 4 4 • 4 4 «3 4 9 1 2 3 4 » • 4 Φ · w - w - << · «· fl * ... tcagtacaac ccgctccgat gccgcatagt 1620 gcccgcgatc cgcaaaaacg cgcgagagtc 1680 gccacggtcc aaagaagacc ccggcaccgc 1740 gaaggatcta gagccaaagg caaagacttg 1800 ttacataaag aacactctct cctcttccag 1860 cacttgaagc tcttcgtctc catcctgaga 1920 tgggtaattc tccccacccc ccaacattcc 1980 aggacttcca gcggcaacac agaaatccca 2040 aggccccaat tcccaaggtt ccctttgctt 2100 aagaaaaggt gtattcgggg acccaccccc 2160 - aacagagcaa gggccgagcc tccggcagtt 2220 gaaggcaagt gaggctggag gcccagggat 2280 gtgtgtgtgt gtgtgtgtgt ctcggggatg 2340 ctttctcctc ccctcacaaa actgagtcca 2400 ggcctcagag tggtcaacag tccctggcca 2460 ctcccctgga gattcctgaa cctgggccag 2520 ccggctgcgt gcggattttg ttgcggtgtc 2580 tccctggcct gccccttaca cctccacaca 2640 cctctcttag ccacacacac ctccagtccc 2700 tggatggggg atgggctagg gaggctgggt 2760 aggtaccccg caaaggagct ggaaggcagg 2820 ctgggcagct tccatcagcc tatactttgg 2880 ccttcccttc tcccgtcact tcgtcctccc 2940 atgcctatgc ccccgcaaac aaccccctcc 3000 gggccggagg ccccccttct cgcctgcggt 3060 gggcgaccgg aggccgtccc ggtgccccgc 3120 ggaagccggg ctcaccaaag gggcgactgg 3180 gggcaggctc cgggggcggg gtcccaggtt 3240 gcggtttggg ctggtttgcc agcctttgga 3300 ctgggagaag acgcagagcg ccgctgggct 3360 tagagccccc tgcatgaggg cgcggtagag 3420 gccgagcttc gcccgcaagc tggggaattc 3480 ttaacagcgc attagagctg cttaacgagg 3540 tcgcccagaa gctaggtgta gagcagccta 3600 ctttgctcga cgccttagcc attgagatgt 3660 tagaagggga aagctggcaa gatcttttac 3720 tactaagtca tcgcgatgga gcaaaagtac 3780 acgaaactct cgaaaaccaa ttagcctttt 3840 cattatatgc actcagcgct gtggggcatt 3900 agcatcaagt cgctaaagaa gaaagggaaa 3960 tacgacaagc tatcgaatta tttgatcacc 4020 ttgaattgat catatgcgga ccagaaaaac 4080 gccgcgcgcg cacgaaaaac aaccacgggt 4140 acgacgacgc ccccgaagag gcggggctgg 4200 gacacacgcg cagactgtcg acggcccccc 4260 acttagacgg cgaggacgtg gcgatggcgc 4320 acacgtcggg ggacggggat tccccgggtc 4380 acggcgctct ggatatggcc gaccccgagt 4440 tcgacgagta cggcgggcag ggggcgcgag 4500 cacctaaatg ctagaggatc cctgtgaagg 4560 acaaactacc cacagagatt taaagctcta 4620 gtgttaaact actgattcta accgcctgtg 4680 aacgggagca gcggcggaac gcccccaacg 4740 catccagcga cgatgaggct accgccgact 4800 gaaaggtaga agaccccaag gaccttccct 4860 tgtttagcaa tagaactctc gcctgcctcg 4920 cgccacacaa gaaaattatg gaaaaatacc 4980 accagccata ccacatttgt agaggtttca 5040 ctgaacctga aacataaaat gaacgcaacc 5100 aacggctaca aacaaagcaa cagcaccaca 5160 cattccagct gtggtccgcc caaacccacc 5220

