MXPA06008698A - Genes associated with canine osteoarthritis and related methods and compositions - Google Patents

Abstract

Described herein is a combination containing polynucleotide molecules that are differentially expressed in osteoarthritis. Also described are methods that may be used for diagnosis and prognosis of osteoarthritis, as well as methods that may be used to screen test substances for effectiveness in treatment modalities for osteoarthritis. Also described are devices and kits that may be used with the described methods.

Description

GENES ASSOCIATED WITH CANINE OSTEOARTHRITIS AND RELATED METHODS AND COMPOSITIONS FIELD OF THE INVENTION This invention relates to the field of degenerative diseases of the joints, such as osteoarthritis. More particularly, the invention relates to novel compositions, devices and methods based on unique profiles of expression of genes associated with osteoarthritis.

BACKGROUND OF THE INVENTION Osteoarthritis (OA), also commonly referred to as degenerative joint disease, is recognized in humans and in all veterinary species (Richardson et al., (1997) Vet. Clin. North Am. 27: 883 -911). OA is a prevalent and debilitating disease in dogs and is often associated with hip dysplasia (Martinez, S. (1997) Osteoarthritis, Vet Clinics of N. Am .: Small Animal Practice 27 (4): 735-758 There is a high degree of similarity between human and human osteoarthritis, which makes it an excellent model in animals for the study of human osteoarthritis, although the causal factors still remain mostly unknown, the disease is characterized by an imbalance in the cartilage matrix degradation that surpasses cartilage matrix synthesis Chondrocyte apoptosis and inflammation may also be related to the disease (Pelletier, J., et al. (2001) Arthri tis &Rheumatism 44 (6) : 1237-1247; Lotz, M. (1999) Osteoarthri tis and Cartilage 7: 389-391.) The disease is typically slow progressing and is characterized by degeneration of the articular cartilage with a loss of both proteoglycan and of collagen and by proliferation of new bone. In addition, an inflammatory response can occur within the synovial membrane. Canine osteoarthritis can arise as a secondary condition that results, in particular, from displeasure of the hip or from desiccant osteochondritis (Martínez, supra). The acquired conditions involve traumatic events that can also lead to osteoarthritis in the dog (Martinez et al., Vet Clin North Am, 27: 759-775, 1997). Treatment modalities for osteoarthritis may include the administration of inflammatory medications as well as the manipulation of fatty acids in the diet (Richardson et al., Supra). The diagnosis of canine osteoarthritis is typically based on symptomatology. Dogs with osteoarthritis show lameness which may have a gradual onset but which can be delegated acutely after exercise. Lameness is exacerbated by rest but decreases after a few minutes of activity. Cold dampened conditions, obesity and prolonged exercise often worsen the signs of lameness (Bederson et al, in Textbook of Veterinary Internal Medicine, 5th Ed., Ettinger et al., Ed., WB Saunders and Co., Philadelphia, 2000, pp. 1862-1886). 'With the emergence of genomic sciences, it has become evident that not only is the regulation of gene expression intimately involved in normal homeostasis, alterations in the differential expression of genes as an aspect in the development of diseases. As a result, the evaluation of gene expression patterns in diseases has increasingly been recognized as important for the understanding of disease processes at the molecular level. (Going et al., European J. Cancer 35: 1895-1904, 1999; Wang et al., Cardiovasc. Res. 35: 414-421). Numerous approaches have emerged to study the comparative expression of genes and current exercise has been found in high throughput analysis methods (for review see Carulli et al, J. Cell. Biochem. Suppl 30/32: 286-296, 1998; Kozian et al., Trends Biotechnol 17: 73-78, 1999). Recent methods developed for high throughput analysis of differential gene expression include, for example, - sequenced EST (Adams et al., Science 252: 1651-1656, 1991; Adams et al., Nature 377: 3-16, 1995), microarray hybridization (Schena et al., Science 270: 467-470, 1995), and differential presentation (Liang et al., Science 257: 967-970, 1992; Welsh et al., Nucleic Acids Res. 20: 4965-4970, 1992). Gene expression in osteoarthritis and particularly in canine osteoarthritis has not yet been studied in depth. Accordingly, there is a need to identify nucleic acid sequences and their encoded proteins that are differentially expressed in osteoarthritis. This information can be useful for the diagnosis of a state of osteoarthritic disease or predisposition to the disease, in a subject, as well as the identification of substances useful in the treatment or prevention of osteoarthritis.

BRIEF DESCRIPTION OF THE INVENTION According to one aspect of the present invention, numerous polynucleotides comprising at least one fragment of a gene have been identified as those that are differentially expressed in osteoarthritic or pre-osteoarthritic subjects as compared to the expression in subjects which are not osteoarthritic or pre-osteoarthritic.

Agree . With an aspect of the present invention, genes that are differentially expressed, fragments of genes and products of genes encoded as well as the expression patterns associated with the group of genes are advantageously used in numerous methods for the. detection of changes in the expression of a gene associated with osteoarthritis, particularly canine osteoarthritis. Additional aspects of the invention relate to methods for the identification of agents useful in the treatment or prevention of osteoarthritis. In accordance with further aspects of the present invention, the compositions, devices and test equipment are provided to facilitate the practice of methods provided in accordance with some embodiments of the invention. Other features and advantages of the present invention will be understood with reference to the detailed description and the examples that follow.

BRIEF DESCRIPTION OF THE FIGURES Figures la-Ib show representative gels used in differential presentation analysis of canine osteoarthritis. The figure shows the differential presentation of osteoarthritic versus normal transcripts loaded in duplicate before the band cut (D = osteoarthritic (patients), N = normal). Figure lb, the same gel after the band cut. Figures 2a-2e show quantitative PCR analysis (cCRP) for transcripts associated with selected AO in canine cartilage. The expression of RNA in arbitrary units is shown (OA AVG = average expression of osteoarthritic cartilage, C AVG = average expression in normal control).

TABLE 1: Relation of gene ID numbers with sequence ID numbers. - - - - 7 - DETAILED DESCRIPTION OF THE ILLUSTRATIVE MODALITIES Definitions The following definitions are provided to facilitate understanding of the present invention: The terms "nucleic acid" or a "nucleic acid molecule", as used herein, refer to any DNA or RNA molecule, whether of single or double chain and, if it is single chain, the molecule of its complementary sequence is in linear or circular form. By presenting nucleic acid molecules, a sequence or structure of a particular nucleic acid molecule can be described herein in accordance with standard conventions to provide the sequence in the 5 'to 3' direction. With reference to nucleic acids according to aspects of the invention, the term "isolated nucleic acid" is sometimes used. This term, when applied to DNA, refers to a DNA molecule that is separated from sequences which are immediately contiguous in the genome as found in the nature of the organism in which it originates. For example, an "isolated nucleic acid" may comprise a DNA molecule inserted into a vector, such as a plasmid or viral vector, or it may be integrated into the genomic DNA of a prokaryotic or eukaryotic cell or into a host organism. When applied to RNA, the term "isolated nucleic acid" refers primarily to an RNA molecule encoded by an isolated molecule of AD? as defined in the above. Alternatively, the term can refer to a molecule of AD? that it has separated sufficiently from other nucleic acids with which it would associate in its natural state (ie, in cells or tissues). An isolated nucleic acid (either ADα or RNA) can be further represented as a molecule produced directly by biological or synthetic means separated from other components present during its production. The terms "percentage of similarity", "percentage of identity" and "percentage of homology", when referring to a particular sequence are used as set forth in the University of Wisconsin GCG software program. - The terms "polynucleotides", "polynucleotide molecule" or "polynucleotide sequence" refers to a chain of nucleotides. It can refer to a DNA or RNA molecule, either single or double chain and, if it is single chain, the molecule of its complementary sequence can be in linear or circular form. Preferably, the chain has from about 50 to 10,000 nucleotides, more preferably from about 150 to 3,500 nucleotides. In some cases, the sequences will be completely complementary (without bad matches) when aligned. In other cases, there may be up to about 30% of bad pairings in the sequences. The term "oligonucleotides", as used herein, refers to sequences, primers and probes of the present invention and is defined as a nucleic acid molecule consisting of two or more ribonucleotides or deoxyribonucleotides, preferably more than 3. The size The exact extent of the olignucleotide will depend on various factors and in particular on the application and use of the oligonucleotide. The term "fragment" refers to a nucleic acid sequence that is preferably of a length of at least about 10 nucleic acids, more preferably of a length of about 40 nucleic acids, more preferably about 100 nucleic acids and encompassing, for example, fragments consisting of nucleic acids 1-100, 300-400, 500-600, 800-900 of NUMBER IDENTIFICATION SEQUENCES: 1-1558 or fragments of similar length at the 3 'end of NUMBER IDENTIFICATION SEQUENCES: 1-1558. A "fragment" can also mean a stretch of at least about 100 consecutive nucleotides containing one or more deletions, insertions or substitutions. A "fragment" can also mean a complete coding sequence of a gene and can include 5 'and 3' untranslated regions. A "fragment" can also refer to polypeptide sequences which preferably are at least about 5 to about 15 amino acids in length, more preferably at least about 10 amino acids in length and which retain some biological activity or immunological activity of a sequence. The term "gene" or "genes" refers to the partial or complete coding sequence of a gene. The term also refers to 5 'or 3' untranslated regions of a transcript. The phrase "gene that is expressed differentially in osteoarthritis" refers to a gene whose amount of mRNA, expressed from said gene, or the amount of gene product translated from mRNA is detectably different, ie, already be greater or lesser, in cells of subjects having osteoarthritis or pre-osteoarthritic subjects compared to the amount of mRNA of a translated gene product in cells of normal subjects which are not osteoarthritic or pre-osteoarthritic. As used herein, the terms "pre-osteoarthritis" or "pre-osteoarthritis" are meant to mean the subject is predisposed to develop osteoarthritis at a later date, but may have no obvious signs or symptoms of osteoarthritis. Preferably, the abundance of transcription or translation products of a gene that is differentially expressed derived from an osteoarthritic or pre-osteoarthritic sample differs by at least about 1.5 times, more preferably at least about 1.2 times, so more preferably at least about 1.3 times and much more preferably at least about 1.4 times, more preferably at least about - 1. 5 times, more preferably at least about 1.6 times and much more preferably at least about 1.75 times, more preferably at least about 2 times, more preferably at least about 3 times, so more preferably at least about 10 times, more preferably at least about 20 times compared to a normal sample. The phrase "gene that is differentially expressed in osteoarthritis" also refers to genes that are not detectable in a normal transcript profile but that are preferably in concentrations of at least about 2 copies per cell, most preferably at less about 3 copies per cell in the osteoarthritic or pre-osteoarthritic transcript profile. The terms "related to osteoarthritis (OA)" and "genes associated with osteoarthritis (OA)" refer to genes that are differentially expressed in osteoarthritis, as defined herein. As used herein, the terms "indicator", "reporter system", "reporter gene" or "reporter gene product" refers to an operational genetic system in which a nucleic acid comprises a gene encoding a product which, when expressed, produces an indicator signal that is easily measurable, for example by biological assay, immunoassay, radioimmunoassay or by colorimetric, fluorogenic, chemiluminescent or other method. The nucleic acid can be RNA or DNA, linear or circular, single- or double-stranded, of antisense or direct polarity and can be operatively linked to the control elements necessary for the expression of the reporter product product. The required control elements will vary according to the nature of the indicator system and whether the reporter gene is in the form of DNA or RNA, but may include, but is not limited to such elements as promoters, enhancers, translational control sequences, signaling polyA addition, transcriptional termination signals and the like. The terms "transform", "transfect" and "transduce" refer to any method or means by which a nucleic acid is introduced into a cell host organism and which can be used interchangeably to infer the same meaning. Such methods include but are not limited to transfection, electroporation, microinjection, PEG fusion and the like. The term "functional" as used herein, implies that the nucleic or amino acid sequence is functional for the assay or purpose mentioned. The phrase "consisting essentially of", when referring to a particular nucleotide or amino acid means a sequence having the properties of a NO IDENTIFICATION SEQUENCE. Dadaist. For example, when used with reference to an amino acid sequence, the phrase includes the sequence by itself and molecular modifications that do not affect the basic and novel characteristics of the sequence. A "vector" is a replicon such as a plasmid, cosmid, bacmid, phage, artificial chromosome (BAC, YAC) or virus in which another genetic sequence or element (either DNA or RNA) can be inserted in a manner that carries out the replication of the sequence or attached element. A "replicon" is any genetic element, for example, a plasmid, cosmid, bacmid, phage, artificial chromosome (BAC, YAC) or virus that is capable of replication primarily under its own control. A replicon can be RNA or AD? and can be single or double chain. The term "probe", as used herein, refers to either a probe for nucleic acid or a probe for a protein. When used in connection with nucleic acids, a "probe" refers to an oligonucleotide, polynucleotide or nucleic acid, either RNA or DNA, as found in nature or in a restriction enzyme digestion or purified in a manner synthetic, which is capable of annealing (annealing) or which hybridizes specifically with a nucleic acid with sequences complementary to the probe. A probe can be single or double chain. The exact length of the probe will depend on many factors including temperature, probe source and method of use. For example, for diagnostic applications, depending on the complexity of the target sequence, an oligonucleotide probe will typically contain about 10-100, preferably about 15-50, and more preferably about 15-25 nucleotides. In some diagnostic applications, a polynucleotide probe preferably contains about 90-1150 nucleotides, more preferably about 300-600 nucleotides, more preferably about 300 nucleotides. The probes herein are selected to be "substantially" complementary to different chains of a particular target nucleic acid sequence. This means that the probes must be sufficiently complementary so that they are capable of "specifically hybridizing" or reassociating with their respective target chains under a set of predetermined conditions. Therefore, the sequence of the probe does not need to reflect the exact complementary sequence of the target. For example, a non-complementary nucleotide fragment can be attached to the 5 'or 3' end of the probe, and the remainder of the probe sequence is complementary to the target chain. Alternatively, non-complementary bases or longer sequences may be interspersed in the probe, provided that the probe sequence has sufficient complementarity with the target nucleic acid sequence to specifically anneal with it. When used in relation to a protein, a "probe" is a substance that binds protein capable of specifically binding to a particular protein or protein fragment with the substantial exclusion of other proteins or protein fragments. Such binding substances can be any molecule to which the protein or peptide specifically binds, which include DNA (for proteins that bind to DNA), antibodies (as described in more detail herein), cell membrane receptors, peptides, cofactors, lectins, sugars, polysaccharides, cells, cell membranes, organelles and organelle membranes. The term "array" refers to an ordered distribution of at least two probes on a substrate. At least one of the probes represents one - control or standard and the other, a probe of diagnostic interest. The distribution from about two to about 40,000 probes in a substrate ensures that the size and signal strength of each labeled complex that is formed between a probe and a nucleic acid sample or a protein binding substance is individually distinguishable. A "hybridization complex" is formed between nucleic acid molecules of a sample when the hydrogen bonds of the purines of a molecule with the pyrimidines of the complementary molecule, for example, the base pairs 5 '-AGTC-3' with 3 '-TCAG-5'. The degree of complementarity and the use of nucleotide analogues affects the efficiency and stringency of the hybridization reactions. The term "specifically hybridizes" refers to the association between two unique nucleic acid molecules of complementary sequence sufficient to allow such hybridization under predetermined conditions generally used in the art (sometimes referred to as "substantially complementary"). For example, the term can refer to the hybridization of a nucleic acid probe. with a substantially complementary sequence contained within a single-stranded DNA or RNA molecule, according to an aspect of the invention, with the substantial exclusion of hybridization of the nucleic acid probe with non-sequence single-stranded nucleic acids. complementary A "sample" is used in the broadest sense as that which contains nucleic acids, proteins, antibodies and the like. A sample may comprise, for example, a body fluid; the soluble fraction of a cell preparation or an aliquot of media in which the cells are grown; a chromosome, an 'organelle or a membrane isolated or extracted from a cell; Genomic DNA, RNA or cDNA in solution or attached to a substrate; a cell; a tissue or a tissue biopsy; a tissue print; a fingerprint, buccal, cutaneous or hairy cells; and similar. The term "standard" refers to a control sample comprising material from a source in a normal biological state (as opposed to that related to OA). A biological state related to OA may include, for example, one in which the source is OA, which is predisposed to develop OA or which has certain biological characteristics of OA. For example, a standard sample may comprise nucleic acids or proteins from a normal subject that is not osteoarthritic or pre-osteoarthritic. Standard samples can also - include samples from normal cells or tissue that has not yet been treated to induce an immune response that can model certain aspects of OA. The term "specific binding" refers to a special and precise interaction between two molecules that depend on their structure, particularly their molecular side groups. For example, the intercalation of a regulator-regulator in the major groove of a DNA molecule, the binding of hydrogen along the main structure between two single-stranded nucleic acids or the union between an epitope of a protein and an agonist , antagonist or antibody. The term "primer", as used herein, refers to a nucleic acid molecule, either RNA or DNA, either single chain or double chain, derived from a biological system, generated by digestion with enzymes of restriction or synthetically produced which, when placed in the proper environment, is capable of acting functionally as a template-dependent nucleic acid synthesis primer. When suitable nucleoside triphosphate precursors or nucleic acids, a polymerase enzyme, suitable cofactors and conditions such as appropriate temperature and pH are present in an appropriate nucleic acid template, the primer can be extended to its 3 'part by the addition of - - nucleotides by the action of a polymerase or similar activity to provide a primer extension product. The primer may vary in length depending on the particular conditions and the requirement of the application. For example, in diagnostic applications according to particular embodiments of the invention, a primer may be an oligonucleotide primer, preferably about 15-25 or more nucleotides long. The primer must have sufficient complementarity with the desired template to prime the synthesis of the desired extension product, ie, it must be capable of reassociating with the desired template chain in a manner sufficient to provide the 3 'hydroxyl portion of the primer in juxtaposition suitable for use at the start of synthesis by a polymerase or a similar enzyme. The sequence of the primer is not required to represent an exact complement of the desired template. For example, a nucleotide sequence can be attached that would not complement the 5 'end of a primer that is otherwise complementary. Alternatively, bases that are non-complementary can be interspersed within the oligonucleotide primer sequence with the proviso that the primer sequence has sufficient complementarity with the desired template chain sequence to functionally provide a template primer primer complex. for the synthesis of the extension product. The amino acid residues described herein are preferred to be in the "L" isomeric form. However, the residues in the isomeric form "D" can substitute for any amino acid residue L, with the proviso that the desired properties of the polypeptide are retained. All sequences of amino acid residues represented herein are adapted to the conventional orientation from left to right, from the terminal amino to the terminal carboxy. A "fragment" or a "portion" of a polypeptide means a stretch of amino acid residues of at least about 5 to 7 contiguous amino acids, often at least about 7 to 9 contiguous amino acids, typically at least about 9 to 13 amino acids contiguous and, more preferably, at least about 20 to 30 or more contiguous amino acids. Fragments of the polypeptide sequence, antigenic determinants or epitopes are useful for inducing immune responses to a portion of the amino acid sequence of a protein. The different "variants" of the polypeptides that are expressed differentially exist in nature. These variants may be alleles - characterized by differences in the nucleotide sequences of the gene encoding the protein or may involve different RNA processing or post-translational modifications. A person skilled in the art can produce variants that have single or multiple amino acid substitutions, deletions, additions or substitutions. These variants may include,. for example: (a) variants with one or more amino acid residues substituted with conservative or non-conservative amino acids, (b) variants in which one or more amino acids are added to the polypeptide, (c) variants in which one or more amino acids include a substituent group, and (d) variants in which the polypeptide is fused with another peptide or polypeptide such as a fusion partner, a protein tag or other chemical portion that can confer useful properties to the polypeptide such as, for example, an epitope for an antibody or a polyhistidine sequence, a portion of biotin and the like. Other polypeptides of the invention may include variants in which amino acid residues of one species are substituted by a corresponding residue of another species, either in conserved or non-conserved positions. In another embodiment, the amino acid residues in non-conserved positions are substituted with conservative or non-conservative residues.

