WO2000060933A9 - Pkc induced targets - Google Patents
Pkc induced targetsInfo
- Publication number
- WO2000060933A9 WO2000060933A9 PCT/US2000/009680 US0009680W WO0060933A9 WO 2000060933 A9 WO2000060933 A9 WO 2000060933A9 US 0009680 W US0009680 W US 0009680W WO 0060933 A9 WO0060933 A9 WO 0060933A9
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- gene
- pkc
- tissue
- nucleic acid
- expression
- Prior art date
Links
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Classifications
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
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- C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
- C12N15/8509—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells for producing genetically modified animals, e.g. transgenic
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/10—Transferases (2.)
- C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K2217/00—Genetically modified animals
- A01K2217/05—Animals comprising random inserted nucleic acids (transgenic)
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K2267/00—Animals characterised by purpose
- A01K2267/03—Animal model, e.g. for test or diseases
Definitions
- the invention features, a method of selecting a gene, e.g., a disease state-related gene.
- the method includes: providing a nucleic acid sample from a PKC-conditioned cell, tissue, or animal; selecting a gene which is expressed, e.g., differentially expressed, in said sample, thereby selecting a gene, e.g., a disease state-related gene.
- Differentially expressed can mean differentially expressed as compared to a control, e.g., a normal or wild type, tissue.
- a PKC-conditioned cell or tissue is a cell or tissue which has an altered, e.g., different from wild-type, normal, or non-disease state tissue, level or expression pattern of PKC. It includes cells in which the level of PKC activity has been altered, e.g., by the addition of exogenous PKC, or by the addition of an inhibitor or antagonist of PKC. In a particularly preferred embodiment it is one wherein an endogenous or exogenous PKC encoding sequence is misexpressed.
- a PKC gene is expressed from a tissue specific promoter, e.g., a vascular tissue specific promoter, e.g., pre-pro-endothelin, or a heart specific promoter, e.g., a myosin heavy chain alpha promoter.
- the promoter can be a heterologous promoter.
- the PKC isoform can be a beta or delta isoform.
- a PKC preferably PKC beta
- a vascular specific e.g., a pre-pro-endothelin, promoter.
- a PKC preferably PKC beta
- a cardiac specific e.g., a myosin heavy chain alpha, promoter.
- a PKC preferably PKC delta
- a vascular specific e.g., a pre-proendothelin, promoter.
- a compound e.g., a polypeptide or small molecule
- transgenic cells e.g., a polypeptide or small molecule
- the effect on the cell, tissue or transgenic cells, or transgenic animal evaluated.
- the effect on the expression of the PKC gene, or a selected gene is evaluated.
- the method further includes determining if a selected gene is expressed in a human tissue, e.g., a normal or disease state tissue, or PKC-conditioned tissue.
- a human tissue e.g., a normal or disease state tissue, or PKC-conditioned tissue.
- the method can include determining if a selected gene is expressed in normal or diseased cardiac, retinal, vascular, or sensory neural tissue. This method can be used to further evaluate the role of a selected gene in a disease. In a preferred embodiment more than one, e.g., 2, 5, 10, or 20, or more, selected gene is analyzed.
- the method further includes expressing a polypeptide encoded by a selected gene.
- the method further includes characterizing the polypeptide, e.g., by a physical characteristic such as molecular weight or sequence, or by a biological or biochemical characteristic such as enzymatic activity, e.g., phosphatase or phosphorylase activity, or by determining if the polypeptide is the substrate for a reaction, e.g., a phosphorylation.
- a physical characteristic such as molecular weight or sequence
- a biological or biochemical characteristic such as enzymatic activity, e.g., phosphatase or phosphorylase activity
- the tissue is from a transgenic animal, e.g., a transgenic mouse.
- the transgenic animal can include a transgene which includes a PKC coding sequence, e.g., a beta or delta isoform coding sequence, functionally coupled to a promoter, e.g., a tissue specific promoter, e.g., a vascular tissue specific promoter, e.g., pre-pro-endothelin, or a heart specific promoter, e.g., a myosin heavy chain alpha promoter.
- a tissue specific promoter e.g., a vascular tissue specific promoter, e.g., pre-pro-endothelin
- a heart specific promoter e.g., a myosin heavy chain alpha promoter.
- the sample is from a cultured cell, e.g., a genetically engineered cell.
- a cultured cell e.g., a genetically engineered cell.