4 ^ ·· ···· · · • 4

4 · 11 • 1 1 11

1

1 1 11

111 and 1 11111911 1

1 1111111 1111 1 11 κ

ccgtcúaccc agatttttcc catagccagt tggatzggggg acgggtitagg gaggccgggt 2760 ccgcgagccc ccaggcggga gcccaccgac aggcaccccg caaaggagcc ggaaggcagg 2820 cccggaaaac cgccccccag actcaggacc ccgggcagcc cccaccagcc cacaccttgg 2880 cccccccgcc ccctaaaccc cccaccccca cccccccccc ccccgccacc ccgccccccc 2940 ccgccccccc agccccgagc ccaaagcccc acgcccacgc ccccgcaaac aacccccccc 3000 cccccaaccc cagcagaacc ccgaggaaag gggccggagg cccccccccc cgcccgcggc 3060 cagagggggc agcgcggcag tcccaagcgg gggcgaccgg aggccgcccc ggcgccccgc 3120 ccgatcaggc caccgggcac aCcgggggcg ggaagccggg cccaccaaag gggcgaccgg 3180 ccccggcagg tgcgggcccc ggtccggccc gggcaggccc cgggggcggg gccccaggtc 3240 acagccccgc ggggggccgg ggggcggccc gcggcccggg ccggcttgcc agccctcgga 3300 gcgaccggga gcatataacc ggagcccccg ccgggagaag acgcagagcg ccgctgggcc 3360 gccgggcctc ccgcctcccc ctcccgcccc tagagcctcc tgcacgaggg cgcggcagag 3420 acccggaccc gccccgcgcc ctgccgcccc gccgagcttc gcccgcaagc cggggaattc 3480 ggacccccgg gaccgaaaga gcccgctaaa gcaaaaaaga agccaccatg tcgctcactt 3540 TGA ccaacaa gaacgcgacc cccgccgccg gcccgggagg cattggcctg gacaccagca 3600 aggagccgcc caagcgcgac cccgccgccc cacaacgccg tgaccgggaa aaccctggcg 3660 ctacccaacc taatcgccct gcagcacatc cccctttcgc cagccggctt catagcgaag 3720 aggcccgcac cgaccgcccc ccccaacagc cgcgcagccc gaatggcgaa 'tggcgccttg 3780 · cctggcctcc ggcaccagaa gcggcgccgg aaagccggcc ggagcgcgat ccccccgagg 3840 ccgataccgc cgtcgccccc ccaaactggc agacgcacgg tcacgatgcg cccatctaca 3900 ccaacgcaac ccatcccacc acggccaacc cgccgtttgc tcccacggag aacccgacgg 3960 gctgccaccc gcccacatct aatgctgatg aaagccggcc acaggaaggc cagacgcgaa 4020 ccacccccga cggcgccaac ccggcgcccc acccgcggcg caacgcgcgc cgggccggcc 4080 acggccagga cagccgcccg ccgcccgaac ccgacccgag cgcaccccca cgcgccggag 4140 aaaaccgccc cgcggcgacg gcgccgcgcc ggagcgacgg cagccacccg gaagaccagg 4200 acacgcggcg gacgagcggc accccccgcg acgccccgcc gccgcacaaa ccgaccacac 4260 aaaccagcga cccccacgcc gccacCcgcc ccaacgacga ccccagccgc gccgaaccgg 4320 aggccgaagc ccagacgcgc ggcgagccgc gcgaccaccc acgggcaaca gcccccccac 4380 ggcagg gcga aacgcaggcc gccagcggca ccgcgccccc cggcggcgaa accaccgacg 4440 agcgcggcgg ccacgccgac cgcgccacac cacgcccgaa cgccgaaaac ccgaaaccgc 4500 ggagcgccga aaccccgaac ccccaccgcg cggcggccga accgcacacc gccgacggca 4560 cgccgaccga agcagaagcc cgcgacgccg gcccccgcga ggcgcggaCc gaaaacggcc 4620 cgctgccgcc gaacggcaag ccgccgccga cccgaggcgc caaccgccac gagcaccacc 4680 ccccgcatgg ccaggccacg gacgagcaga cgacggcgca ggacaccccg ccgacgaagc 4740 agaacaaccc caacgccgcg cgcCgcccgc accacccgaa ccacccgccg cggcacacgc 4800 cgcgcgaccg ccacggcccg cacgcggcgg acgaagccaa caccgaaacc cacggcaCgg 4860 cgccaacgaa cccgccgacc gacgacccgc gccggccacc agcgacgagc gaacgcgCaa 4920 cgcgaacggc gcagcgcgac cgcaaccacc cgagcgcgac cacccggccg ccggggaacg 4980 aaccaggcca cggcgccaac cacgacgcgc cgcaccgccg gaccaaaccc gccgaccccc 5040 cccgcccggc acagcacgaa ggcggcggag ccgacaccac ggccaccgac accacccgcc 5100 cgacgcacgc gcgcgcggac gaagaccagc cccccccggc cgcgccgaaa cggcccacca 5160 aaaaacggcc cccgccaccc ggagagacgc gcccgccgac cccccgcgaa cacgcccacg 5220 cgacgggcaa C agccccggc ggccccgcca aacaccggca ggcgccccgc cagcaccccc 5280 gttcacaggg cggccccgcc tgggaccggg cggaccagcc gccgaccaaa cacgacgaaa 5340 acggcaaccc gcggccggcc cacggcggcg accccggcga cacgccgaac caccgccagc 5400 cccgcacgaa cggcccggcc cccgccgacc acacgccgca cccagcgccg acggaagcaa 5460 aacaccagca gcagcccccc cagccccgcc cacccgggca aaccaccgaa gcgaccagcg 5520 aacacccgcc ccgccacagc gacaacgagc ccccgcaccg gacggcggcg ccggacggca 5580 agccgccggc aagcggcgaa gcgcccccgg acgccgcccc acaaggcaaa cagccgaccg 5640 aaccgcccga accaccgcag ccggagagcg ccgggcaacc ccggcccaca gcacgcgcag 5700 cgcaaccgaa cgcgaccgga cggccagaag ccgggcacac cagcgcccgg cagcagcggc 5760 gcccggcgga aaaccccagc gcgacgcccc ccgccacgcc ccacgccacc ccgcacccga 5820 ccaccagcga aacggacccc cgcaccgagc cgggcaacaa gcgccggcaa cccaaccgcc 5880 agccaggccc ccccccacag ccgcggaccg gcgacaaaaa acaaccgccg acgccgcCgc 5940 gcgaccagcc cacccgcgca ccgccggaca acgacaccga cgcaagcgaa gcgacccgca 6000 ccgaccccaa cgcccgggcc gaacgccgga aggcggcggg ccaccaccag gccgaagcag 6060 cgccgccgca gcgcacg GCA gacacacccg ccgacgcggc gccgaccaca accgcccacg 6120 cgcggcagca ccaggggaaa accccactca ccagccggaa aacccaccgg accgacggca 6180 gcggccaaac ggcgaccacc gccgacgccg aagcggcgag cgacacaccg cacccggcgc 6240 ggaccggccc gaaccgccag ccggcgcagg cagcagagcg ggcaaaccgg cccggaccag 6300 ggccgcaaga aaaccacccc gaccgcccca ccgccgcccg ccccgaccgc cgggacccgc 6360