- Techniques for obtaining these variants include genetics (deletions, deletions, mutations, etc.), chemical and enzymatic techniques that are known to a person skillfully skilled in the art. To the extent of such allelic variations, analogs, fragments, derivatives, mutants and modifications, including alternative forms of nucleic acid processing and alternative post-translational modification forms result in derivatives of a differentially expressed polypeptide that retains any of the biological properties of the differentially expressed polypeptide and are included within the scope of this invention. The term "isolated protein" or "isolated and purified protein" refers primarily to a protein produced by the expression of an isolated nucleic acid molecule according to an aspect of the invention. Alternatively, this term may refer to a protein that has been sufficiently separated from other proteins with which it would naturally associate so that it exists in a "substantially pure" form. "Isolated" does not mean to exclude artificial or synthetic mixtures with other compounds or materials, or the presence of impurities that do not interfere with the fundamental activity and that may be present, for example, due to incomplete purification, addition of stabilizers or elaboration of compounds, for example, in immunogenic preparations or pharmaceutically acceptable preparations. The term "substantially pure" refers to a preparation comprising at least about 50-60% by weight of a given material (eg, nucleic acid, protein, etc.). More preferably, the preparation comprises at least about 75% by weight and. much more preferably about 90-95% of the given compound. The purity is measured by methods suitable for the given material (for example chromatographic methods, agarose or polyacrylamide gel electrophoresis, CLAP analysis and the like). The term "tag", "tag sequence" or "protein tag" refers to a chemical moiety, whether nucleotide, oligonucleotide, polynucleotide or amino acid, a peptide or protein or other chemical substance, that when added to another sequence , provides additional utility or confers useful properties, particularly in the detection or isolation of said sequence. Thus, for example, a homopolymeric nucleic acid sequence or a nucleic acid sequence complementary to a retention oligonucleotide can be added to a probe primer or sequence to facilitate the subsequent isolation of an extension product or hybridized product. In the case of protein labels, histidine residues (for example 4 to 8 consecutive histidine residues) can be added to either the amino or carboxyterminal part of a protein to facilitate isolation of the protein by chelating metal chromatography. Alternatively, the amino acid, peptide, protein or fusion partner sequences that represent epitopes or binding determinants reactive with specific molecules of antibody or other molecules (e.g., reporter epitope, c myc epitope, transmembrane epitope or virus hemagglutinin protein) of influenza A, protein A, cellulose binding domain, calmodulin binding protein, maltose binding protein, chitin binding domain, glutathione S transferase and the like) can be added to proteins to facilitate the isolation of the protein by procedures such as affinity or immunoaffinity chromatography. The chemical label portions include molecules such as biotin which can be added to either nucleic acids or proteins and facilitates isolation or detection by interaction by avidin reagents and the like. Numerous other label portions are known and can be considered, by one skilled in the art as contemplated within the scope of this definition. An "antibody" or "antibody molecule" is any immunoglobulin, uding antibodies and fragments thereof, which bind to a specific antigen. The term udes polyclonal, monoclonal, chimeric and bispecific antibodies. As used herein, an antibody or antibody molecule contemplates both an intact immunoglobulin molecule and an immunologically active portion and an immunoglobulin molecule such as those portions known in the art as Fab, Fab ', F (ab') 2 and F (v). As used herein, the term "subject" or "patient" refers to both humans and animals, unless it is specified that the "subject" or "patient" is an animal or a human. The animal subjects are preferably vertebrates and more preferably mammals. The "therapeutic modality" refers to any means of treatment or prevention of a disease, condition or disorder. In one aspect of the present invention, numerous genes that are differentially expressed in osteoarthritic subjects have been identified in comparison with non-osteoarthritic subjects. These genes and gene fragments as well as their encoded proteins and fragments can be used, for example, in a variety of diagnostic and prognostic assays as well as assays useful in the screening of test substances to determine efficacy in treatment modalities for osteoarthritis. In some embodiments of the invention, the expression of at least one gene that is differentially expressed can be measured. In preferred embodiments, the expression of two or more genes that are differentially expressed can be measured by providing an expression pattern of "gene or gene expression profile." More preferably, the measurement of a multiplicity of genes which are differentially expressed can be carried out by providing additional information for a gene or profile expression pattern In various embodiments of the present invention, changes in gene expression can be measured in one or both of two ways: (1) by measuring transcription through the detection of mRNA produced by a particular gene, and (2) by measuring translation through the detection of protein produced by a particular transcript.Decreased or increased expression can be measured a-RNA level using any of the methods well known in the art for the quantification of polynucleotides such as, for example, PCR (including, without limitation, R T-PCR and PCRq), ribonuclease protection, Northern blotting and other hybridization methods. Genes that are examined or interrogated according to the present invention typically are in the form of mRNA or ARγm subjected to reverse transcription. The genes can be cloned or amplified. Cloning itself does not seem to divert the representation of genes within a population. However, it may be preferable to use polyA + AR? as a source, since it can be used, for minor processing stages. In accordance with aspects of the present invention, 1558 genes whose functions are closely related to osteoarthritis (OA) have been identified. The association is determined by comparing the expression of genes in normal tissue and tissue from subjects in whom OA has been diagnosed. The genes identified in this way fall into two broad categories. The first category comprises known genes, many of which up to now the OA association has not been appreciated. These genes are included in Table 6 together with their corresponding gene ID numbers and their identification sequence numbers. According to another aspect of the invention, a second category comprises nucleic acid segments that do not demonstrate homology with previously identified sequences. In this way, it is considered that this category includes one or more novel genes. A preferred embodiment of the invention relates to a nucleic acid-isolated molecule comprising a novel OA-associated gene, mRNA or cDNA produced from the gene associated with OA. One aspect of the present invention relates to a combination of 1558 polynucleotide molecules that are differentially expressed in an osteoarthritic subject or in a pre-osteoarthritic subject as compared to expression in subjects which are not osteoarthritic or pre-osteoarthritic. In one embodiment of the invention described herein, the segments of the 1558 related genes OA of canine cartilage are obtained by using differential presentation. The nucleotide sequences of these polynucleotides are set forth in NUMBER IDENTIFICATION SEQUENCES: 1-1558 (Table 1 shows the relationship between the NUMBER IDENTIFICATION SEQUENCE and the gene ID number). The BLAST analysis of these sequences identified homologies with a number of nucleic acid sequences previously identified (Table 2). These include a number of nucleic acid sequences previously identified with unidentified homologies for known genes. The BLAST analysis also identified sequences that show homology with previously identified genes; the information includes gene names as well as database access numbers for respective homologs of these - which are given in Tables 2A and 2B, Table 2A CaMax: ENSG00000122912 ENST00000265870 CARRIER PROTEIN 10 q21.3 1009c SEVERE DISEASE (GDC) (AUTOANTÍGENO DE GRAVE ILLNESS) (GDA) (HOMOLOGO DE PROTEÍNA CARRIERA DE SOLUTO MITOCONDRIAL). [Source: SWISSPROT; Acc: P16260] CaMax: 1011a CaMax: 1011a CaMax: 1013a CaMax: ENSG00000109775 ENST00000264689 q35.1 1015d CaMax: 1016b CaMax ENSG00000160961 ENST00000292530 PROTEIN 333 OF FINGER 19 P13.12 1019a OF ZINC. [Source: SWISSPROT; Acc: Q96 JL9] CaMax ENSG00000064989 ENST00000264152 PRECURSOR OF Q32.1 1020a TYPE 1 RECEPTOR OF THE PEPTIDE RELATED TO THE GENE OF CALCITON INA (RECEPTOR CGRP TIP0 1). [Source: WISSPROT; Ace: Q16 602] - - - - CaMax: ENSG00000064205 ENST00000190983 PRECURSOR OF l 20 q13.12 1366a PROTEIN SIMILAR TO FACTOR OF CONNECTIVE TISSUE GROWTH (CTGF-L) (WNT1 INDUCED SIGNALING PROTEIN 2) (WISP-2) (PROTEIN 58 RELATED TO THE FACTOR OF GROWTH OF CONNECTIVE TISSUE). [Source: SWISSPROT; Acc: 076 076] CaMax: ENSMUSG0000003 ENSMUST0000003 F3 1368a 0871 3159 CaMax: ENSG00000119326 ENST00000325551 CATENINA (PROTE1NA q31.3 1371a ENST00000325580 ASSOCIATED WITH CADERINE) a-SIMILAR 1; a-CATUUNA. [Source: RefSeq; Acc: NM_0037 98] CaMax: ENSG00000083771 ENST00000218546 M PHASE OF 13 q12.11 137b FOSFOPROTEIN 8 (FRAGMENT). [Source: SWISSPROT; Acc: Q99 549] CaMax: 1381a CaMax: 1381a - CaMax: ENSG00000067208 ENST00000263785 INTEGRATION SITE p22.1 1383a VIRAL ECOTRÓPICO 5; GEN 4S OF NEUROBLASTOMA STAGE. [Source: RefSeq: Acc: NM_0056 65] CaMax: ENSG00000099194 ENST00000266053 ACIL-COA SATURASE 10 q24.31 1384a (EC 1.14.19.1) (ESTEAROIL-COA DESATURASE) (FATTY ACID DESATURASE) (d (9) -DESATURASE). [Source: SWISSPROT; Acc-OOO 767] CaMax: 1391a CaMax: 1394b CaMax: ENSG00000111912 ENST00000229634 COACTIVADOR 7 DE q22.32 1397b ENST00000318575 NUCLEAR RECEIVER; PROTEIN OF 140 KDA ASSOCIATED TO ESTRÓGENO RECEPTOR. [Source: RefSeq; Acc: NM_1817 82] CaMax: 1399a CaMax: 13a CaMax: 1400a CaMax: ENSG00000109756 ENST00000264431 PDZ DOMAIN WHAT q32.1 1401c CONTAINS THE GUANINE NUCLEOTIDE EXCHANGE FACTOR (GEF) 1; RA (RAS / RAP1A- ASSOCIATING) -GEF; PDZ DOMAIN CONTAINING THE GUANINE NUCLEOTIDE EXCHANGE FACTOR (GEF) 1; RA (RAS / RAP1A- ASSOCIATING) -GEF; PDZ DOMAIN CONTAINING A GUANINE NUCLEOTIDE EXCHANGE FACTOR (GEF) 1. [Source: RefSeq; Acc: NM_014247] CaMax: 1403a CaMax: 1406a CaMax: ENSG00000166170 ENST00000299204 REGULATOR 5 CHAPERON 14 q32.32 1409b MOLECULAR OF THE BAG FAMILY (BAG-5). [Source: SWISSPROT; Acc: Q9 UL15] CaMax: ENSG00000058272 ENST00000261207 SUBUNIDAD 12A 12 q21.2 1411a ENST00000312727 PROTEIN REGULATOR (INHIBITOR) PHOSPHATASE 1; MYOSIN PHOSPHATASE; SUBUNITY 1 OBJECTIVE. [Source: RefSeq; Acc: NM_0024 80] CaMax: ENSG00000139688 ENST00000267164 FACTOR IIF START OF 13 q14.12 174a TRANSCRIPTION, SUBUNIDAD ß (TFIlF-ß) (FACTOR RAP30 OF START OF TRANSCRIPTION). [Source: SWISSPROT; Acc: P13 984] CaMax: 174a CaMax: 1750a CaMax: 1751a CaMax: 1755a CaMax: 1755a CaMax: ENSG00000104852 ENST00000221448 RIBONUCLEOPROTEIN OF 19 q13.33 1758a 70 KDA NUCLEAR SMALL U1 (U1 SNRNP 70 KDA) (SNRNP70) (U1-70K) . [Source: SWISSPROT; Acc: P08 621] CaMax: ENSG00000179454 ENST00000324772 BTB DOMAIN (POZ) WHICH 14 q21.1 1759b CONTAINS 5. [Source: RefSeq; Acc: NM_0176 58] CaMax: 1760c CaMax: 1760c CaMax: - - CaMax: ENSG00000134215 ENST00000280840 PROTEIN VAV-3. P13.3 1950a [Source: SWISSPROT; Acc: Q9 UKW4] CaMax: ENSG00000138386 ENST00000321041 NGFI-A UNION PROTEIN 1 q32.2 1953a (EGR-1 UNION PROTEIN 1) (PROTEIN P54 TRANSCRIPTIONAL REGULATOR). [Source: SWISSPROT; Acc: Q13 506] CaMax: 1954e CaMax: ENSG00000157077 ENST00000287722 INTERACTION PROTEIN p32.3 1961T ENST00000287727 HOMOLOGA DECAPENTAPLÉGICA AGAINST MOTHERS (MADH INTERACTION PROTEIN) (SMAD ANCHOR FOR ACTIVATION OF - RECEIVER) (RECEIVER ACTIVATION ANCHOR) (HSARA) ( SERINE PROTEASE NOVEDOSE) (NSP). [Source: SWISSPROT; Acc: 095 405] CaMax: ENSG00000080469 ENST00000328494 CONVEYOR 2 FROM p21.32 1967th ENST00000190846 ANTIGEN PEPTIDE (APT2) (CONVEYOR TAP2 PEPTIDE) (CONVEYOR PSF2 PEPTIDE) (FACTOR 2 PEPTIDE SUPPLY) (PSF-2) (PEPTIDE) CARRIER INVOLVED IN ANTIGEN PROCESSING 2 IN) [Source: SWISSPROT; Acc: Q03 519] CaMax: ENSG00000138063 ENST00000260632 PROTEIN PELUNO. p14 1968a [Source: RefSeq; Acc: NM_0206 51] - - - CaMax: ENSG00000117868 ENST00000251527 q36.3 2222b CaMax: 2223a CaMax: ENSG00000116690 ENST00000251819 PROTEOGLYCAN 4; FACTOR q31.1 2224a MEGACARIOCITO STIMULATOR; PROTEOGLYCAN 4 (PROTEIN OF THE SURFACE AREA) ARTICULATED IN FACTOR STIMULANT OF MEGACARIOCITO); SYNDROME OF CAMPTODACTILIA-ARTOPATÍA- PERICARDITIS DE JACOBS; CAMPTODATILIA, ARTROPATÍA, COXA VARA, PERICARDITIS SYNDROME [source: RefSeq; acc: NM_005807] CaMax: ENSG00000151544 ENST00000281234 10 Q25.3 2225b CaMax: ENSMUSG0000003 ENSMUST0000003 HORMONE RECEPTOR 15 D1 2234a 8760 8856 THERROTROPIN RELEASER (TRH-R) (TIROLIBERINE RECEIVER). [source: SWISSPROT; Acc: P21 761] CaMax: 2235a CaMax: 2235a CaMax: ENSG00000172572 ENST00000325802 PHOSPHODIESTERASE AT 3 ', 5'-C'CLICA 12 p12.2 2238a INHIBITED BY CGMP (EC 3.1.4.17) (FOSFODIESTERESA TO INHIBITED BY CYCLIC GMP) (CGI -PDE A), [source: SWISSPROT; Acc: Q14432] CaMax: ENSG00000167996 ENST00000301775 HEAVY CHAIN OF FERRITINE 11 q12.3 106a (SUB-UNIT H OF FERRITINA) [source: SWISSPROT; Acc: P02794] CaMax: ENSG00000167996 ENST00000301775 HEAVY STRENGTH OF FERRITINE 11 q12.3 106a (Sub-unit H of FERRITINA) [source: SWISSPROT; Acc: P02794] CaMax: 360a CaMax: 360a CaMax: ENSG00000182944 ENST00000331029 EWS PROTEIN JOINING RNA 22 q12.2 364a ENST00000329871 (ONCOGEN EWS) (EWING SARCOMA RUPTURE REGION 1 PROTEIN) [source: SWISSPROT; Acc: Q01844] CaMax: 370a CaMax: ENSG00000136938 ENST00000277182 MEMBER B OF THE FAMILY OF q22.33 374a FOSFOPROTEIN 32 NUCLEAR RICH IN ACID LEUCINE (PROTEIN PHAPI2) (PROTEIN SSP29 DYEED WITH SILVER) (ACID PROTEIN RICA IN LEUCINE) [source: SWISSPROT; Acc: Q92688] CaMax: 375d CaMax: 379a CaMax: ENSG00000145216 ENST00000306932 FIP1-SIMILAR1; REARRECOGNIZED IN q12 38a ENST00000273816 HIPEROSINOFILIA [source: RefSeq; Acc: NM_030917] CaMax: - - - - - - - - - - Table 2B - - - - - - - - - - - - - - One embodiment of the invention relates to a combination comprising two or more polynucleotide molecules that are selected from NUMBER IDENTIFICATION SEQUENCES 1558 or fragments thereof. Preferably, the combination comprises about 10 or more polynucleotide molecules, more preferably about 50 or more polynucleotide molecules, more preferably about 200 or more polynucleotide molecules, more preferably about 400 or more polynucleotide molecules, and much more preferably about 1000 or more polynucleotide molecules. In a preferred embodiment, the invention relates to a combination of 396 differentially expressed polynucleotide molecules whose sequences are represented by NUMBER IDENTIFICATION SEQUENCES: 1-396. Table 3 identifies a list of gene sequences determined from clinical samples that are to be differentially expressed in OA versus normal subjects to such a degree that it is statistically significant (p <0.05). Table 3 includes the gene IDs, expression values, standard deviations and multiples of expression difference (OA versus normal). Preferably, the combination comprises two or more of the polynucleotide molecules selected from the NUMBER IDENTIFICATION SEQUENCES: 1-396 or fragments of the same. In a particularly preferred embodiment, the invention relates to a combination of 217 polynucleotide molecules that are differentially expressed, whose sequences are represented by SEQUENCES. IDENTIFICATION NUMBERS: 1-217. Table 4 identifies a sequence listing determined from clinical samples that are to be differentially expressed in OA versus normal subjects to such a degree that it is highly significant (p <; 0.01). Table 4 includes the gene IDs, expression values, standard deviations and multiples of expression difference (OA versus normal). Preferably, the combination comprises two or more of the polynucleotide molecules selected from the NUMBER IDENTIFICATION SEQUENCES: 1-217 or fragments thereof. In accordance with one aspect of the invention, one or more oligonucleotide or polynucleotide probes for interrogating a sample can be prepared using the sequence information set forth herein for any of the 1558 fragments of. isolated genes (SEQUENCES OF IDENTIFICATION NUMBERS: 1-1558). According to another aspect of the invention, probes can be prepared using the sequence information available for any of the genes or gene fragments identified therein. The probes must be of sufficient length to specifically hybridize substantially exclusively with appropriate complementary genes or transcripts. Preferably, the oligonucleotide probes will be at least about 10, 12, 14, 16, 18, 20 or 25 nucleotides in length. In some embodiments, larger probes of at least about 30, 40, 50, 60, 70, 80, 90 or 100 nucleotides are desirable and in some embodiments larger probes of about 100 nucleotides may be suitable. Preferably, a connection of two or more nucleic acid probes is provided to detect the expression of gene products that are differentially expressed in OA, more preferably a collection of approximately 10 or more probes, more preferably a collection of approximately 50 or more probes more preferably a collection of approximately 200 or more probes, more preferably a collection of approximately 400 or more probes, more preferably a collection of approximately 1000 or more probes. In a preferred embodiment of the invention, one or more oligonucleotide or polynucleotide probes can be prepared using the sequence information which. is established in any of the NUMBER IDENTIFICATION SEQUENCES: 1-396. Preferably, one or more oligonucleotide or polynucleotide probes can be prepared using the sequence information that is established for any of the NUMBER IDENTIFICATION SEQUENCES: 1-217. In some preferred embodiments of the present invention, immobilized nucleic acid probes can be used for the rapid and specific detection of nucleic acid molecules and their expression patterns. Typically, a nucleic acid probe is linked to a solid support and a target nucleic acid (eg, a genomic nucleic acid, an amplicon or, more commonly, an amplified mixture) that is hybridized to the probe. Either the probe, the target or both can be labeled, typically with a fluorophore or other label, such as streptavidin. When the label is labeled, hybridization can be detected upon detection of a fluorescent linkage. When the probe is labeled, hybridization is typically detected by extinguishing the label. When the probe and label are labeled, hybridization detection is typically performed by monitoring a color shift resulting from the proximity of the two labels attached. A variety of labeling strategies, brands and the like are known in the art, particularly for fluorescence based application. Another aspect of the invention relates to one or more probes comprising polypeptide binding agents that specifically bind to polypeptides produced by expression of one or more nucleic acid molecules comprising sequences that are selected from the SEQUENCE OF IDENTIFICATION NUMBERS: 1- 1558 or fragments thereof. According to another aspect of the invention, protein binding probes can be prepared using the sequence information available for any of the genes or gene fragments identified in Table 2. Preferably, a collection of two or more polypeptide probes for detecting the expression of gene products that are differentially expressed in OA is provided, more specifically a collection of approximately 10 or more probes, more preferably a collection of approximately 50. or more probes, more preferably a collection of approximately 200 or more probes, more preferably a collection of approximately 400 or more probes, and most preferably a collection of approximately 1000 or more probes. In a preferred embodiment of the invention, probes comprising polypeptide binding agents linked especially to polypeptides produced by nucleic acid molecules comprising sequences that are selected from SEQUENCES OF IDENTIFICATION NUMBERS: 1-396. In a particularly preferred embodiment, the probes comprise polypeptide binding agents specifically linked to polypeptides produced by nucleic acid molecule comprising the sequences that are selected from NUMBER IDENTIFICATION SEQUENCES: 1-217. The technical assays that can be used to determine the levels of a protein in a sample are also known to those skilled in the art. Such assay methods include radioimmunoassays, competitive binding assays, Western blot analysis and ELISA assays. In. Test methods using antibodies, polyclonal and monoclonal antibodies are suitable for use in the present invention. Such antibodies can be immunologically specific for a particular protein, or an epitope of the protein, or a fragment. of protein, as will be understood by those skilled in the art. Methods for producing polyclonal and monoclonal antibodies immunologically specific for a protein or peptide are also known in the art. Preferred embodiments of the present invention may use antibodies for the detection and quantification of proteins produced by expression of the polynucleotides described herein. Although the proteins can be detected by immunoprecipitation, affinity separation, Western blot analysis and the like, a preferred method uses the ELISA-type methodology, wherein the antibody is immobilized on a solid support and a target protein or peptide is exposed to the antibody immobilized. Either the probe, the target or both can be marked. A variety of marking strategies, brands and the like are known in the art. In particularly preferred embodiments of the invention, expression patterns or profiles of a plurality of genes that are differentially expressed in osteoarthritis are observed using arrays of probes to detect nucleic acids or target proteins. In one embodiment, arrays of oligonucleotide or polynucleotide probes can be used, whereas in another embodiment, arrays of antibodies or other proteins that specifically bind to gene products that are differentially expressed can be used. Such arrays are commercially available (for example through Affymetrix, Inc., Applied Biosystems, Inc., Agilent, Inc.), or can be made by adapting them, according to known methods, such as, for example, on-site synthesis. on a solid support or union of presintetized probes to a solid support via microprinting techniques. In preferred embodiments nucleic acid arrays or protein binding probes are made adapted to specifically detect transcripts or proteins produced by two or more of the 1558 genes that are differentially expressed or fragments of genes described herein. In one embodiment of the invention, arrays of nucleic acid or protein binding probes are made adapted to specifically detect transcripts or proteins produced by two or more of the genes or gene fragments identified in Table 2. In a preferred embodiment, arrays of nucleic acid or protein binding probes can be made adapted to specifically detect transcripts or proteins produced by two or more of the 396 genes or fragments of genes that are differentially expressed identified in Table 3. In a Preferred embodiment, nucleic acid arrays or protein binding probes are made adapted to specifically detect transcripts or proteins produced by two or more of the 217 genes that are differentially expressed or fragments of genes identified in Table 4. Preferably a collection of two or more nucleic acid or polypeptide probes to detect the expression of gene products that are differentially expressed in OA are immobilized on a support at separate sites, more preferably a collection of approximately 10 or more probes, most preferably a collection of approximately 50 or more probes, more preferably A collection of approximately 200 or more probes is most preferable, most preferably a collection of approximately 400 or more probes, and much more preferably a collection of approximately 1000 or more probes.