- Such cells include cells which have been modified, e.g., by the introduction of a construct which directs the production of PKC.
- the construct can be introduced by viral vector, e.g., retroviral adenoviral vector.
- the construct can include a PKC coding sequence, e.g., a beta or delta isoform coding sequence, functionally coupled to a promoter, e.g., a tissue specific promoter, e.g., a vascular tissue specific promoter, e.g., pre-pro-endothelin, or a heart specific promoter, e.g., a myosin heavy chain alpha promoter.
- a tissue specific promoter e.g., a vascular tissue specific promoter, e.g., pre-pro-endothelin
- a heart specific promoter e.g., a myosin heavy chain alpha promoter.
- an isoform e.g., the beta isoform is misexpressed, e.g., overexpressed.
- This isoform can be used to find a selected gene which is related to cardiopathy, retinopathy, or sensory neuropathy.
- an isoform e.g., the delta isoform is misexpressed, e.g., overexpressed. This isoform can be used to find a selected gene which inhibits, or is correlated with the inhibition, of tissue growth, e.g., unwanted tissue growth characterized by restenosis.
- Methods of the invention allow for the identification of genes, e.g., selected genes, the expression of which is modulated, e.g., increased or decreased, by a PKC.
- the invention includes a population of nucleic acids, the expression of which is modulated by a PKC.
- the population can be placed on a substrate or otherwise disposed such that individual selected nucleic acids are positionally distinguishable. For example, they can be placed on a two dimensional array, e.g., a chip, a titer plate, or a membrane, and used to profile gene expression from tissue, e.g., normal, disease state, or treated tissue.
- RNA, or cDNA, from control and treated tissue can be used to determine the effect of a treatment, e.g., a drug candidate, on expression of selected genes. Similar methods can be performed with proteins. E.g., proteins encoded by selected genes can be analyzed or used as probes of the components of control and experimental tissue.
- the invention also includes transgenic animals discussed herein, e.g., transgenic mice, e.g., mice in which a PKC gene is expressed from a tissue specific promoter, e.g., a vascular tissue specific promoter, e.g., pre-pro- endothelin, or a heart specific promoter, e.g., a myosin heavy chain alpha promoter.
- a tissue specific promoter e.g., a vascular tissue specific promoter, e.g., pre-pro- endothelin
- a heart specific promoter e.g., a myosin heavy chain alpha promoter.
- the promoter can be a heterologous promoter.
- the invention also includes a tissue from such an animal.
- the invention also includes genetically engineered cell into which is introduced a PKC gene expressed from a tissue specific promoter, e.g., a vascular tissue specific promoter, e.g., pre-pro-endothelin, or a heart specific promoter, e.g., a myosin heavy chain alpha promoter.
- a tissue specific promoter e.g., a vascular tissue specific promoter, e.g., pre-pro-endothelin, or a heart specific promoter, e.g., a myosin heavy chain alpha promoter.
- the promoter can be a heterologous promoter.
- the nucleic acid can be introduced, e.g., by a viral vector, e.g., a retro viral or adenoviral vector.
- the invention also included preparations of selected nucleic acid described herein.
- Fig. 1 is a depiction of the effect of PKC isoforms on VEGF-induced BREC growth determined as ng/well of DNA.
- PKC beta enhances VEGF- induced growth;
- Fig. 2 is a depiction of the effect of PKC isoforms on VEGF-induced BREC migration determined as cells/field;
- Fig. 3 is a depiction of the effect of PKC isoforms on VEGF-induced Tube formation determined as total tube length, mm/field.
- PKC delta isoforms have a specific effect on tube formation o endothelial cells and suggests delta has a specific role in the differentiation of blood vessels.
- heterologous promoter is a promoter which is not naturally associated with a gene or a purified nucleic acid.
- a “purified” or “substantially pure” or isolated “preparation” of a polypeptide means a polypeptide that has been separated from other proteins, lipids, and nucleic acids with which it naturally occurs.
- the polypeptide is also separated from substances, e.g., antibodies or gel matrix, e.g., polyacrylamide, which are used to purify it.
- the polypeptide constitutes at least 10, 20, 50 70, 80 or 95% dry weight of the purified preparation.
- the preparation contains: sufficient polypeptide to allow protein sequencing; at least 1, 10, or 100 ⁇ g of the polypeptide; at least 1, 10, or 100 mg of the polypeptide.
