Patent Application
20090217398
A Sequence Listing is submitted on duplicate compact discs labeled CFR (computer readable form), Copy 1 and Copy 2. The contents of the CFR, Copy 1, and Copy 2 compact disks are the same. The Sequence Listing information on the CFR, Copy 1, and Copy 2 compact disks are identical. The Sequence Listing is in a file named “8123.txt.” The file was created on Feb. 24, 2006 at 3:13 PM and contains 188 KB of data. The file was created using an IBM PC compatible computer running the Windows 2002 operating system. The Sequence Listing in 8123.txt is incorporated herein by reference in its entirety.
1. Field of the Invention
The present invention relates generally to genes differentially expressed in animals and particularly to genes differentially expressed in fat animals compared to lean animals.
2. Description of the Related Art
It is generally accepted in the scientific community that genes play a role in animal development and that the regulation of gene expression plays a key role in the development of some diseases or conditions that affect an animal's health and well being. Similarly, the differential expression of genes is one factor in the development of such diseases and conditions and the evaluation of gene expression patterns has become recognized as crucial to understanding the development and control of such diseases and conditions at the molecular level. To advance the understanding of genes and their relationship to disease, a number of methods have been developed for studying differential gene expression, e.g., DNA microarrays, expressed tag sequencing (EST), serial analysis of gene expression (SAGE), subtractive hybridization, subtractive cloning and differential display (DD) for mRNA, RNA-arbitrarily primed PCR (RAP-PCR), Representational Difference Analysis (RDA), two-dimensional gel electrophoresis, mass spectrometry, and protein microarray based antibody-binding for protein.
Gene expression in fat animals compared to lean animals has not been thoroughly investigated. Therefore, a need exists to identify genes and proteins encoded by genes that are differentially expressed in fat animals compared to lean animals. Such genes, proteins, and their fragments would be useful for formulating a prognosis that an animal is likely to become fat, developing a diagnosis that an animal is fat, screening substances to determine if they are likely to be useful for modulating the amount of adipose tissue on an animal, and using such substances to modulate the amount of adipose tissue on an animal.
Fat animals can be defined as those animals having an excess of body adipose tissue. Generally, animals such as humans, canines, and felines weighing more than 15% of their ideal body weight are considered fat. The most common cause of an animal being fat is an over consumption of food that results in an excess intake of calories. However, there are other factors that can increase an animal's chances for being fat, e.g., lifestyle, health, eating habits, breed, spaying, and neutering. Also, the incidence of animals becoming fat generally increases with age due to a general decrease in metabolic rate and in physical activity. Surveys estimate that 25% of dogs in the United States that visit veterinary clinics are fat to the point of being obese. Studies have shown that fat animals are significantly more at risk for diseases such as arthritis, heart disease, respiratory disease, diabetes, bladder cancer, hypothyroidism, and pancreatitis.
Modulating the amount of adipose tissue on an animal, including preventing an animal from becoming fat or treating a fat animal to reduce the amount of adipose tissue on the animal or treating a lean animal to increase the amount of adipose tissue in the animal, is difficult. Increasing the amount of adipose tissue on an animal usually involved increasing the amount of food consumed. The most effective and easiest way to prevent an animal from becoming fat or to reduce the amount of fat on an animal is with dietary restriction and exercise. However, it is often difficult to ensure compliance with diet and exercise programs. Other methods involve the use of drugs such as phentermine, fenfluramine, sibutramine, orlistat, and phenylpropanolamine. Unfortunately, side effects occur with these drugs. For example, the administration of fenfluramine and phentermine for the treatment of human obesity can result in cardiac valve damage in humans. Sibutramine can increase blood pressure and orlistat may have unpleasant gastrointestinal side effects.
Given the problems with current methods for dealing with adipose tissue on an animal, there is a continuing need for new methods and compositions useful for formulating a prognosis that an animal is likely to become fat, developing a diagnosis that an animal is fat, screening substances to determine if they are likely to be useful for modulating the amount of adipose tissue on an animal, and using such substances to modulate the amount of adipose tissue on an animal.
It is, therefore, an object of the present invention to provide one or more genes or gene segments that are differentially expressed in fat animals compared to lean animals.
It is another object of the present invention to provide combinations of two or more polynucleotides or polypeptides that are differentially expressed in fat animals compared to lean animals.
It is another object of the present invention to provide compositions of two or more polynucleotide or polypeptide probes suitable for detecting the expression of genes differentially expressed in fat animals compared to lean animals and devices such as substrate arrays containing the probes.