Since chondrocytes represent the cellular component of cartilage, a tissue affected by OA, the construction of an arrangement of chondrocytes may represent a useful tool to study the gene expression profile of osteoarthritic chondrocytes. The use of the differential presentation for selection of transcripts is used by the present invention to enrich the clones represented in an array for transcripts associated with OA. In one aspect of the invention, methods are provided for determining nucleic acids associated with OA in a sample. Preferably, a combination comprising one or more polynucleotide molecules that are selected from the NUMBER IDENTIFICATION SEQUENCES: 1-1558, more preferably they are selected from the NUMBER IDENTIFICATION SEQUENCES: 1-396, more preferably they are selected of the IDENTIFICATION SEQUENCES NUMBERS: 1-217 are used to prepare probes that hybridize with nucleic acids from a test sample, forming hybridization complexes that are detected and compared with those of a standard, so that the differences between the sample and standard hybridization complexes are indicative of the differential expression of nucleic acids in the sample. In a preferred embodiment, the nucleic acid probes are made to specifically detect transcripts or fragments thereof by one or more of the genes or gene fragments identified in Table 2. In some preferred embodiments, the nucleic acids in the sample are they can amplify before hybridization. In another aspect of the invention methods are provided for testing polypeptides associated with OA in a sample. Preferably, a nucleotide of the NUMBER IDENTIFICATION SEQUENCES is selected: 1-1558, more preferably they are selected from the NUMBER IDENTIFICATION SEQUENCES: 1-396, more preferably they are selected from the NUMBER IDENTIFICATION SEQUENCES: 1-217 and they are used to prepare protein binding probes which specifically bind to translation products of those polypeptides or fragments thereof. These probes are reacted with the test sample, forming binding complexes that are detected and compared with those of a standard, such that differences between the sample and the standard binding complexes are indicative of differential expression of polypeptides in the sample. In a preferred embodiment, protein binding probes are made to specifically detect polypeptides or fragments thereof produced by one or more of the genes or gene fragments identified in Table 2. In accordance with some preferred embodiments of the invention, The assays described herein for the detection of transcription and translation products associated with OA can be used in methods useful for determining a diagnosis or prognosis of osteoarthritis in a patient. According to one embodiment of the invention, a typical diagnostic test will comprise obtaining a sample of cells or tissues from a patient in which the expression of a gene associated with OA is expected to occur. Such cells or tissue includes, but is not limited to, cartilage tissue and chondrocytes. The sample is then analyzed for either 1) increased or decreased expression of one or more selected genes, via mRNA or protein detection, or 2) a particular gene expression profile, for example via a gene or protein array technology , as described herein. Such a diagnostic procedure can lead to the determination of whether the indications of osteoarthritis are present in the patient. In another embodiment of the invention, the diagnostic methods described herein may also be extended to provide prognostic information regarding the recovery of the patient from the OA disease, or to monitor the progress of the patient in response to a therapeutic regimen. In this situations, the diagnostic test is performed at intervals during the course of patient recovery treatment, and a change in the expression of a target gene or a particular change in the pattern of gene expression is indicative of the level of recovery or improvement of the patient. In one aspect of the invention, tests are provided to identify substances effective in the treatment modalities for osteoarthritis. In one embodiment of the invention, there is provided a method for measuring the effect of a test substance on the expression profile of genes that are differentially expressed in osteoarthritis, comprising the steps of: a) obtaining a standard expression profile from a first sample by measuring the transcription or translation products of two or more genes that correspond to two or more genes or gene fragments identified in Tables 1 and 2 in the absence of the test substance; b) obtain a test expression profile from a second sample by measuring the transcription or translation products of two or genes, or fragments of "genes identified in Tables 1 or 2 that are expressed in the test substance; ) comparing the standard expression profile of the test expression profile, wherein a change in the test expression profile compared to the standard expression profile is indicative of an effect of the test substance on the expression profile of genes that are differentially expressed in osteoarthritis, compared to a non-osteoarthritic condition, preferably two or more of the genes or gene fragments that correspond to two or more of the genes or gene fragments identified in Table 3 (SEQUENCES IDENTIFICATION NUMBERS: 1-396.) More preferably, two or more of the genes or gene fragments correspond to two or more of the genes or gene fragments identified s in table 4 (IDENTIFICATION SEQUENCES NUMBERS: 1-217). In some preferred embodiments, the samples are obtained from cultured cells. In this case, the standard expression profile is obtained from cells that have not been contacted with the test substance, while the test expression profile is obtained from cells that have been contacted with a test substance. The test compounds can include proteins, polypeptides, nucleic acids, small molecule pharmaceuticals, vitamins, minerals, fatty acids, polysaccharides, extracts, nutriseptics, pharmaceutical substances and nutrients) and the like. In a preferred embodiment, test compounds are nutrients that can be added to foods or other substances in the diet, or that can be taken as a dietary supplement. As exemplified herein, such nutrients include, but are not limited to, fatty acids such as omega-3 fatty acids (e.g., eicosapentaenoic acid) and omega-6 fatty acids (e.g., arachidonic acid), glucosamine, chondroitin sulfate, and vitamin D derivatives such as l, 25-dihydroxyvitamin D3 and 24R, 25-dihydroxyvitamin D3). One type of assay according to one embodiment of the invention involves measuring the activity of the protein encoded by one of the OA-associated genes mentioned above, in the presence or absence of a candidate substance. Such activity assays are well known in the art. If a cell-free activity assay is available for the selected protein, such assay is simply carried out on the purified protein in the presence or absence of the tested substance. The candidate substances are selected based on their ability to positively or negatively regulate the activity of the purified protein. It should be noted that assays of this type can be carried out, for example, in a recombinant cellular system as described in the following. They can also be performed, for example, in a system without cells, in some cases. For such in vitro activity assays, it is desirable to have a source of the protein of interest purified. One or more of the protein products of the aforementioned genes are commercially available, or can be purifiable in significant amounts from an appropriate biological source, for example cultured cells. Alternatively, proteins can be produced recombinantly from an isolated gene or cDNA by expression in a suitable prokaryotic or eukaryotic expression system and subsequently purified, as is also well known in the art. Another embodiment of the invention comprises in vitro cell assays for expression of genes associated with OA or activity of their encoded proteins. For these embodiments, a nucleic acid construct comprising an OA-associated gene is introduced into the host cells according to an aspect of the invention. In a preferred embodiment, mammalian cell lines are used. Host cells contemplated for use include, but are not limited to NIH3T3, CHO, HeLa and COS, as well as non-mammalian cells such as yeast, bacteria and insect cells. The coding sequences are operably linked to appropriately regulate suitable expression elements for the particular host cell to be used. Methods for introducing nucleic acids into host cells are well known in the art. Such methods include, but are not limited to transfection, transformation, calcium phosphate precipitation, electroporation and lipofection. Recombinant cells can be used to identify compounds which modulate the expression of genes or activity associated with OA, of their encoded proteins. For gene expression assays, it is preferred to prepare an artificial construct comprising the promoter of a selected OA-associated gene, operably linked to a mimic gene. The indicator construct can be introduced into a cultured cell which includes, without limitation, the standard host cell lines described above or other suitable cells, for example, cartilage related cells, such as chondrocytes. The assay is performed by monitoring the expression of the reporter gene in the presence or absence of a test compound. The candidate substances are selected on the basis of their ability to positively or negatively affect the expression of the gene. In another embodiment of the invention, the genes associated with OA and the gene fragments described herein can be used to manipulate the genome of non-human animal subjects, The methods of manipulating the genomes of a wide variety of animals are known per se. Those skilled in the art Such methods include, without limitation, the production of transgenic animals and with an inactivated gene In a preferred embodiment of the invention, a gene or gene fragment identified in Table 2 is used to prepare a construct. which is used to break or "block" the corresponding endogenous gene in an animal and thus produce an animal that has a null mutation for said gene locus.In some embodiments, the animals exhibit a reduction or complete elimination of the expression of one or more genes having a nucleic acid sequence that is selected from the NUMBER IDENTIFICATION SEQUENCES: 1-1558. In addition, the animals exhibit a reduction or complete elimination of the expression of one or more genes having a nucleic acid sequence that is selected from the SEQUENCES OF IDENTIFICATION NUMBERS: 1-396. In some embodiments, the animals exhibit a reduction or complete elimination of the expression of one or more genes having a nucleic acid sequence that is selected from the NUMBER IDENTIFICATION SEQUENCES: 1-217. In other embodiments, the animals show a reduction or complete elimination of the expression of one or more genes shown in Table 6. The transgenic animals are preferably mammals. In some embodiments, the transgenic animals are rodents (e.g., mice and rats). In other modalities, the animals are, for example, goats, cats, dogs, cows, pigs, sheep, horses, non-human primates, rabbits and guinea pigs. In some embodiments, small interfering RNAs are used to functionally interrupt genes. Briefly, expression of the gene is inhibited by a short interfering RNA (AR? Is), AR? of interference (RNAi) or an expression-obstructed or post-transcriptional silencing gene (PTGS) (see, for example, Ketting et al (2001) Genes Develop 15: 2654-2659). The AR? Molecules can be directed to AR? M homologous molecules for destruction by separation of the AR? M molecule within the region encompassed by the AR? Is molecule. Accordingly, AR? S capable of targeting and separating AR? M from the gene products shown in Table 6 can be used to decrease or eliminate the expression of one or more of these genes. In other modalities, ARs are able to direct and separate the mRNA from one or more genes shown in Table 1 (NUMBER IDENTIFICATION SEQUENCES: 1-1558) can be used to decrease or eliminate the expression of one or more of these genes . In another embodiment of the invention, the genes associated with OA and the gene fragments described herein are used to design molecules that can be used to interfere with the expression of one or more genes associated with OA, such molecules can include, without limitation, RNA interference probes and antisense molecules.