XT?

• ·

cattgtcaga catgtatacc ccgtacgtct ccccgagcga aaacggtctg cgctgcggga cgcgcgaatt gaattatggc ccacaccagc ggcgcggcga cttccagtcc aacatcagcc gccacagcca acagcaaccg atggaaacca gccatcgcca tctgctgcac gcggaagaag gcacatggcc gaacatcgac ggtctccata tggggattgg tggcgacgac tcctggagcc cgtcagtatc ggcggaatta cagctgagcg ccggtcgcta ccatcaccag ttggtctggt gtcaaaaata ataataaccg gcaggccatg tctgaaagca ttcgcgtaag gaaatccatt acgtactatt taaaaaacac aaacttttgg atgttcggtt tattcttttt ctcttacttt tttatcatgg gagcctactt cccgcttttc ccgatttggc cacacgacat caaccatatg agcaaaagcg atacgggtat tatttttgcc gctatttctc tgttgtcgct attattccaa ccgccgctgg tctgctttct gacaaactcg gcctcgactc cagactgaga acttcagggt gagtttgggg acccttgatt gttctttctt tttcgctatt gaaaaattca tgttatatgg agggggcaaa gttttcaggg tgttgtttag aatgggaaga tgtcccttgt atcaccatgg accctcatga taatttcgtt tccttcactt tctactctgt cgacaaccat tgtctcctct tattttcttt tcattttctg taactttttt cgttaaactt tagcttgcat ttgtaacgaa tttttaaatt caetttcgtt tatttgtcag attgtaagta ctttctctaa tcactttttt tccaaggcaa tcagggtaat tatattgtac ttcagcacag tcttagagaa caattgttat aattaaatga taaggtagaa catttctgca tataaattct ggctggcgtg gaaatattct tattggtaga aacaactaca tcccggcaat catcctgcct ttctctttat ggttacaatg atatacactg tttgagatga ggataaaata ctctgagccc aaaccgggcc cctctgccaa ccatgttcat gccttcttct ttttcctaca gctcctgggc aacgtgctgg ttgttgtgct gtctcatcat tttggcaaag aattcactcc tcaggtgcag gccgcctatc agaaggtggt ggctggtgcg gccaacgccc tggctcacaa ataccaccga GATC