It is a further object of the present invention to provide methods and compositions for detecting the differential expression of one or more genes differentially expressed in fat animals compared to lean animals in a sample.
It is another object of the present invention to provide a method for measuring the effect of a test substance on the expression profile of one or more genes differentially expressed in fat animals compared to lean animals as a method for screening a test substance to determine if it is likely to be useful for modulating the amount of adipose tissue on an animal.
It is another object of the invention to provide methods for formulating a prognosis that an animal is likely to become fat or developing a diagnosis that an animal is fat.
It is another object of the invention to provide methods and compositions for modulating the expression of one or more genes differentially expressed in fat animals compared to lean animals or for modulating the amount of adipose tissue on an animal.
One or more of these other objects are achieved using novel combinations of 295 polynucleotide probes representing 254 genes and gene segments that are differentially expressed in fat animals compared to lean animals. The polynucleotides are used to produce compositions, probes, devices based on the probes, and methods for determining the status of polynucleotides differentially expressed in fat animals compared to lean animals useful for achieving the above-identified objects, e.g., prognosing and diagnosing conditions relating to animal adipose tissue and for screening substances to determine if they are likely to be useful for modulating the amount of adipose tissue on an animal. Such substances, once identified, may be used to modulate the amount of adipose tissue on an animal. Various kits comprising combinations of probes, devices utilizing the probes, and substances are also provided.
It is also an object of this invention to provide methods for using “class predictor” gene profiles to differentiate between fat and lean animals. Class predictor technology can be used to facilitate the clinical diagnosis of an animal's body type, e.g., class prediction can be used in a blood-based test to make a positive determination as to whether an animal is fat or lean or has the propensity to become fat or lean. This and other objects disclosed herein may be achieved using novel combinations of 65 polynucleotide probes identified herein that can act as class predictors for fat and lean animals using blood samples taken from fat and lean animals. These class predictor genes can be used e.g., to develop blood-based test kits to predict if an animal is fat or has the propensity to become fat or they can be used to predict if a lean animal can maintain its leanness. Class predictors can also be used to define the body condition score of an animal and as such may have various useful applications in veterinary clinics.
It is also a further object of this invention to provide methods for using class predictor gene profiles to accurately identify fat animals and follow their progression at the biochemical level and indicate whether their gene expression profiles are consistent with being fat or lean.
It is also an object of this invention to provide methods to modulate the amount of adipose tissue in an animal in vivo by administration of one or more active ingredients that are shown in vitro to modulate the expression of genes involved in fat metabolism.
Further objects of the invention include use of the polynucleotides, probes, active ingredients and class predictor data disclosed herein in the manufacture of compositions, devices and kits as described herein, e.g., for modulating the expression of one or more genes differentially expressed in fat animals compared to lean animals or for modulating the amount of adipose tissue on an animal, for detecting the expression of genes differentially expressed in fat animals compared to lean animals and for predicting or diagnosing the body condition score of an animal, including the identification of fat animals from lean animals, and in methods for detecting the expression of genes differentially expressed in fat animals compared to lean animals, for modulating the expression of one or more genes differentially expressed in fat animals compared to lean animals, for measuring the effect of a test substance on the expression profile of one or more genes differentially expressed in fat animals compared to lean animals, for screening a test substance to determine if it is likely to be useful for modulating the amount of adipose tissue on an animal, for formulating a prognosis that an animal is likely to become fat or developing a diagnosis that an animal is fat or for modulating the amount of adipose tissue on an animal.
Other and further objects, features, and advantages of the present invention will be readily apparent to those skilled in the art.
The term “animal” means a human or other animal, including avian, bovine, canine, equine, feline, hicrine, murine, ovine, and porcine animals, that has adipose tissue. When the term is used in the context of comparing fat to lean animals, the animals that are compared are animals of the same species and possibly of the same race or breed. In preferred embodiments, the animal is a canine or feline, most preferably a canine.
The term “antibody” means any immunoglobulin that binds to a specific antigen, including IgG, IgM, IgA, IgD, and IgE antibodies. The term includes polyclonal, monoclonal, monovalent, humanized, heteroconjugate, antibody compositions with polyepitopic specificity, chimeric, bispecific antibodies, diabodies, single-chain antibodies, and antibody fragments such as Fab, Fab′, F(ab′)2, and Fv, or other antigen-binding fragments.