Another aspect of the invention features compositions of matter to facilitate the practice of the tests described in the foregoing. These compositions may comprise collections of two or more probes or primers for use in the detection of genes, gene fragments or encoded proteins, related to OA, which are differentially expressed, in accordance with certain aspects of the invention. In some embodiments, the compositions may comprise collections of two or more oligonucleotides or polynucleotides that specifically hybridize with nucleic acid molecules that are selected from NUMBER IDENTIFICATION SEQUENCES: 1-1558. Preferably, the compositions may comprise collections of two or more oligonucleotides or polynucleotides that specifically hybridize with nucleic acid molecules that are selected from NUMBER IDENTIFICATION SEQUENCES: 1-396. More preferably, the compositions may comprise collections of two or more oligonucleotides or polynucleotides that specifically hybridize with nucleic acid molecules that are selected from NUMBER IDENTIFICATION SEQUENCES: 1-217. Preferably, the composition may comprise collections of two or more oligonucleotides or polynucleotides that specifically hybridize to genes or gene fragments that are selected from the genes and gene fragments identified in Table 2. The collection may comprise a primer pair to amplify the sequence . In some preferred embodiments the amplification can be performed using polymerase chain reaction (PCR), more preferably quantitative PCR (cCRP). In a preferred embodiment, the collection comprises a larger plurality of probes, for example about 10, 50, 200, 400, 1000 or more probes, each of which hybridizes specifically to a part or all of the sequences of the IDENTIFICATION SEQUENCES NUMBERS: 1-1558. In a preferred embodiment, the nucleic acid probes are immobilized on a solid support. In a particularly preferred embodiment they are immobilized in an array format, more preferably in a miniature or microarray. Such microarrays are known in the art and are sometimes referred to as "DNA chips", "microchips", "biological chips" and other similar terms and may contain the complete array of genes or gene fragments altered by OA, in addition to those presented in Tables 1 and 2. In another embodiment, these compositions comprise two or more substances that bind proteins capable of specifically binding proteins or protein fragments encoded by the genes and gene fragments identified in Tables 1 and 2. In one embodiment preferred, the binding substances are antibodies and the library comprises two or more antibodies to detect two or more proteins or peptides encoded by NUMBER IDENTIFICATION SEQUENCES: 1-1558, respectively. Preferably, these compositions comprise two or more protein binding substances capable of specifically binding protein or protein fragment encoded by the genes and gene fragments of NUMBER IDENTIFICATION SEQUENCES: 1-396. More preferably, these compositions comprise two or more protein binding substances capable of specifically binding proteins or protein fragments encoded by the genes and gene fragments of the NUMBER IDENTIFICATION SEQUENCES: .1-217. Such binding substances may be any molecule to which the protein or peptide specifically binds including DNA (for proteins that bind to DNA), antibodies, cell membrane receptors, peptides, cofactors, lectins, sugars, polysaccharides, cells, cell membranes, organelles and organelle membranes. In a preferred embodiment, the collection comprises a larger plurality of antibodies, for example about 10, 50, 200 ,. 400, 1000 or more, each of which binds immunospecifically with part or all of a protein or peptide encoded by genes or gene fragments identified in Tables 1 or 2. In a preferred embodiment, the antibodies are immobilized on a solid support. In a particularly preferred embodiment, they are immobilized in an array format, more preferably in a miniature or microarray, as described above for oligonucleotide probes and may contain the complete array of proteins altered by OA, in addition to genes or fragments of genes identified in Tables 1 and 2. Another embodiment of the present invention relates to substances and compounds identified in any of the methods described herein that have an effect on the expression profile of genes that are differentially expressed in OA. Preferably, such substances will be effective in the treatment or prevention of OA. Another additional aspect of the invention represents test equipment for use in one or more assays described herein. One type of kit comprises one or more pairs of primers for amplifying nucleic acids which correspond to the genes associated with OA and gene fragments described herein. The team may further comprise samples of total mRNA derived from tissue of various physiological states, for use as controls. The team may also comprise buffers, nucleotide bases and other compositions to be used in hybridization or amplification reactions. Each solution or composition can be contained in a bottle or bottle and all jars are kept in closed confinement in a box for commercial sale. Another type of protein comprises one or more nucleic acids or protein binding probes wherein the nucleic acid probe hybridizes specifically with a gene or gene fragment associated with OA, according to certain aspects of the invention, or the binding probe of protein, which binds specifically to a protein encoded by the gene or gene fragment associated with OA. Preferably the protein probe is an antibody which is immunologically specific for the protein encoded by the gene or gene fragment associated with OA. In preferred embodiments, the nucleic acid or the probes that bind protein are immobilized on a solid support. In a particularly preferred embodiment, the device comprises immobilized arrays of nucleic acids or probes that "bind to protein, arrays comprising probes specific for a plurality of genes or gene fragments associated with OA described herein, or proteins encoded by the These kits also contain appropriate control samples of mRNA or protein from tissues of known physiological state, to be used as controls in the assays, they may also comprise buffers and reagents to carry out the tests, each solution, reagent or composition in the group. can be contained in a bottle or bottle and all jars are kept in confinement in a box for commercial sale Preferably, the devices should further comprise instructions for performing a gene expression assay In another aspect, the invention provides a method to alter the biological profile of cells from induce a change in the profile of gene expression for cells with respect to the genes involved in OA. The method involves administering to the cells an effective amount of a compound that alters the expression of one or more genes that have a nucleic acid sequence that is selected from the SEQUENCES OF IDENTIFICATION NUMBERS: 1-1558. In some embodiments, the compound affects the extent of one or more genes having a nucleic acid sequence that is selected from "NUMBER IDENTIFICATION SEQUENCES: 1-396." In some embodiments, the compound affects the expression of one or more genes having a nucleic acid sequence that is selected from the NUMBER IDENTIFICATION SEQUENCES: 1-217 In other embodiments, the compound affects the expression of one or more genes having the gene products shown in Table 6. invention also provides a method of affecting the expression of genes involved in OA comprising exposing cells to an effective amount of a compound that modulates the expression of one or more genes having a sequence that is selected from the SEQUENCES OF IDENTIFICATION NUMBERS: 1- 1558. In some modalities, the compound affects the expression of one or more genes having a nucleic acid sequence that is selected from SEQUENCES OF IDENTIFICATION NUMBERS: 1-396. In some embodiments, the compound affects the expression of one or more genes having a nucleic acid sequence that is selected from NUMBER IDENTIFICATION SEQUENCES: 1-217. In other embodiments, the compound affects the expression of one or more genes which have the gene products shown in Table 6. In other embodiments, the cells are cells associated with symptoms of osteoarthritis. In other modalities, the cells are chondrocytes. In other embodiments, the compounds are administered to cells in vitro. In other embodiments, the compounds are administered to the cells in vivo. The compounds can be administered to the subjects via any route of administration. Preferably, the subjects are vertebrates. More preferably, the subjects are mammals which include dogs, cats and humans.