- a “purified preparation of cells”, as used herein, refers to, in the case of plant or animal cells, an in vitro preparation of cells and not an entire intact plant or animal. In the case of cultured cells or microbial cells, it consists of a preparation of at least 10% and more preferably 50% of the subject cells.
- small molecule includes peptides, peptidomimetics, or non-peptidic compounds, such as organic molecules, having a molecular weight less than 2000, preferably less than 1000. Methods described herein can be used to screen small molecules.
- transgene means a nucleic acid sequence (encoding, e.g., one or more subject PKC isoform), which is partly or entirely heterologous, i.e., foreign, to the transgenic animal or cell into which it is introduced, or, is homologous to an endogenous gene of the transgenic animal or cell into which it is introduced, but which is designed to be inserted, or is inserted, into the animal's genome in such a way as to alter the genome of the cell into which it is inserted (e.g., it is inserted at a location which differs from that of the natural gene or its insertion results in a knockout).
- a transgene can include one or more transcriptional regulatory sequences and any other nucleic acid, such as introns, that may be necessary for optimal expression of the selected nucleic acid, all operably linked to the selected nucleic acid, and may include an enhancer sequence.
- transgenic cell refers to a cell containing a transgene.
- a "transgenic animal” is any animal in which one or more, and preferably essentially all, of the cells of the animal includes a transgene.
- the transgene can be introduced into the cell, directly or indirectly by introduction into a precursor of the cell, by way of deliberate genetic manipulation, such as by microinjection or by infection with a recombinant virus.
- This molecule may be integrated within a chromosome, or it may be extra chromosomally replicating DNA.
- tissue-specific promoter means a DNA sequence that serves as a promoter, i.e., regulates expression of a selected DNA sequence operably linked to the promoter, and which effects expression of the selected DNA sequence in specific cells of a tissue, such as vascular or heart tissue.
- tissue-specific promoter means a DNA sequence that serves as a promoter, i.e., regulates expression of a selected DNA sequence operably linked to the promoter, and which effects expression of the selected DNA sequence in specific cells of a tissue, such as vascular or heart tissue.
- tissue-specific promoter means a DNA sequence that serves as a promoter, i.e., regulates expression of a selected DNA sequence operably linked to the promoter, and which effects expression of the selected DNA sequence in specific cells of a tissue, such as vascular or heart tissue.
- the term also covers so-called “leaky” promoters, which regulate expression of a selected DNA primarily in one tissue, but cause expression in other tissues as well.
- “Misexpression”, as used herein, refers to a non-wild type pattern of gene expression, at the RNA or protein level. It includes: expression at non-wild type levels, i.e., over or under expression; a pattern of expression that differs from wild type in terms of the time or stage at which the gene is expressed, e.g., increased or decreased expression (as compared with wild type) at a predetermined developmental period or stage; a pattern of expression that differs from wild type in terms of decreased expression (as compared with wild type) in a predetermined cell type or tissue type; a pattern of expression that differs from wild type in terms of the splicing size, amino acid sequence, post-transitional modification, or biological activity of the expressed polypeptide; a pattern of expression that differs from wild type in terms of the effect of an environmental stimulus or extracellular stimulus on expression of the gene, e.g., a pattern of increased or decreased expression (as compared with wild type) in the presence of an increase or decrease in the strength of the stimulus.
- the invention includes, vectors, preferably expression vectors, containing a nucleic acid identified by a method described herein.
- a vector can include a nucleic acid in a form suitable for expression of the nucleic acid in a host cell.
- the recombinant expression vector includes one or more regulatory sequences operatively linked to the nucleic acid sequence to be expressed.
- the term "regulatory sequence” includes promoters, enhancers and other expression control elements (e.g., polyadenylation signals). Regulatory sequences include those which direct constitutive expression of a nucleotide sequence, as well as tissue-specific regulatory and/or inducible sequences.
- the design of the expression vector can depend on such factors as the choice of the host cell to be transformed, the level of expression of protein desired, and the like.
- the expression vectors of the invention can be introduced into host cells to thereby produce proteins or polypeptides.
- host cell which includes a nucleic acid molecule identified by a method described herein.
- host cell and “recombinant host cell” are used interchangeably herein. Such terms refer not only to the particular subject cell but to the progeny or potential progeny of such a cell. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term as used herein.