The term “array” means an ordered arrangement of at least two probes on a substrate. At least one of the probes is a control or standard and at least one of the probes is a diagnostic probe. The arrangement of from about two to about 40,000 probes on a substrate assures that the size and signal intensity of each labeled complex formed between a probe and a sample polynucleotide or polypeptide is individually distinguishable.
The term “body condition score” (BCS) means a method for body composition analysis based upon an animal's body size and shape. Several methods are known to skilled artisans, e.g., methods disclosed in U.S. Pat. No. 6,691,639 and in the reference entitled “Small Animal Clinical Nutrition”, 4th Edition, in Chapter 13 (ISBN 0-945837-05-4).
The term “Body Mass Index” (BMI) means an animal's weight (in kilograms) divided by its height (in meters) squared.
The term “Class Predictor” as used herein refers to a genomic, proteomic or metabolomic profile that is generated using supervised learning methods employing algorithms such as, but not limited to, Weighted Voting, Class Neighbors, K-Nearest Neighbors and Support Vector Machines from a group of pre-defined samples (“the training set”) to establish a prediction rule that then can be applied to classify new samples (“the test set”).
The term “DEXA” means body composition analysis dual-energy X-ray absorptiometry.
The term “differential expression” or “differentially expressed” means increased or unregulated gene expression or means decreased or down-regulated gene expression as detected by the absence, presence, or at least two-fold change in the amount of transcribed messenger RNA or translated protein in a sample.
The term “fat” as applied to an animal means any animal that is determined to have an excess amount of body adipose tissue or an animal that is prone to developing an excess amount of body adipose tissue using techniques and methods known to health care providers and other skilled artisans. An animal is prone to becoming fat if the animal has an inclination or a higher likelihood of developing excess adipose tissue when compared to an average animal in the general population. Generally, without limiting the definition, an animal is considered fat if (1) the animal has a BMI of 25 or more (a number considered to include “overweight” and “obese” in some methods of characterizing animal conditions), (2) the animal's weight is 15% or more than its “ideal” body weight as defined by health care professionals or related skilled artisans, (3) an animal's percent body fat is 27% or more as determined by DEXA, or (4) an animal has a body condition score of more than 3 as determined by skilled artisans using the method disclosed in “Small Animal Clinical Nutrition”, 4th Edition, in Chapter 13 (ISBN 0-945837-05-4) or its equivalent using other BCS methods.
The term “fat-associated genes” means all or a subset of the genes identified by SEQ ID NOs:1-295, particularly the 254 genes identified herein as differentially expressed in fat animals compared to lean animals.
The term “fold” when used as a measure of differential gene expression means an amount of gene expression in an animal that is a multiple or a fraction of gene expression compared to the amount of gene expression in a comparison animal, e.g., a fat animals compared to a lean animal. For example, a gene that is expressed three times as much in the animal as in the comparison animal has a 3 fold differential gene expression and a gene that is expressed one-third as much in the animal as in the comparison animal also has a 3 fold differential gene expression.
The term “fragment” means (1) an oligonucleotide or polynucleotide sequence that is a portion of a complete sequence and that has the same or similar activity for a particular use as the complete polynucleotide sequence or (2) a peptide or polypeptide sequence that is a portion of a complete sequence and that has the same or similar activity for a particular use as the complete polypeptide sequence. Such fragments can comprise any number of nucleotides or amino acids deemed suitable for a particular use. Generally, oligonucleotide or polynucleotide fragments contain at least about 10, 50, 100, or 1000 nucleotides and polypeptide fragments contain at least about 4, 10, 20, or 50 consecutive amino acids from the complete sequence. The term encompasses polynucleotides and polypeptides variants of the fragments.
The term “gene” or “genes” means a complete or partial segment of DNA involved in producing a polypeptide, including regions preceding and following the coding region (leader and trailer) and intervening sequences (introns) between individual coding segments (exons). The term encompasses any DNA sequence that hybridizes to the complement of gene coding sequences.
The term “genes differentially expressed in fat animals” means genes from which the amount of mRNA expressed or the amount of gene product translated from the mRNA is detectably different, either more or less, in tissue from fat animals as compared to lean animals.