The change in gene expression is preferably a difference of at least 1.01 times. More preferably it is at least a difference of 1.05, 1.10, 1. 25, 1.50, 1.75, 2.0, 2.25, 2.50, 2.75, 3.0, 3.25, 3.50, 3.75, 4.0 times or more. It has been shown that chondroitin sulfate has an effect on a wide variety of genes associated with OA, as shown in detail in Tables 7-12. It has been found that glucosamine also has an effect on a variety of genes associated with OA, as shown in detail in Tables 13-18. La, 25-dihydroxyvitamin D3 and 24R, 25-dihydroxyvitamin D3 also affect the expression of genes related to OA, as shown in Tables 19-20. It has also been shown that eicosapentaenoic acid (EPA) and arachidonic acid (AA) affect genes related to OA, as shown in Tables 21-23. The following examples are provided to describe the invention in greater detail. They are intended to illustrate, but not to limit the invention.

EXAMPLE 1 Extraction of RNA from Chondrocytes Normal cartilage and osteoarthritic chondrocytes from dogs (frozen instantly with N2) are obtained and stored at -80 ° C. Osteoarthritic chondrocytes originating from dogs diagnosed clinically with osteoarthritis undergo total hip replacement. 300 to 500 mg are ground in N2 (pestle and mortar) and transferred to a clean pre-cooled 50 ml tube. Trizol (2 ml / 100 mg) is added and the mixture is homogenized using a Polytron equipment for 2 x 30 seconds and 1 minute (high speed). The homogenate is then centrifuged at 10,000 x g for 10 minutes at 4 ° C. The supernatant is separated and 0.2 volume of chloroform is added to the supernatant, swirled and centrifuged at 10,000 x g for 15 minutes at 4 ° C. The upper aqueous phase is separated and 5 volumes of 4M guanidine thiocyanate, 25 mM sodium citrate, 0.5% sarcosil, 0.1 M beta-mercaptoethanol and 0.475 volumes of 100% ethanol are added to the upper aqueous phase. The solution is then applied to Qiagen aqueous RNA minicoluids (cat # 74104), using a vacuum manifold (according to the manufacturer's instructions) "for further purification of the RNA.The purified? AR is then precipitated with ethanol for concentrate , is resuspended in water with DEPC and treated with deoxyribonuclease I to eliminate residual AD.Deoxyribonuclease treatment equipment D? A-freeMR Ambion (cat # 1906) is used for treatment with deoxyribonuclease I, according to the instructions The RNA is quantified in a Beckman DU 640B spectrophotometer at 260 nm (Beckman Coulter, Inc., 4300 N. Harbor Boulevard, PO Box 3100, Fullerton, CA 92834-3100) The absorbance of 1 to 260 nm is equivalent at 40 μg RNA / ml Typical yields are from 0.65 to 0.8 μg / μl The quality of the ARα is determined by absorbance at 260 nm / 280 nm with a typical ratio of 1.7-2.0. by electrophoresis on a 1% agarose gel / formaldehyde / Tris-borate-EDTA (TBE), pH 7.8 as a buffer (90 mM Tris, 90 mM boric acid, 2 mM EDTA). Are approximately 1 to 3.5 μg of AR loaded? (2 to 5 μl) after they are mixed with 15 μl of gel loading solution (10 mM Tris, pH 7.5, 1 mM EDTA, 0.02% bromophenol blue, 10% glycerol). The gel is run at 50 volts for 3-4 hours, stained with SYBR Green 1 (Molecular Probes, Inc., PO Box 22010, Eugene, OR 97402-0469, 4849 Pitchford Ave., Eugene OR 97402-9165) to a dilution of 1: 10,000 for 30 minutes in the dark and is scanned using a Hitachi FMBIO II fluorescent scanner at 505 nm (Hitachi Genetic Systems, 1201 Harbor Bay Parkway Ste. 150, Alameda, CA 94502) L Example 2 Differential Presentation The fluorescent differential display is made using one of three primers anchored in combination with 1 of 80 arbitrary primers (GenHunter). In total, 240 PCR reactions are carried out. The reactions are separated using PAGE and visualized using a fluorescent scanner (EMBIOII)., Hitachi). Bands representing genes that are differentially expressed are cut, re-amplified and run on agarose gel to verify size. Subsequently they are subcloned (PCR-TRAP, GenHunter) and sequenced. The differential display is performed using a GenHunter RNAimage ™ or differential display system of AR? M green fluorescent R? Aspectra ™ 1 (GenHunter Corporation, 624 Grassmere Park Drive, Suite 17,? Ashville, T? 37211). Are approximately 200 ng of RA subjected to reverse transcription? in the next reaction (final concentration): RT buffer (25 mM Tris-Cl, pH 8. 3, 37.6 mM KCl, 1.5 mM MgCl 2, 5 mM DTT), 625 μM of each of the TP, 50 pmol of the H-TnG primer (GenHuntér) (5'-AAGCTTTTTTTTTTTG 3 ') (SEQUENCE OF IDENTIFICATION NUMBER 1559 ) or of primer H-T1XG (GenHunter) (5 '-AAGCTTTTTTTTTTTX 3') (SEQUENCE OF IDENTIFICATION NUMBER: 1560) or of the primer H-T1XA (GenHunter) (5 'AAGCTTTTTTTTTTTA 3') (SEQUENCE OF IDENTIFICATION NUMBER 1561) in a total volume of 19 μl. Add 1 μl (100 units / μl) of MMLV reverse transcriptase 10 minutes in the 37 ° C stage in a thermal cycler (GeneAmp PCR System 9700, PE Applied Biosystem 9700, PE Applied Biosystem, 850 Lincoln Center Dr., Foster CA 94404 ) and the following reaction is performed: 5 minutes 65 ° C, 60 minutes 37 ° C, 5 minutes 75 ° C followed by a retention at 4 ° C. Two μl of reverse transcription reaction are used in the following polymerase chain reaction: PCR buffer (10 mM Tris-Cl, pH 8.4, 50 M-KCl, 1.5 mM MgCl 2, 0.001% gelatin), 50 μm of each dNTP, 5 pmol of fluorescein-labeled H-TnG primer (GenHunter) (primer labeled with fluorescein, 5 'AAGCTTTTTTTTTTTG 3') (SEQUENCE OF IDENTIFICATION NUMBER: 1562), or primer H-T1XC marcaco with fluorescein (GenHunter) (primer labeled with fluorescein, 5 'AAGCTTTTTTTTTTTC 3') (SEQUENCE OF IDENTIFICATION NUMBER: 1563) or primer H-TX1A marked with fluorescein (GenHunter ) (fluorescein labeled primer 5 'AAGCTTTTTTTTTTTA 3') (SEQUENCE OF IDENTIFICATION NUMBER: 1564) one of the primers H-AP provided in the 200 pM kit, 1 unit Amplitaq DNA polymerase (PE 'Applied Biosystem, 850 Lincoln Center Dr ., Foster CA 94404) in a total of 20 μl. The following thermocycler reaction is used: 40 cycles of 15 seconds at 94 ° C, 2 minutes at 40 ° C, 30 seconds at 72 ° C followed by 5 minutes at 72 ° C and retention at 4 ° C. μl of each PCR sample is mixed with 5 μl of dextran blue loaded buffer and 10 μl of deionized formamide and subjected to electrophoresis in a 6% polyacrylamide gel at 55 watts for 3 hours in TBE buffer. The gel is scanned using a Hitachi FMBIO II eguipo at 505 nm. The cDNA bands are differentially expressed and cut with a razor, placed in a 1.5 ml tube, rinsed with 10-0 μl of sterile water for 10 minutes and then boiled for 15 minutes. The tubes are centrifuged for 2 minutes at 10,000 x g and the supernatant is transferred to a new tube. 10 μl of 3M sodium acetate, 5 μl of glycogen (10 mg / ml) and 450 μl of 100% ethanol are added to the supernatant and the tubes are placed at -80 ° C overnight. The samples are centrifuged at 10,000 x g for 10 minutes at 4 ° C and the supernatant is removed. The cDNA pellets are washed with cold 85% ethanol (-20 ° C), centrifuged as above for 1 minute and the supernatant is removed. The cDNA pellets are resuspended in 10 μl of sterile water. 4 μl of the cDNA extracts are amplified as in the previous PCR reaction with the following exceptions: 40 μl of total reaction volume, 20 μm of each of dNTP; 200 pM of unlabeled primer H-TnG, H-TuC, or H-TnA (GenHunter) and 2 units of Amplitag DNA polymerase (PE Applied Biosystems). The PCR conditions are the same as in the previous. 15 μl of the amplified cDNA extracts are mixed with 3 μl of 6X labeled dye (bromophenyl blue 0.25%, xylene cyanol FF 0.25%, glycerol 30%) and subjected to electrophoresis on a 1.5% agarose gel. The gel is run at 100 volts for 2 to 3 hours in TBE buffer, it is dyed, it is visualized as in the previous. The bands are cut with a razor and the cDNA is extracted according to the Qiagen ™ QIAgen gel extraction kit (Qiagen, Inc., 28159 Avenue Stanford, Valencia, CA 91355). To each gel coverslip 300 μl of QX1 buffer and 10 μl of QIAXEMR II suspension are added to a 1.5 ml tube and incubated at 50 ° C for 10 minutes. The tubes are swirled every two minutes during incubation. The tubes are centrifuged at 10, 000 x g for 30 seconds and the supernatant is discarded. The pellets are washed once with 500 μl of QX1 buffer and twice with PE buffer (by swirling and centrifuging as indicated above for each wash) .The sediment is air dried for 10 minutes and 20 μl of sterile water The tubes are incubated for 5 minutes at room temperature and the cDNA is eluted by centrifugation as done in the above, for 30 seconds.The supernatant is then transferred to a new 1.5 ml tube and stored at - 20 ° C.