- Vector DNA can be introduced into host cells via conventional transformation or transfection techniques.
- transformation and “transfection” are intended to refer to a variety of art- recognized techniques for introducing foreign nucleic acid (e.g., DNA) into a host cell, including calcium phosphate or calcium chloride co-precipitation, DEAE-dextran-mediated transfection, lipofection, or electroporation
- a host cell of the invention can be used to produce (i.e., express) a protein. Accordingly, the invention further provides methods for producing a protein identified by a method described herein using the host cells of the invention. In one embodiment, the method includes culturing the host cell of the invention (into which a recombinant expression vector encoding a protein has been introduced) in a suitable medium such that a protein is produced. In another embodiment, the method further includes isolating a protein from the medium or the host cell.
- the invention provides methods for identifying modulators, i.e., candidate or test compounds or agents (e.g., proteins, peptides, peptidomimetics, peptoids, small molecules or other drugs) which bind to proteins encoded by a nucleic acid identified by a method described herein, have a stimulatory or inhibitory effect on, for example, expression or activity, or have a stimulatory or inhibitory effect on, for example, the expression or activity of a substrate.
- modulators i.e., candidate or test compounds or agents (e.g., proteins, peptides, peptidomimetics, peptoids, small molecules or other drugs) which bind to proteins encoded by a nucleic acid identified by a method described herein, have a stimulatory or inhibitory effect on, for example, expression or activity, or have a stimulatory or inhibitory effect on, for example, the expression or activity of a substrate.
- Compounds thus identified can be used to modulate the activity of target gene products (e.g.
- the invention provides assays for screening candidate or test compounds which are substrates of a protein or polypeptide or a biologically active portion thereof. In another embodiment, the invention provides assays for screening candidate or test compounds which bind to or modulate the activity of a protein encoded by a nucleic acid identified by a method described herein or polypeptide or a biologically active portion thereof.
- the test compounds of the present invention can be obtained using any of the numerous approaches in combinatorial library methods known in the art.
- an assay is a cell-based assay, or an assay which includes cell-free cellular components, in which a protein or biologically active portion thereof is contacted with a test compound, and the ability of the test compound to modulate its activity is determined.
- test compound to modulate binding of a protein encoded by a nucleic acid identified by a method described herein to a compound, e.g., a substrate, or to bind to the gene can also be evaluated.
- nucleic acid sequences identified herein can be used as polynucleotide reagents.
- these sequences can be used e.g., to: (i) map their respective genes on a chromosome e.g., to locate gene regions associated with genetic disease or to associate the gene with a disease; or (ii) identify or evaluate an individual from a minute biological sample (tissue typing).
- Genes can be mapped to chromosomes by preparing PCR primers
- primers can then be used for PCR screening of somatic cell hybrids containing individual human chromosomes. Only those hybrids containing the human gene corresponding to the gene sequences will yield an amplified fragment.
- Sequences can be used to identify individuals from biological samples using, e.g., restriction fragment length polymo ⁇ hism (RFLP).
- RFLP restriction fragment length polymo ⁇ hism
- an individual's genomic DNA is digested with one or more restriction enzymes, the fragments separated, e.g., in a Southern blot, and probed to yield bands for identification.
- the sequences of the present invention are useful as additional DNA markers for RFLP (described in U.S. Patent 5,272,057).
- the sequences of the present invention can also be used to determine the actual base-by-base DNA sequence of selected portions of an individual's genome.
- the nucleotide sequences described herein can be used to prepare two PCR primers from the 5' and 3' ends of the sequences.
- primers can then be used to amplify an individual's DNA and subsequently sequence it.
- Panels of corresponding DNA sequences from individuals, prepared in this manner, can provide unique individual identifications, as each individual will have a unique set of such DNA sequences due to allelic differences.
- Allelic variation occurs to some degree in the coding regions of these sequences, and to a greater degree in the noncoding regions.
- Each of the sequences described herein can, to some degree, be used as a standard against which DNA from an individual can be compared for identification purposes. Because greater numbers of polymo ⁇ hisms occur in the noncoding regions, fewer sequences are necessary to differentiate individuals.
- the noncoding sequences of SEQ ID NO: 1 can provide positive individual identification with a panel of perhaps 10 to 1,000 primers which each yield a noncoding amplified sequence of 100 bases. If predicted coding sequences, such as those in SEQ ID NO:3 are used, a more appropriate number of primers for positive individual identification would be 500-2,000.