The term “homolog” means (1) a polynucleotide, including polynucleotides from the same or different animal species, having greater than 30%, 50%, 70%, or 90% sequence similarity to a polynucleotide identified by SEQ ID NOs:1-295 and having the same or substantially the same properties and performing the same or substantially the same function as the complete polynucleotide, or having the capability of specifically hybridizing to a polynucleotide identified by SEQ ID NOs:1-295 under stringent conditions or (2) a polypeptide, including polypeptides from the same or different animal species, having greater than 30%, 50%, 70%, or 90% sequence similarity to a polypeptide identified by the expression of polynucleotides identified by SEQ ID NOs:1-295 and having the same or substantially the same properties and performing the same or substantially the same function as the complete polypeptide, or having the capability of specifically binding to a polypeptide identified by the expression of polynucleotides identified by SEQ ID NOs:1-295. Sequence similarity of two polypeptide sequences or of two polynucleotide sequences is determined using methods known to skilled artisans, e.g., the algorithm of Karlin and Altschul (Proc. Natl. Acad. Sci. USA 87:2264-2268 (1990)). Such an algorithm is incorporated into the NBLAST and XBLAST programs of Altschul et al. (J. Mol. Biol. 215:403-410 (1990)). To obtain gapped alignments for comparison purposes, Gapped Blast can be utilized as described in Altschul et al. (Nucl. Acids Res. 25: 3389-3402 (1997)). When utilizing BLAST and Gapped BLAST programs, the default parameters of the respective programs (e.g., XBLAST and NBLAST) are used. See http://ww.ncbi.nlm.nih.gov.
The term “hybridization complex” means a complex that is formed between sample polynucleotides when the purines of one polynucleotide hydrogen bond with the pyrimidines of the complementary polynucleotide, e.g., 5′-A-G-T-C-3′ base pairs with 3′-T-C-A-G-5′. The degree of complementarily and the use of nucleotide analogs affect the efficiency and stringency of hybridization reactions.
The term “in conjunction” means that a drug, food, or other substance is administered to an animal (1) together in a composition, particularly food composition, or (2) separately at the same or different frequency using the same or different administration routes at about the same time or periodically. “Periodically” means that the substance is administered on a dosage schedule acceptable for a specific substance. “About the same time” generally means that the substance (food or drug) is administered at the same time or within about 72 hours of each other. “In conjunction” specifically includes administration schemes wherein substances such as drugs are administered for a prescribed period and compositions of the present invention are administered indefinitely.
The term “lean” as applied to an animal means any animal that is determined not to be fat using techniques and methods known to health care providers and other skilled artisans. Generally, without limiting the definition, an animal is considered lean if (1) the animal has a BMI of less than 25 or (2) the animal's weight is less than 15% more than its “ideal” body weight as defined by health care professionals or related skilled artisans, (3) an animal's percent body fat is less than 27% as determined by DEXA, or (4) an animal has a body condition score of 3 or less as determined by skilled artisans using the method disclosed in “Small Animal Clinical Nutrition”, 4th Edition, in Chapter 13 (ISBN 0-945837-05-4) or it equivalent using other BCS methods.
The term “modulating the amount of adipose tissue on an animal” means causing the animal to lose adipose tissue, causing the animal to gain adipose tissue, or causing the animal to maintain the amount of adipose tissue on the animal if the animal is prone to gaining or losing adipose tissue. Thus, modulating the amount of adipose tissue on an animal encompasses preventing a lean animal from becoming fat and treating a fat animal to reduce the amount of adipose tissue on the animal, as well as treating a lean animal to add adipose tissue in appropriate circumstances, e.g., when treating a lean animal that is determined by skilled artisans to be so underweight that the addition of adipose tissue is desirable. Conventional methods may be used to assess the amount of adipose tissue on an animal, as well as to determine the animal's lean muscle mass and/or bone mineral content, information which may be of relevance in such an assessment.
The term “polynucleotide” or “oligonucleotide” means a polymer of nucleotides. The term encompasses DNA and RNA (including cDNA and mRNA) molecules, either single or double stranded and, if single stranded, its complementary sequence in either linear or circular form. The term also encompasses fragments, variants, homologs, and alleles, as appropriate for the sequence, that have the same or substantially the same properties and perform the same or substantially the same function as the original sequence. The sequences may be fully complementary (no mismatches) when aligned or may have up to about a 30% sequence mismatch. Preferably, for polynucleotides, the chain contains from about 50 to 10,000 nucleotides, more preferably from about 150 to 3,500 nucleotides. Preferably, for oligonucleotides, the chain contains from about 2 to 100 nucleotides, more preferably from about 6 to 30 nucleotides. The exact size of a polynucleotide or oligonucleotide will depend on various factors and on the particular application and use of the polynucleotide or oligonucleotide. The term includes nucleotide polymers that are synthesized and that are isolated and purified from natural sources. The term “polynucleotide” is inclusive of “oligonucleotide.”