The purified amplified gel cDNA is subcloned according to the PCR-TRAP cloning system type "11 from GenHunter (GenHunter Corporation, 624 Grassmere Park Drive, Suite 17, Nashville, TN 37211). 5 μL of purified cDNA is added gel and amplified at 300 ng of the PCR-TRAPMR vector, ligase buffer (50 mM Tris-Cl, pH 7.8, 10 M MgCl 2, 10 mM DTT, 10 mM ATP, 5 μg BSA) and 200 units of T4 AD? ligase, they are mixed and incubated overnight at 16 ° C. The competent GH cells (E. coli from (lac-pro) ara thi (f80dlacZdelM15)) are transformed by ligation reaction by mixing 10 μl of reaction to 100 μl of competent GH cells, on ice, in 1.5 ml tube The tubes are incubated on ice for 45 minutes, thermally shocked at 42 ° C for 2 minutes and then incubated on ice for 2 minutes. of LB broth (Luria-Bertani, Difco) to each tube and the tubes are incubated at 37 ° C for 1 hour, with shaking (250 rpm). place 200 μl of these transformations on plates of LB-agar-tet (LB-agar, Difco, tetracycline, 20 μg / ml) and incubate overnight at 37 ° C. The colonies are verified to determine the insertion using the PCR protocol of colony lysate GenHunter. The colonies are taken with a clean pipette tip and placed in 50 μl of colony lysate buffer (GenHunter, TE with 0.1% Tween 20) in a microcentrifuge tube. The tubes are boiled for 10 minutes, centrifuged at 10,000 x g for 2 minutes and the corresponding lysate (supernatant) is transferred to a new microcentrifuge tube. 2.0 μl of lysate is added to the PCR buffer, 20 μM each of dNTP, 200 pmol each of Lgh (5 'CGACAACACCGATAATC) (IDENTIFICATION SEQUENCE NUMBER: 1565) and Rgh (5' GACGCGAACGAAGCAAC) (SEQUENCE OF IDENTIFICATION NUMBER: 1566) as primers and 1 unit of Amplitag DNA polymerase (PE Applied Biosystems) in a total volume of 20 μl. The following thermocycler reaction is used: 30 cycles of 94 ° C for 30 seconds, 52 ° C for 40 seconds and 72 ° C for 1 minute followed by 72 ° C for 5 minutes and a retention at 4 ° C. The PCR products are analyzed in a 1.5% agarose gel as in the above. 3-5 ml of LB broth are inoculated with appropriate colonies and incubated overnight at 37 ° C, at 250 rpm. The plasmids are isolated according to the QlAprep Qiagen plasmid protocol. The bacteria are sedimented (10,000 x g, 30 seconds) using 2 x 1.5 ml of inoculated broth and the supernatant is removed. The pelleted bacteria are resuspended with 250 μl of P1 buffer, 250 μl of P2 buffer is added afterwards and the tubes are mixed by gentle inversion. 350 μl of N3 buffer is added, the tubes are mixed by gentle inversion and then centrifuged for 10 minutes. The supernatants are added to a QlAprep column and centrifuged for 30 seconds. The filtrates are discarded, 0.5 ml of PB buffer is added and the tubes are centrifuged for 30 seconds. The columns are washed with 0.75 ml of PE buffer and centrifuged for 30 seconds. The filtrates are discarded and the tubes are centrifuged for an additional 1 minute. The DNA is eluted from the column by adding 50 μl of sterile water to the column. The column is incubated at room temperature for 1 minute and then centrifuged for 1 minute. The resulting supernatant contains plasmid DNA quantified as above (absorbance of 1 to 260 nm equals 50 -μg / ml) with a typical yield of 350 μg / ml and a ratio at 260 nm / 280 nm of 1.8. Sequencing reactions using 200 to 500 ng of plasmid DNA in the ABI Prism BigDye Terminator Cycle Seguencing Ready reaction type (PE "Applied Biosystems, 850 Lincoln Center Dr., Foster CA 94404) 0.8 μl primer (0.16 μm) is added of final concentration of either Lgh or Rgh, GenHunter) to the ADβ plasmid, together with 4.0 μl of terminator reaction mixture (which contains AD? polymerase AmpliTag, FS, deoxynucleoside triphosphates, MgCl2, Tris-HCl buffer, pH 9.0, dichloro-labeled A-Dye terminator (R6G), dichloro-labeled C-Dye terminator (ROX), dichloro-labeled G-Dye terminator (R110) and T-Dye terminator labeled with dichlor (TAMRA)) and brought to a final volume of 10 μl with sterile water. The following thermocycler reaction is used: 25 cycles of 96 ° C for 10 seconds, 50 ° C for 5 seconds, 60 ° C for 4 minutes followed by a retention at 4 ° C. The terminators with unincorporated dye are separated from the sequencing reactions according to the DyeEx centrifugation protocol of Qiagen. The separate DyeEx centrifugation columns are placed in 2.0 ml microcentrifuge tubes and centrifuged at 750 x g for 3 minutes. The columns are placed in new tubes, sequencing reactions are added to the columns and centrifuged, as in the above, for 3 minutes. The eloat is placed at 74 ° C until dry. 5 μl of formamide / dextran blue (5: 1 ratio) are added to each of the dried sequencing pellets. Then 1.5 μl to 2.0 μl is added to a 5% polyacrylamide gel (in TBE buffer) in an automated Perkin Elmer ABI Prism 377 DNA sequencer. Each clone of isolated plasmid is sequenced 2-6 times (2-6 sequencing reactions) different, 1-3 times for each primer, Lgh or Rgh). The sequence files of the ABI 377 sequencer are transferred to the Wisconsin Package of the Genetic Computer Group and a corresponding consensus sequence is determined. Approximately 1750 clones are isolated using differential presentation. All genes that appear to be expressed differentially are selected. Figure 1 shows a polyacrylamide gel representation image. Panel A represents a gel before cutting a band and panel B represents the same gel after cutting the band. The sizes of the clones distributed from 90 p.b. up to 1150 p.b. with an average size of 300 p.b. After filtering sequences for redundant sequences, dimers, fragments of E. coli, fragments <; 100 p.b., etc., remain 1558. The sequences that are obtained (NUMBER IDENTIFICATION SEQUENCES: 1-1558) are shown in Table 1, which is appended hereto and which forms part of the present specification. The sequences obtained are submitted to the program BLAST against the public domain genomes of human, mouse and dog to get a first hit. The coincidence that covers more than 50% of the sequence and an expected value (E value) less than 0.002 is considered a success at this stage. The first hits are used to extend the sequence using the respective genome. The sequences extend 2 kb to the 5 'or 3' side of the hit. The extended sequences are then used for BLAST analysis against public domain protein databases (Ensembl, swissprot / trembl). The respective hits (those with an expected value less than 0.002 in this case) are consolidated and used for annotations. In some cases, this strategy does not provide successes, and in these situations the original sequences are directly subjected to BLAST against swissprot / trembl or Ensembl proteins. In this case, correct answers are considered when the expected value is less than 0.002. The results of the BLAST analysis (compared to 1/28/04 of the sequences isolated by differential presentation are shown in Table 2, which is annexed to the present one and which forms part of the present specification. column further to the left by the gene ID designations (number of clones) used by the inventors herein Many sequences coincide with a description of a previously identified gene, the description column also includes the database source and the access number of the corresponding database Table 2 includes additional information on the number of databases which include the ensemble gene IDs, the IDs of the ensemble transcript, the swissprot / ensemble, OMIM (Mendelian inheritance in line in man), RefSeg, Pfam, InterPro and HUGO The information is also shown with respect to the chromosome number (#) and location of the chromosome for many of the sequences. The column marked "signal peptide" indicates the sequence for which a predicted signal peptide is produced in the amino acid sequence; the column marked "TMHMM" (hidden transmembrane Markov model) indicates sequences for which a predicted transmembrane region occurs in a protein sequence. Table 6 includes a list of clones demonstrating homology with previously identified genes.

Table 6 CaMax: SITE 5 OF ECOTROPIC VIRAL INTEGRATION; GENE OF PHASE 911 1383a 4S OF NEUROBLASTOMA. [Source: RefSeq: Acc: M_005665] CaMax: ACIL-COA DESATURASE (EC 1.14.19.1) (ESTEAROIL-COA 1330 1384a DESATURASE) (FATTY ACID DESATURASE) (d (9) -DESATURASE). [Source: SWISSPROT; Acc: O00767] CaMax: NUCLEAR RECEIVER 7 COACTIVATOR; PROTEIN OF 140 KDA 858 1397b ASSOCIATED WITH ESTRÓGENO RECEPTOR. [Source: RefSeq; Acc: NM_181782] CaMax: PDZ DOMAIN CONTAINING EXCHANGE FACTOR 306 1401c GUANINE NUCLEOTIDE (GEF) 1; RA (RAS / RAP1A-ASSOCIATE) - GEF; PDZ DOMAIN CONTAINING THE GUANINE NUCLEOTIDE EXCHANGE FACTOR (GEF) 1; RA (RAS / RAP1A-ASSOCIATE) - GEF; NUCLEOTIDE GUANINA EXCHANGE FACTOR CONTAINING PDZ DOMAIN (GEF) 1. [Source: RefSeq; Acc: NM_014247] CaMax: MOLECULAR CHAPERON 5 REGULATOR OF THE BAG FAMILY. 853 1409b [Source: SWISSPROT; Acc: Q9UL15] CaMax: SUBUNITY 12A REGULATORY (INHIBITOR) PROTEIN 1247 1411a PHOSPHATASE 1; SUBUNIDAD 1 OBJECTIVE OF MIOSINAFOSFATASA. [Source: RefSeq; Acc: NM_002480] CaMax: PROTEIN 8 OF PROGRESSION OF CELLULAR CYCLE. 981 1420c [Source: RefSeq; Acc: NM_0207739] CaMax: PROTEIN 1 OF CHANGE OF THE PLACE OF TISSUE LYMPHOMA 1248 1421a LYMPHOID ASSOCIATED WITH MUCOSA (EC 3.4.22.-) (TRANSLOCATION ASSOCIATED WITH LYMPHOMA OF MALTA) (PARACASPASA). [Source: SWISSPROT; Acc: Q9UDY8] CaMax: PRECURSOR 1 PROTEIN SIMILAR TO SPARC (PROTEIN 145 143.2c ENDOTHELIAL VENGE HIGH) (HEVINA) (MAST 9). [Source: SWISSPROT; Acc: Q14515] - - CaMax: POTENTIAL HELICASE WITH ZINC FINGER DOMAIN. 1305 2056d [Source: SWISSPROT; Acc: P42694] CaMax: PALADINE; CG1-151 PROTEIN. 1307 2070a [Source: RefSeq; Acc: NM_016081] CaMax: ATPASA IA POTENTIAL PHOSPHOLIPID CONVEYOR (EC 99 2074b 3.6.3.1) (ATPASA II CHROMAHYL GRAPH) (ATPASA CLASS I TYPE 8A MEMBER 1). [Source: SWISSPROT; Acc: Q9Y2Q0] CaMax: SEPTINE 7 (PROTEIN HOMOLOGIST CDC10). 1098 2078a [Source: SWISSPROT; Acc: Q16181] CaMax: WD REPEAT SEQUENCE AND FYVE DOMAIN CONTAINING A 113 2083e ISOFORMA 1; SR1 PROTEIN OF FOSFOINOSITIDE UNION; WD40 AND FYVE DOMAIN CONTAINING 1. [Source: RefSeq; Acc: NM_020830] CaMax: FIBRILLINE PRECURSOR 1. 1104 2100b [Source: SWISSPROT; Acc: P35 555] CaMax: PROTEIN 345 DE ZINC FINGER (PROTEIN HZF10 DE FINGER DE 989 2110a ZINC). [Source: SWISSPROT; Acc: Q14585] CaMax: PROTEIN 1 ASSOCIATED WITH RESISTANCE MULTIMEDICAMENTOS. 505 211b [Source: SWISSPROT; Acc: P33 527] CaMax: TRANSPORTER 4 OF PEPTIDE-HISTIDINE. 1557 2123a [Source.-RefSeq; Acc: NM_145648] CaMax: EFFECTOR 1 OF SMALL PROTEIN OF CDC42. 990 2129a [Source: RefSeq; Acc: NM_020239 CaMax: PROTEOGLUCANO 4; STIMULATING FACTOR OF 215 2224a MEGACARIOCITOS; PROTEOGLUCAN 4 (PROTEIN OF ARTICULAR SURFACE AREA, STIMULATING FACTOR OF MEGACARIOCITOS); JACOBS SYNDROME OF CAMPTODACTILIA- ARTHROPATHY-PERICARDITIS; CAMPTODACTILIA, ARTROPATHY, COXA VARA, PERICARDITIS SYNDROME. [Source: RefSeq; Acc: NM_005807] CaMax: THERROTROPIN RELEASING HORMONE RECEPTOR (TRH-R) 994 2234a (TIROLIBERINE RECEPTOR) [Source: SWISSPROT; Acc: P21761] CaMax: Phosphodiesterase 3 ', 5'-CICLICA INHIBITED BY CGMP (EC 3.1.4.17) 996 2238a (FOSFODIESTERASE TO INHIBITED BY CYCLIC GMP) (CGI-PDE A). [Source: SWISSPROT; Acc: Q14432] CaMax: FOSFODIESTERASE A 3 ', 5'-CICLICA INHIBITED BY CGMP (EC 188 2241a 3.1.4.17) (FOSFODIESTERASE TO INHIBITED BY CYCLIC GMP) (CGI-PDE A). [Source: SWISSPROT; Acc: Q14432] CaMax: FOSFODIESTERASE TO S'.d'-CICUCA INHIBITED BY CGMP (EC 996 2238a 3.1.4.17) (FOSFODIESTERASE TO INHIBITED BY CYCLIC GMP) (CGI-PDE A). [Source: SWISSPROT; Acc: Q14432] CaMax: TROMBOSPONDIN PRECURSOR 1 999 2267a [Source: SWISSPROT; Acc: P07996] CaMax: FOSFODIESTERASE A 3 ', 5'-CICLICA INHIBITED BY CGMP (EC 188 2241a 3.1.4.17) (FOSFODIESTERASE TO INHIBITED BY CYCLIC GMP) (CGI-PDE A). [Source: SWISSPROT; Acc: Q14432] CaMax: TROMBOSPONDIN PRECURSOR 1 206 2374a [Source: SWISSPROT; Acc: P07996] - - EXAMPLE 3 Preparation of microarray Microarray probes are generated by isolated PCR amplification clones in differential presentation. The probes are tapped in duplicate on slides coated with poly-L-lysine using a GMS417 array (Affymetrix). Osteoarthritic cartilage samples are obtained from the femoral heads of dogs that have been clinically diagnosed as having experienced total hip replacement. The RNA is hybridized to the slides using the HC ExpressArray eguip (Digene) and visualized using a GMS418 scanner (Affymetrix). The Imagene program (Biodiscovery) was used to find the points and the subsequent analysis of the data was done using GeneSight (Biodiscovery). Expression levels are represented after subtraction of the background, logarithmic transformation (base 2) and normalization of the global slide signal. Preparation of microarray clone Cultivation blogs are inoculated 1. 5 ml Maicent broth (MB) plus 50 mg / ml tetracycline, with appropriate clones from glycerol concentrates and grown overnight with shaking at 37 ° C. These cultures are used to inoculate a second culture blog that is grown - - for about 6 hours with agitation at 37 ° C. These six-hour cultures are used to inoculate two culture blogs in duplicate which are grown overnight, with shaking at 37 ° C. The cultures are centrifuged to pellet the isolated cells and plasmids using the 96-well culture system (Qiagen). Plasmid concentrations are determined using a spectrophotometer by measuring the absorbance at 260 nm. All plasmid clones of cDNA are amplified in duplicate using the following PCR reaction (final concentration): 10X PCR buffer (10 mM Tris-HCl, pH 8.3, 50 mM KCl, 2.5 mM MgCl20, 500 μM each of dNTP, 600 nM of Rgh primer, 600 nM of Lgh primer, 1 μl (5 units / μl) of Tag Eppendorf polymerase and 1 μl (-100 ng / μl) of cDNA template in a total of 100 μl The reaction is carried out under the following conditions: 9 ° C for 30 seconds, 52 ° C for 40 seconds, 72 ° C for 1 minute, for 40 cycles, followed by 72 ° C for 5 minutes and 4 ° C - retention. The amplified products are checked in 1.5% agarose gel and purified using the Minelute protocol (Qiagen). The 200 μl of PCR is added to the filter plate and the vacuum is turned on to extract all the PCR reagents and the liguid, leaving only the cDNA attached to the filter. 30 μl of molecular grade water are added to the filter plate and incubated at room temperature on an orbital shaker at 900 rpm for 5 minutes. The supernatant containing the purified DNA is aspirated from the filter plate. The cDNAs are dried for 2 h or until completion, in a speed vac system at 45 ° C. 30 μl of Corning Universal Printing buffer is added to all cDNAs and resuspended overnight at room temperature on an orbital shaker. Transfer 2 μl for concentration analysis and add the appropriate amount of the Corning Universal Printing buffer to a final concentration of 200 to 500 ng / μl. The plates are stored at -20 ° C and after each arrangement print. Clone Arrangement The microscope slides (Goldseal cat # 3010) are immersed in 10% NaOH (Fisher cat # S318-500), 57% EtOH solution and incubated at room temperature on an orbital shaker at rpm for 2 hours. The slides are rinsed with Milli-Q 5X water for 30 seconds each time. While the slides remain in the last rinse water, a poly-L-lysine 10% (Sigma cat # P892Ó), PBS IX 10% (GibcoBRL cat # 70013-032) is brought to 700 ml using Milli-Q water in containers of plastic. The coverslips are immersed in coating solution and incubated at room temperature on an orbital shaker at 50 rpm for 1 hour. The slides are rinsed in Milli-Q 5X water for 30 seconds each time and rotated at 500 rpm for 1 minute. The slides are incubated in an oven at 55 ° C for 10 min and kept in the desiccator for at least 14 days for a period not longer than 3 months before the arrangement. The cDNA clones are distributed using the GMS 417 distributor (Affymetrix). All slides are placed in a desiccator at room temperature to allow them to settle overnight. The slides are rehydrated on boiling Milli-Q water (steam) and dried quickly with the DNA side up, in a heating blog at 80 ° C. To ensure efficient cross-linking, the slides are baked for 2 hours at 80 ° C in an oven and then cross-linked with Stratalinker (120 mJ, Stratagene). All slides are stored in a desiccator at room temperature until used for cDNA hybridization. Hybridization of cDNA microarray All RNA samples are subjected to reverse transcription using the following reaction: 5X superscript II primer buffer (Invitrogen), 1 μl (1 pmol / μl) RT primer (Genisphere), 1 μl Superase-InMR ribonuclease inhibitor, 1 μl of 10 M each of dNTP, 2 μl of 0.1 M DTT, 1 μl of Superscript II and 5 μg of total RNA. The reaction is carried out at 42 ° C for 2 h. The reaction is stopped by adding 3.5 μl of 0.5 M NaOH / 50 mM EDTA and by incubating at 65 ° C for - minutes. The reaction is neutralized by adding 5 μl of 1 mM Tris-HCl, pH 7.5. 101.5 μl of 10 mM Tris, pH 8, 1 mM EDTA are added and the cDNA is purified and concentrated following the protocol of Microcon YM-30 (Millipore). The concentrated cDNA is brought to a final volume of 10 μl with nuclease-free water and the following reagents are added: 20 μl of 2X hybridization buffer (Genisphere), 2 μl of LNA-blogger and 8 μl of nuclease-free water, for a total of 40 μl. The hybridization mixture is heated at 80 ° C for 10 minutes and loaded onto a microarray slide on the edge of a ÜfterSlip eguipment. The slide is then placed in a GeneMachines double hybridization chamber and placed in a water bath at 60 ° C overnight. The next day the slides are processed according to the 3DNA Array 350 protocol (Genisphere). Briefly, the slides are washed (2XSSC-.2% SDS, 2X SSC, .2X SSC), dried by centrifugation at 1000 rpm for 1 min and the retention hybridization of 3DNA is performed. The slides are washed (2XSSC- .2% SDS, 2X SSC, .2X SSC) are dried by centrifugation at 1000 rpm for 1 min and are scanned using a GMS 418 array scanner (Affymetrix). Microarray analysis The scanned images represent RNA transcripts attached to specific clones and are quantified and verified for point quality control using the Imagene analysis program (BioDiscovery). The quantized images are analyzed using the Genesight analysis software (BioDiscovery). The analysis represents the subtraction of the background that surrounds the points, averaging the replicas of the points, the deletion of clone information represents clone hybridization signals no greater than 200 above the background in all samples, the logarithmic transformation (base 2) and the overall normalization of each slide (the values are expressed as average point intensity percent).