- a panel of reagents from nucleotide sequences described herein is used to generate a unique identification database for an individual, those same reagents can later be used to identify tissue from that individual.
- positive identification of the individual, living or dead can be made from extremely small tissue samples.
- the present invention also includes diagnostic assays, prognostic assays, and monitoring clinical trials are used for prognostic (predictive) pu ⁇ oses to thereby treat an individual.
- the invention provides, a method of determining if a subj ect is at risk for a disorder related to a lesion in or the misexpression of a gene which encodes a protein encoded by a nucleic acid identified by a method described herein.
- Such disorders include, e.g., a disorder associated with the misexpression a protein encoded by a nucleic acid identified by a method described herein.
- the method includes one or more of the following: detecting, in a tissue of the subject, the presence or absence of a mutation which affects the expression of a gene identified by a method described herein, or detecting the presence or absence of a mutation in a region which controls the expression of the gene, e.g., a mutation in the 5' control region; detecting, in a tissue of the subject, the presence or absence of a mutation which alters the structure of a gene identified by a method described herein; detecting, in a tissue of the subject, the misexpression of a gene identified by a method described herein, at the mRNA level, e.g., detecting a non-wild type level of a mRNA ; detecting, in a tissue of the subject, the misexpression of the gene, at the protein level, e.g., detecting a non-wild type level of a protein encoded by a nucleic acid identified by a method described herein.
- the method includes: ascertaining the existence of at least one of: a deletion of one or more nucleo tides from the gene; an insertion of one or more nucleotides into the gene, a point mutation, e.g., a substitution of one or more nucleotides of the gene, a gross chromosomal rearrangement of the gene, e.g., a translocation, inversion, or deletion.
- detecting the genetic lesion can include: (i) providing a probe/primer including an oligonucleotide containing a region of nucleotide sequence which hybridizes to a sense or antisense sequence from the gene or naturally occurring mutants thereof or 5' or 3' flanking sequences naturally associated with the gene; (ii) exposing the probe/primer to nucleic acid of the tissue; and detecting, by hybridization, e.g., in situ hybridization, of the probe/primer to the nucleic acid, the presence or absence of the genetic lesion.
- detecting the misexpression includes ascertaining the existence of at least one of: an alteration in the level of a messenger RNA transcript of the gene; the presence of a non-wild type splicing pattern of a messenger RNA transcript of the gene; or a non-wild type level of the gene.
- Methods of the invention can be used prenatally or to determine if a subject's offspring will be at risk for a disorder.
- the method includes determining the structure of the gene, an abnormal structure being indicative of risk for the disorder.
- the method includes contacting a sample form the subject with an antibody to the protein or a nucleic acid, which hybridizes specifically with the gene.
- the presence, level, or absence of protein or nucleic acid identified by a method described herein in a biological sample can be evaluated by obtaining a biological sample from a test subject and contacting the biological sample with a compound or an agent capable of detecting the protein or nucleic acid (e.g., mRNA, genomic DNA) that encodes the protein such that the presence of protein or nucleic acid is detected in the biological sample.
- a biological sample includes tissues, cells and biological fluids isolated from a subject, as well as tissues, cells and fluids present within a subject.
- a preferred biological sample is serum.
- the level of expression of the gene can be measured in a number of ways, including, but not limited to: measuring the mRNA encoded by the _ gene; measuring the amount of protein encoded by the gene; or measuring the activity of the protein encoded by the gene.
- the level of mRNA corresponding to the gene in a cell can be determined both by in situ and by in vitro formats.
- the invention features, a method of analyzing a plurality of capture probes.
- the method can be used, e.g., to analyze gene expression.
- the method includes: providing a two dimensional array having a plurality of addresses, each address of the plurality being positionally distinguishable from each other address of the plurality, and each address of the plurality having a unique capture probe, e.g., a nucleic acid or peptide sequence; contacting the array with a nucleic acid, or a protein encoded by a nucleic acid, identified by a method described herein, preferably purified, nucleic acid, preferably purified, polypeptide, preferably purified, or antibody, and thereby evaluating the plurality of capture probes.
- Binding e.g., in the case of a nucleic acid, hybridization with a capture probe at an address of the plurality, is detected, e.g., by signal generated from a label attached to the nucleic acid, polypeptide, or antibody.