6420

6480

6540

6600

6660

6720

6780

6840

6900

6960

7020

7080

7140

7200

7260

7320

7380

7440

7500

7560

7620

7664 <210> 14 <211> 20 <212> DNA <213> Artificial Seguence

<223> primer corresponding to tTA segence <400> 14 cgagggcctg ctcgatctcc <210> 15 <211> 20 <212> DNA <213> Artificial Seguence <220>

<223> primer corresponding to 3 'untranslated region of tTA segence <400> 15 ggcattccac cactgctccc

<210> 16 <211> 21 <212> DNA <213> Homo sapiens <400> 16

gagcaccctc ctcatgacct c <21o> 17 <211> 22 <212> DNA <213> Escherichia coli

• <400> 17 gttggtgtag atgggcgcat cg

<210> 18 <211> 21 <212> DNA <213> Escherichia coli <400> 18

gcggggtctc aggttacagc c <210> 19 <211> 22 <212> DNA <213> Artificial Sequence <220>

<223> PCR primer corresponding to tet activator <400> 19 cgcccagaag ctaggtgtag ag

<210> 20 May <211> 20 May <212> DNA <213> Artificial Sequence <220> <223> PCR primer for tet <400> 20 May

activator cggccatatc cagagcgccg

<210> 21 <211> 23 <212> DNA <213> Homo sapiens <400> 21

gccctctggc ctgctggctc atg

<210> 22 <211> 24 <212> DNA <213> Homo sapiens <400> 22nd

caggagagtc ttgcctgtat cctc

<210> 23 <211> 25 <212> DNA <213> Mus muscalis <400> 23

aggagggagc tgacagatac actcc

Claims (27)