EXAMPLE 4 Analysis of expression using microarrays RNA is extracted from cartilage, as described above. The microarray analysis (described above) is performed on 8 samples of osteoarthritic cartilage from dogs that have been clinically diagnosed that have undergone total chain replacement and 8 samples of normal cartilage. A standard T test (two categories) is performed on the final hybridization signals for osteoarthritic characterization of cartilage samples (p <0.05 and p <0.01, the results are shown in tables 3 and 4, respectively). TABLE 3 - - - - - - - - - - - The use of differential display to isolate gene transcripts has enabled the present inventors to develop a microarray chip enriched in transcripts involved in osteoarthritis. The use of this chip to analyze samples from dogs with osteoarthritis: (1) confirms the results of the differential presentation, and (2) allows an additional characterization of osteoarthritis in dogs at the molecular level. The transcripts analyzed by PCRc (discussed in the above) have validated the differential expression analysis from the microarray.

EXAMPLE 5 Quantitative polymerase chain reaction. (CCRP) Confirmation of changes in RNA transcripts is performed using quantitative PCR. Inverse transcriptase reactions are performed using Super Script II reverse transcriptase for RT-PCR (Invitrogen) according to the manufacturing instructions. Is 1 μg of AR added? to 1.5 μl of 10 mM dTP TP, 1.5 μl of random hexamers and 0.6 μl of Oligo dT primers and brought to a final volume of 15 μl. The samples are incubated at 68 ° C for 10 minutes and then cooled at 4 ° C for at least 1 minute using the GeneAmp PCR System 9700 (Applied Biosystems). A portion (0.25X) of the above reaction is separated and used as a RT minus reaction (negative control that does not contain Super ScriptMR II reverse transcriptase). Using the same Super ScriptMR II reverse transcriptase machine, a master mix containing 3 μl of 10X RT buffer, 6 μl of 25 mM MgCl 2, 3 μl of 0.1 M DTT is prepared and 1.5 μl of ribonuclease inhibitor is made. A portion (0.25X) is separated and 0.375 μl of H20 is added for the RT minus samples. To the rest of the master mix is added 1.125 μl of Super ScriptMR II reverse transcriptase for RT positive reactions. All reactions are then incubated at 42 ° C for 1 hour, boiled at 95 ° C for 5 minutes and then reduced to 4 ° C using the GeneAmp PCR System 9700. The samples are then diluted 1 part for reaction RT to 29 parts of H20 to create a cDNA concentrate for experimentation. The primers and 5 'nuclease assay probes are designed based on the sequences selected from the differential presentation using Primer ExpressTM. Vl .5 (Applied Biosystems ExpressTM Primer Tutorial for Real Time Quantitative PCR Primer and Probé Design Tutorial). Minor groove bonding probes (ABI Custom Oligo Synthesis Factor) are requested from ABI. All oligonucleotides are reconstituted with buffer TE- - pH = 8.0 (Ambion) to a concentrated concentration of 100 μM and then diluted with TE buffer to a concentrated working concentration of 5 μM. TaqManMR and Universal PCR Master Mix (Applied Biosystems) are used for quantitative PCR reactions, according to the manufacturer's instructions. The primer concentration is 300 μM each and the concentration of the probe is 200 μM (determined optimal from previous experiments). 4 μl of RT and RT reactions are used less for quantitative PCR reactions. All positive reactions are performed in triplicate, and negative controls are performed alone. The standard PCRc conditions are used as described in TaqMan ™ Universal Master Mix (Applied Biosystems, 50.0 ° C for 2 minutes, 95.0 ° C for 10 minutes and 40-50 cycles of 95 ° C for 15 seconds and then 60 ° C for 1 minute) to 0.5 volumes. The samples are run on an ABI Prism 7700 Sequence Detector, using the program ABI sequence detector Program vl .7a. All samples are run alone against each primer / probe set to determine which standard curve should be used. Standard curves are generated using serial dilutions of hepatic RNA or RNA from experimental samples. Alternatively, if the samples are not found within the - intervals of the curve, the sample with the lower Ct (cycle threshold) is subjected to reverse transcription and a serial dilution of 1:10 is used as the standard curve for that primer / probe set. The values are normalized to concentrations of G3PDH (glyceraldehyde-3-phosphate dehydrogenase) determined by quantitative PCR. The inductions are calculated from each normalized value of the smallest sample. The error bars represent standard error of the means. Table 5 shows the primers and probes used for the PCRc analysis.

TABLE 5 - EXAMPLE 6 Analysis by PCRc of dog cartilage with OA PCRc was performed as described, in the above in 6 samples of osteoarthritic cartilage from clinically diagnosed dogs who had undergone total hip replacement and 8 samples of normal cartilage. The results are shown in Figure 2 (A-E).

EXAMPLE 7 Microarray analysis of treated OA samples A. Culture of chondrocyte cells in vitro Dog cartilage is digested in a shaking water bath at 37 ° C using the following enzymes: trypsin (0.25%) for 25 minutes, hyaluronidase (150 U / ml) for 1 hour and collagenase (0.78%) overnight. The digested cartilage is filtered to obtain chondrocytes. Dripping from a syringe of 10 cc of Dulbecco's modified Eagle's medium (DMEM) + 2.4% alginate (with low melting point) + cells, into calcium chloride (102 mM) to form "spheres". The chondrocyte spheres are cultured in DMEM / F12 + P / S (100 'U / ml penicillin and 100 μg / ml streptomycin) + fetal bovine serum (FBS) 10%. The medium is changed every third day. At the end of the treatment (see below) the chondrocyte spheres are dissolved in 55 mM sodium citrate and 30 mM EDTA. The suspensions are centrifuged at 1800 rpm for 10 minutes. The cells are washed with phosphate buffer and centrifuged again at 1800 rpm for 5 minutes. Add 1 ml of lysis binding solution (AmbionMR RNAqueous ™) to the sediments of isolated canine chondrocytes, mix thoroughly and store at -20 ° C until RNA isolation is performed. B. Isolation of AR? cell culture The samples are swirled and homogenized using a Quiashredder column (Qiagen) according to the manufacturer's instructions. The homogenized lysate is harvested and an equal volume of 64% ethanol is added thereto. This mixture is then applied to a R? Aqueous ™ filter cartridge, 700 μl at a time and centrifuged for 1 minute at 10,000 rpm. The cartridge is washed using 700 μl of wash solution # 1 and 500 μl of wash solution # 2/3 with centrifugation at 10,000 rpm for 1 minute for each wash. The filter cartridge is dried by centrifugation 10,000 rpm) for 1 minute. Does the RA elute? 3 times by centrifugation (as in the above) using 30 μl aliquots of the elution solution 95-100 ° C. The AR? The resultant is treated with deoxyribonuclease and quantified as established - - previously. After RNA release, the RNA is prepared from the microarray hybridization as previously established. C. Statistical analysis of the cell culture microarray The data are transformed to logarithm base 2. The data are normalized using quantile normalization.