- the capture probes can be a set of nucleic acids from a selected sample, e.g., a sample of nucleic acids derived from a control or non-stimulated tissue or cell.
- the method can include contacting the nucleic acid, polypeptide, or antibody with a first array having a plurality of capture probes and a second array having a different plurality of capture probes.
- the results of each hybridization can be compared, e.g., to analyze differences in expression between a first and second sample.
- the first plurality of capture probes can be from a control sample, e.g., a wild type, normal, or non-diseased, non-stimulated, sample, e.g., a biological fluid, tissue, or cell sample.
- the second plurality of capture probes can be from an experimental sample, e.g., a mutant type, at risk, disease-state or disorder-state, or stimulated, sample, e.g., a biological fluid, tissue, or cell sample.
- the method can be used to detect SNPs, as described above.
- the invention features, a method of analyzing a plurality of probes.
- the method is useful, e.g., for analyzing gene expression.
- the method includes: providing a two dimensional array having a plurality of addresses, each address of the plurality being positionally distinguishable from each other address of the plurality having a unique capture probe, e.g., wherein the capture probes are from a PKC-conditioned cell or subject; contacting the array with one or more inquiry probe, wherein an inquiry probe can be a nucleic acid, polypeptide, or antibody; providing a two dimensional array having a plurality of addresses, each address of the plurality being positionally distinguishable from each other address of the plurality, and each address of the plurality having a unique capture probe, e.g., wherein the capture probes are from a cell or subject which are not PKC-conditioned; and thereby evaluating the plurality of capture probes.
- Binding e.g., in the case of a nucleic acid, hybridization with a capture probe at an address of the plurality, is detected, e.g., by signal generated from a label attached to the nucleic acid, polypeptide, or antibody.
- the invention features, a method of analyzing a plurality of probes or a sample.
- the method is useful, e.g., for analyzing gene expression.
- the method includes: providing a two dimensional array having a plurality of addresses, each address of the plurality being positionally distinguishable from each other address of the plurality having a unique capture probe, contacting the array with a first sample from a cell or subject which is PKC-conditioned; providing a two dimensional array having a plurality of addresses, each address of the plurality being positionally distinguishable from each other address of the plurality, and each address of the plurality having a unique capture probe, and contacting the array with a second sample from a cell or subject which is not PKC-conditioned; and comparing the binding of the first sample with the binding of the second sample.
- Binding e.g., in the case of a nucleic acid, hybridization with a capture probe at an address of the plurality, is detected, e.g., by signal generated from a label attached to the nucleic acid, polypeptide, or antibody.
- the same array can be used for both samples or different arrays can be used. If different arrays are used the plurality of addresses with capture probes should be present on both arrays.
- the invention features, a method of analyzing a nucleic acid or protein encoded by, a nucleic acid identified by a method described herein, e.g., analyzing structure, function, or relatedness to other nucleic acid or amino acid sequences.
- the method includes: providing a nucleic acid or amino acid sequence; comparing the sequence with one or more preferably a plurality of sequences from a collection of sequences, e.g., a nucleic acid or protein sequence database; to thereby analyze the sequence.
- the method can include evaluating the sequence identity between a sequence and a database sequence.
- the method can be performed by accessing the database at a second site, e.g., over the internet.
- the invention features, a set of ohgonucleotides, useful, e.g., for identifying SNP's, or identifying specific alleles of a nucleic acid identified by a method described herein.
- the set includes a plurality of ohgonucleotides, each of which has a different nucleotide at an interrogation position, e.g., an SNP or the site of a mutation.
- the ohgonucleotides can be provided with differential labels, such that an ohgonucleotides which hybridizes to one allele provides a signal that is distinguishable from an ohgonucleotides which hybridizes to a second allele.
- genes and the protein products thereof which are related to a disease state. These selected genes or proteins can serve as a point of intervention or as diagnostic or drug discovery tool.
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Abstract
Description
Claims
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AU47982/00A AU4798200A (en) | 1999-04-12 | 2000-04-12 | Pkc induced targets |
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US12888599P | 1999-04-12 | 1999-04-12 | |
US60/128,885 | 1999-04-12 |
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WO2000060933A9 true WO2000060933A9 (en) | 2002-02-14 |
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US5747340A (en) * | 1994-06-03 | 1998-05-05 | Syntex (U.S.A.) Inc. | Targeted gene expression using preproendothelin-1 promoters |
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