PATENT CLAIMS A transgenic non-human mammal or its progeny with somatic and germ cells comprising in a stable integrated form a recombinant gene encoding a polypeptide, comprising an enzymatically active, matrix degrading enzyme, wherein said recombinant gene is selectively expressed in chondryocytes of said mammal and said expression causes pathological symptoms characteristic of degenerative cartilage disease 2. A transgenic animal or its progeny with somatic and germ cells comprising a stably integrated first recombinant gene encoding a polypeptide selected from the group consisting of MMP-1, MMP-2, MMP-3, MMP-7, MMP-8, MMP- 9, MMP-10, MMP-13 MMP-14, MMP-15, MMP-16, MMP-17 and enzymatically active variants thereof. The transgenic animal as defined in claim 2, wherein said first recombinant gene is selectively expressed in synovial chondriocytes of said animal. The transgenic animal defined in claim recombinant gene encodes MMP-13. 2, wherein said first 5. The transgenic animal is human. as defined in the claim 4, wherein said MMP-13 The transgenic animal as defined in claim 4, wherein said first recombinant gene encodes a variant of the MMP-13 polypeptide comprising the enzymatically active proMMP-13. The transgenic animal as defined in claim 6, wherein the recombinant gene comprises a sequence encoding MMP-13 given in SEQ. ID NO: 1. * 1 ·· · · · · · · · · · · · · · · · · · · · · · ·. • · »·« «« «« «« «« «« The transgenic animal as defined in claim 2, wherein said transgenic animal is selected from the group consisting of mouse, rat, rabbit, sheep, cow, goat and pig. The transgenic animal as defined in claim 8, wherein said animal is a mouse. animal The transgenic animal as defined in claim 2, wherein the expression of said recombinant gene is under the control of a first regulatable promoter. The transgenic animal as defined in claim 10, wherein said first regulatable promoter comprises tet07. The transgenic animal as defined in claim 11, wherein said promoter has the sequence shown in SEQ ID NO: 2. The transgenic animal as defined in claim 10, further comprising a second recombinant gene encoding a polypeptide that regulates said first promoter. The transgenic animal as defined in claim 12, wherein the expression of said second recombinant gene is under the control of a second controllable promoter. The transgenic animal as defined in claim 13, wherein said second controllable promoter comprises sequences derived from a type II collagen promoter that causes the selective expression of said second recombinant gene in the joint tissue. 16. Transgenic mouse or its progeny with somatic and germ cells, containing in a stably integrated form: (i) a first recombinant gene comprising a sequence encoding a different MMP-13 polypeptide comprising MMP-13 *, wherein said sequence is operably linked to the tetO7 promoter; and (ii) a second recombinant gene encoding a tTA protein operably linked to a sequence derived from the type II collagen promoter. The transgenic mouse as defined in claim 16, wherein expression of said recombinant genes in the joint tissue causes pathological symptoms characteristic of degenerative joint degenerative diseases. An isolated nucleic acid encoding an enzymatically active proMPM-13, wherein said nucleic acid has a sequence selected from the group consisting of the sequence described in SEQ ID NO: 1, its sequence-conserved mutants and functionally-conserved mutants. A recombinant cloning vector comprising the nucleic acid defined in claim 18. A host cell comprising the vector as defined in claim 19. A method for producing a polypeptide comprising culturing a cell as defined in claim 20 under conditions suitable for expression of said enzymatically active proMMP-13. A method for inducing phenotypic changes associated with degenerative cartilage disease in a mammal, comprising maintaining the mammal as defined in claim 1 under conditions wherein said recombinant gene is selectively expressed in the joint tissue of said mammal. A method for inducing phenotypic changes associated with degenerative cartilage disease in a mammal, comprising maintaining the mammal as defined in claim 2 under conditions wherein said recombinant gene is selectively expressed in the joint tissue of said mammal. A method for inducing phenotypic changes associated with degenerative cartilage disease in mice, including maintenance The mice defined in claim 16 for a predetermined period without tetracycline or biologically active analogs thereof. 25. A method for determining the ability of a substance to counteract degenerative cartilage disease, said method comprising: (i) administering a known dose of the agent to the transgenic animal as defined in claim 1 under conditions where phenotypic indicators associated with degenerative cartilage disease manifest; (ii) monitoring the development of phentotypic indicators of cartilage degenerative disease for a predetermined time after drug administration; and (iii) comparing the range of phenotypic indicators in the treated animal relative to the control non-administered transgenic animal, wherein any difference in the nature or extent of the phenotypic indicators or any difference in time necessary for the development of the phenotypic indicators indicates the possibility that the substance acts against degenerative cartilage disease. 26. A method for determining the ability of a substance to counteract degenerative cartilage disease, said method comprising: (i) maintaining the transgenic animal as defined in claim 16 for a predetermined period of time without tetracycline or a tetracycline analogue, wherein said maintaining causes one or more phenotypic indicators of cartilage degenerative disease to appear in said animal; (ii) administering to the animal a known dose of said agent; (iii) monitoring the development of said phenotypic indicators for a predetermined period of time after administration of the substance; and (iii) comparing the range of phenotypic indicators in the animal treated with the substance compared to the control transgenic animal not exposed to the substance, where any difference in the nature or range of phenotypic indicators indicates the ability of the substance to counteract degenerative cartilage disease. 5 ^ · «* ··· A method for determining the ability of a substance to counteract degenerative cartilage disease, the method comprising: (i) providing a transgenic animal as defined in claim 16, which is maintained in the presence of a tetracycline or tetracycline analogue to suppress the expression of said first recombinant gene; (ii) substantially co-administering (a) administering a known dose of said agent to said animal and (b) stopping said tetracycline; (iii) monitoring the development of phenotypic indicators of degenerative cartilage disease for a predetermined time after administration of the agent; and (iv) a comparison of the range of phenotypic indicators in the animal treated with the substance compared to the control transgenic animal not exposed to the substance, where any difference in the nature or range of the indicators, or any difference in time necessary for the development of the indicators ability of the substance to counteract degenerative cartilage disease.