After normalization, a concordance relationship is calculated. Genes that are expressed differentially are determined using the paired t-test (a = 0.05) for EPA versus AA; EPA calculated versus calculated AA; Chondroitin sulfate and glucosamine 100 μg versus control, 100 μg versus 10 μg and 10 μg versus control. Genes that are differentially expressed are determined by first using proportions of calculated AA versus AA and EPA calculated versus EPA, followed by paired t-test (a = 0.05) for the proportions of AA 'calculated / AA and EPA calculated / EPA . The genes that are expressed differentially follow a trend for all analyzes of glucosamine and chondroitin sulfate and are determined for each treatment pair where the response to treatment results in increases or decreases, the same direction, in the whole of the three samples. Genes that are differentially expressed are determined using the t test of two modified Welch samples for both 1.25 D3 versus control and 24.25 D3 versus control (a = 0.05). 1. Treatment with chondroitin sulfate Chondrocytes are treated with chondroitin sulfate based on the recognition of chondroitin sulfate as a joint nutrient. The chondrocyte spheres are treated with 100 μg / ml, 10 μg / ml or 0 μg / ml (control) of chondroitin sulfate (n = 3 = for 1 week in DMEM / F12 - + P / S + 10% FBS) The medium is changed every third day. week, the chondrocyte spheres are dissolved in 55 mM sodium citrate and 30 mM EDTA.The suspensions are centrifuged at 1800 rpm for 10 minutes.The cells are washed with phosphate buffer and centrifuged again at 1800 rpm for 5 minutes. Add 1 ml of lysis binding solution (Ambion ™ RNAqueous ™) to the isolated dog chondrocyte pellet, mix thoroughly and store at -20 ° C until RNA isolation is performed. chondroitin due to poor correlation with the rest of the array data This reduces this analysis to n = 3. The results are shown in Tables 7-12. - 3 - - - - - - . - 2. Treatment with Glucosamine The treatment with glucosamine is used to determine the effect of this nutrient on the health of the joint on the differential expression of genes associated with OA. The chondrocyte spheres are treated with 100 μg / ml, 10 μg / ml or 0 μg / ml (control) of glucosamine (n = 3) for 1 week in DMEM / F12 + P / S + 10% FBS. The medium is changed every third day. After one week, the chondrocyte spheres dissolve in 55 mM sodium citrate and 30 mM EDTA. The suspensions are centrifuged at 1800 rpm for 10 minutes. The cells are washed with phosphate buffer and centrifuged again at 1800 rpm for 5 minutes. 1 ml of binding solution (Ambion ™ RNAgueous ™) is added to the isolated dog chondrocyte pellet, mixed thoroughly and stored at -20 ° C until the RNA isolation is performed. The results are shown in Tables 13-18. 3. Treatment with the, 25-dihydroxyvitamin D3 (1,25 D3) and 24R, 25-dihydroxyvitamin D3 (24, 25 D3). Treatment with 1,25 D3 and 24R, 25D3 is applied to chondrocytes based on their known effects on prostaglandin production and differential responses to vitamin D3 metabolites in chondrocytes to determine the effect of these compounds on gene expression associated with OA. The chondrocyte spheres are treated with 1.25 D3 10"7M or 24.25 D3 10" "7M for 24 hours or without vitamin D (the equivalent ethanol is added for control) (n = 3) in DMEM / F12 + P / S + 10% FBS After 24 hours, the chondrocyte spheres are dissolved in 55 mM sodium citrate and 30 mM EDTA.The suspensions are centrifuged at 1800 rmp for 10 minutes.The cells are washed with phosphate buffer and centrifuged again at 1800 rpm for 5 minutes Add 1 ml of lysis binding solution (AmbionMR RNAqueous ™) to the isolated dog chondrocyte pellet, mix thoroughly and store at -20 ° C until RNA isolation is performed. Results are shown in tables 19 and 20. - 996a 14.73 14.52 0.22 1.16 - - 4. Treatment with eicosapentaenoic acid (EPA) and arachidonic acid (AA) Chondrocytes are treated with eicosapentaenoic acid (EPA) and arachidonic acid (AA) based on the recognition in the literature that EPA acts as an anti-inflammatory. AA is used as a control to represent a typical Western diet. Chondrocytes are enriched with 50 μM EPA or 50 μM AA (using albumin as a carrier) for two weeks in DMEM / HAMS + P / S + 10% FBS. The medium is changed every third day. Each set (n = 3) is divided and half is treated with conditioned medium of stimulated monocytes and neutrophils (SMNCM) for a week with medium that is changed every third day. SMNCM is prepared by isolating monocytes and neutrophils from whole dog blood using NycoPrep ™ according to the manufacturer's instructions. Monocytes and neutrophils are stimulated with lipopolysaccharide (20 ng / ml) for 72 hours. The resulting supernatant is used as SMNCM in cell culture experimentation (the SMNCM constitutes 10% of the medium used during the experimentation). The chondrocyte spheres are dissolved in 55 mM sodium citrate and 30 mM EDTA. The suspensions are centrifuged at 1800 rpm for 10 minutes. The cells are washed with phosphate buffer and centrifuged again at 1800 rpm for 5 minutes. 1 ml of lysis-binding solution (Ambion ™ RNAqueous ™) is added to the pellet of isolated dog chondrocytes, mixed thoroughly and stored at -20 ° C until RNA isolation can be performed. A sample of the calculated treatment of EPA / AA is separated due to the little relationship with the rest of the settlement data. This reduces these analyzes to n = 3. The results are shown in Tables 21-23.

- Experiments show that various treatments can alter the expression of genes associated with OA. In some cases, the effect on gene expression is statistically significant (p 0.05). 'In other cases, although it can not be demonstrated that the change is statistically significant due to. the variability of expression, there is a definite tendency for the expression to change only in one direction (either increased expression or diminished expression). This unidirectional change is considered to be both biologically relevant and significant. In some cases, it is considered that the down regulation of the expression of certain genes will have a beneficial biological effect on OA. For other genes, the increased expression will have a beneficial biological effect. This invention allows the identification of genes that are related to the beneficial effects, as demonstrated by the regulation of compounds known to be involved, for example, in anti-inflammatory procedures. The invention also allows the. identification of new compounds which may have beneficial effects based on their regulation of the gene expression of the OA-associated genes described in this invention. The results show that one can alter the biology of cells with different treatments and have a direct impact on gene expression of genes associated with OA. The invention allows a rapid and powerful screening of the compounds to identify candidate and preventive treatments of OA in animals, particularly humans. The descriptions of each patent, patent application, publication and access number to database sequences mentioned or described in this document are hereby incorporated by reference in their entirety. Various modifications of the invention, in addition to those described herein, will be apparent to those skilled in the art from the foregoing description. Such modifications are also intended to be within the scope of the appended claims.

Claims (24)

- CLAIMS

1. A combination comprising a plurality of polynucleotide molecules wherein the polynucleotide molecules are differentially expressed in an osteoarthritic subject or in a pre-osteoarthritic subject, as compared to expression in subjects which are not osteoarthritic or pre-osteoarthritic.

2. A combination as described in claim 1, wherein the plurality of polynucleotide molecules comprises two or more molecules that are selected from the NUMBER IDENTIFICATION SEQUENCES: 1-1558 or fragments thereof.

3. Method for detecting differential expression of nucleic acids in a sample, comprising the steps of: a) hybridizing a combination comprising a plurality of polynucleotide molecules, wherein the polynucleotide molecules are differentially expressed in an osteoarthritic subject or in a "pre-osteoarthritic subject compared to the expression in subjects who are not osteoarthritic or pre-osteoarthritic with nucleic acids from the sample and in this way one or more hybridization complexes are formed, b) detect the hybridization complexes, and c) compare the hybridization complexes with those of a standard wherein - the differences between the standard and the hybridization complexes of the sample indicate differential expression of nucleic acids in the sample

4. Method as described in claim 3, wherein the Polynucleotide molecules hybridize with nucleic acid sequences that are selected from the IDENTIFICATION SEQUENCE

S NUMBERS: 1-1558 or fragments thereof. The method as described in claim 3, wherein the polynucleotide molecules hybridize with nucleic acid sequences that are selected from sequences of genes identified in Table 2 or fragments thereof.

6. Method for detecting differential expression of polypeptides in a sample, comprising the steps of: a) reacting a combination comprising a plurality of molecules that bind proteins with polypeptides of the sample and thus allow the production of the specific binding, wherein the proteins bound by the protein-binding molecules are differentially expressed in an osteoarthritic subject or in a pre-osteoarthritic subject as compared to the expression in subjects which are not osteoarthritic or pre-osteoarthritic; b) detect the specific binding; and c) comparing the specific binding in the sample with that of a standard, where the differences between the standard and the specific binding of the sample indicate differential expression of polypeptides in the sample.

The method as described in claim 3 or 6, which further comprises the step of treating the sample with at least one compound, wherein the comparison with a standard is indicative of whether the treatment with the test compound altered the expression differential of nucleic acids or polypeptides in the sample.

8. Composition of matter comprising a collection of two or more probes for detecting the expression of genes that are differentially expressed in osteoarthritic or pre-osteoarthritic subjects as compared to subjects who are not osteoarthritic or pre-osteoarthritic, wherein the probes comprise two or more than: a) nucleic acid molecules that hybridize specifically with two or more of the genes or gene fragments identified in Tables 1 and 2, or fragments thereof; or b) polypeptide binding agents that specifically bind to polypeptides produced by expression of two or more nucleic acid molecules comprising sequences that are selected from one or more genes or gene fragments identified in Tables 1 and 2, or fragments of the same. -

9. Composition as described in claim 8, wherein the genes or gene fragments comprise the NUMBER IDENTIFICATION SEQUENCES: 1-1558 or fragments thereof.

10. Composition as described in claim 8, wherein the gene or gene fragments comprise genes or gene fragments identified in Table 2.

11. Device for detecting the expression of a plurality of genes that are differentially expressed in osteoarthritis, comprising a substrate to which it is attached , in known places, a plurality of probes, wherein the probes comprise: a) a plurality of oligonucleotides or polynucleotides, each of which hybridizes specifically with a different sequence which is selected from any of the SEQUENCES OF • IDENTIFICATION NUMBERS: 1 -1558 or fragments thereof; or b) a plurality of polypeptide binding agents, each of which specifically binds to a different polypeptide or fragment thereof, produced by expression of a nucleic acid molecule comprising a sequence that is selected from the genes or fragments of genes comprising any of the NUMBER IDENTIFICATION SEQUENCES: 1-1558 or fragments thereof.

12. Method for measuring the effect of a test compound on the expression of one or more genes that are differentially expressed in osteoarthritis, comprising the steps of: a) measuring standard expression by measuring the transcription or translation products of one or more of genes or gene fragments comprising any of the NUMBER IDENTIFICATION SEQUENCES: 1-1558 or fragments thereof, in a standard sample in the absence of test compound; b) measuring the test expression by measuring the transcription or translation products of one or more of the genes or gene fragments which comprise any of the NUMBER IDENTIFICATION SEQUENCES: 1-1558 or fragments thereof, in a test sample in the presence of the test compound; c) comparing the standard expression with the test expression, wherein a change in the test expression compared to the standard expression is indicative of an effect on the test compound on the expression of the genes which are differentially expressed in osteoarthritis, compared to a non-osteoarthritic condition.

13. Method as recited in claim 12, wherein the measurement utilizes a composition of matter comprising a plurality of probes, wherein the probes comprise two or more of the genes or gene fragments which comprise any of the SEQUENCES OF IDENTIFICATION NUMBERS: 1-1558 or fragments thereof. The method as described in claim 12, wherein the standard and test samples are obtained from at least one mammalian subject. 15. Method for measuring the effect of a test compound on the expression of an OA-associated gene, wherein the gene is selected from the group consisting of the genes identified in Table 6, the method comprising measuring the production of products from transcription or translation made by gene expression in the presence or absence of the test compound, wherein a change in the production of the transcription or translation products in the presence of the test compound is indicative of an effect of the test compound on the expression of the gene. 16. Method according to claim 15, characterized in that gene expression is measured by providing a DNA construct comprising a sequence encoding a reporter gene operably linked to transcriptional regulatory sequences of an OA-associated gene, and measuring the formation of a reporter gene product in the presence or absence of the test compound. 17. Method to diagnose or develop a prognosis for a subject who shows signs of osteoarthritis, the method includes: a) obtaining a sample of the subject; b) measuring in the sample the production of transcription or translation products produced by the expression of one or more genes associated with OA or fragments of genes comprising: any of the NUMBER IDENTIFICATION SEQUENCES: 1-1558 or fragments thereof; c) compare the transcription or translation products of the sample with those of a standard, where a difference in the expression of any of the genes or gene fragments associated with OA are indicative of osteoarthritis. 18. Apparatus for detecting the presence of osteoarthritis or predisposition for osteoarthritis in a subject, comprising one or more oligonucleotides of at least about 10 consecutive nucleotides of a sequence that is selected from the sequences that hybridize to two or more genes or fragments of genes, which comprises any of the NUMBER IDENTIFICATION SEQUENCES: 1-1558 or fragments thereof, wherein the oligonucleotides specifically bind to nucleic acids that are differentially expressed in an osteoarthritic subject or in a subject predisposed to osteoarthritis, in comparison with expression in subjects who are not osteoarthritic or predisposed to osteoarthritis. 19. Equipment for testing the expression of genes - which are differentially expressed in osteoarthritis, comprising a container containing a collection of two or more probes, wherein the probes comprise one or more of: a) oligonucleotides or polynucleotides that hybridize specifically with two or more genes or gene fragments that comprise any of the NUMBER IDENTIFICATION SEQUENCES: 1-1558, or fragments thereof; or b) polypeptide binding agents that specifically bind polypeptides produced by expression of two or more genes or gene fragments comprising any of the NUMBER IDENTIFICATION SEQUENCES: 1-1558 or fragments thereof; and instructions for performing a gene expression assay. 20. Method for modulating the expression of a gene associated with osteoarthritis in a cell by administering an effective amount of a compound under appropriate conditions to alter the expression of at least one gene associated with osteoarthritis having a sequence that is selected from the SEQUENCES IDENTIFICATION NUMBERS: 1-1558 or fragments thereof. 21. Method for regulating the expression of a gene associated with osteoarthritis in a cell by administering an effective amount of a compound under appropriate conditions to alter the expression of at least one gene associated with osteoarthritis having a gene product identified in the Table The method as described in claim 21 or 22, wherein the compound is a vitamin, mineral, nutraceutical, a pharmaceutical substance of a stick molecule, protein, polypeptide, nucleic acid, fatty acid or polysaccharide. 23. The method as described in claim 23, wherein the compound is eicosopentaenoic acid, arachidonic acid, glucosamine, chondroitin sulfate, IV, 25-dihydrovitamin D3, or 24R, 25-dihydroxyvitamin D3. 24. Method for identifying compounds that regulate genes associated with osteoarthritis, which comprises: - a) measure the standard expression by measuring the transcription or translation products of one or more of the genes or gene fragments which comprise any of the NUMBER IDENTIFICATION SEQUENCES: 1-1558 or fragments thereof, in a standard sample in the absence, of a test compound; b) measuring the test expression by measuring the transcription or translation products of one or more of the genes or gene fragments which comprise any of the NUMBER IDENTIFICATION SEQUENCES: 1-1558, or fragments thereof, in a sample of test in the presence of the test compound; and c) comparing the standard expression with the test expression, wherein a change in the test expression compared to the standard expression is indicative of an effect of the test compound on the expression of genes that are differentially expressed in osteoarthritis in comparison with a non-osteoarthritic condition.