The term “polypeptide,” “peptide,” or “protein” means a polymer of amino acids. The term encompasses naturally occurring and non-naturally occurring (synthetic) polymers and polymers in which artificial chemical mimetics are substituted for one or more amino acids. The term also encompasses fragments, variants, and homologs that have the same or substantially the same properties and perform the same or substantially the same function as the original sequence. The term encompass polymers of any length, preferably polymers containing from about 2 to 1000 amino acids, more preferably from about 5 to 500 amino acids. The term includes amino acid polymers that are synthesized and that are isolated and purified from natural sources.
The term “probe” means (1) an oligonucleotide or polynucleotide, either RNA or DNA, whether occurring naturally as in a purified restriction enzyme digest or produced synthetically, that is capable of annealing with or specifically hybridizing to a polynucleotide with sequences complementary to the probe or (2) a peptide or polypeptide capable of specifically binding a particular protein or protein fragment to the substantial exclusion of other proteins or protein fragments. An oligonucleotide or polynucleotide probe may be either single or double stranded. The exact length of the probe will depend upon many factors, including temperature, source, and use. For example, for diagnostic applications, depending on the complexity of the target sequence, an oligonucleotide probe typically contains about 10 to 100, 15 to 50, or 15 to 25 nucleotides. In certain diagnostic applications, a polynucleotide probe contains about 100-1000, 300-600, nucleotides, preferably about 300 nucleotides. The probes herein are selected to be “substantially” complementary to different strands of a particular target sequence. This means that the probes must be sufficiently complementary to specifically hybridize or anneal with their respective target sequences under a set of predetermined conditions. Therefore, the probe sequence need not reflect the exact complementary sequence of the target. For example, a noncomplementary nucleotide fragment may be attached to the 5′ or 3′ end of the probe, with the remainder of the probe sequence being complementary to the target sequence. Alternatively, noncomplementary bases or longer sequences can be interspersed into the probe provided that the probe sequence has sufficient complementarity with the sequence of the target polynucleotide to specifically anneal to the target polynucleotide. A peptide or polypeptide probe may be any molecule to which the protein or peptide specifically binds, including DNA (for DNA binding proteins), antibodies, cell membrane receptors, peptides, cofactors, lectins, sugars, polysaccharides, cells, cell membranes, organelles and organellar membranes.
The term “sample” means any animal tissue or fluid containing, e.g., polynucleotides, polypeptides, antibodies, metabolites, and the like, including cells and other tissue containing DNA and RNA. Examples include adipose, blood, cartilage, connective, epithelial, lymphoid, muscle, nervous, sputum, and the like. A sample may be solid or liquid and may be DNA, RNA, cDNA, bodily fluids such as blood or urine, cells, cell preparations or soluble fractions or media aliquots thereof, chromosomes, organelles, and the like.
The term “single package” means that the components of a kit are physically associated in or with one or more containers and considered a unit for manufacture distribution, sale, or use. Containers include, but are not limited to, bags, boxes, bottles, shrink wrap packages, stapled or otherwise affixed components, or combinations thereof. A single package may be containers of individual food compositions physically associated such that they are considered a unit for manufacture, distribution, sale, or use.
The term “useful variations” means (1) for a polynucleotide, the complements of the polynucleotide; the homologs of the polynucleotide and its complements; the variants of the polynucleotide, its complements, and its homologs; and the fragments of the polynucleotide, its complements, its homologs, and its variants and (2) for a polypeptide, the homologs of the polypeptide; the variants of the polypeptide and its homologs; and the fragments of the polynucleotide, its homologs, and its variants.
The term “virtual package” means that the components of a kit are associated by directions on one or more physical or virtual kit components instructing the user how to obtain the other components, e.g., in a bag containing one component and directions instructing the user to go to a website, contact a recorded message, view a visual message, or contact a caregiver or instructor to obtain instructions on how to use the kit.
The term “standard” means (1) a control sample that contains tissue from a lean animal if a fat animal is being tested or tissue from a fat animal if a lean animal is being tested or (2) a control sample that contains tissue from a lean or fat test animal that has not been exposed to a test substance being examined in the corresponding lean or fat animal to determine if the test substance causes differential gene expression, as appropriate for the context of its use.
The term “stringent conditions” means (1) hybridization in 50% (vol/vol) formamide with 0.1% bovine serum albumin, 0.1% Ficoll., 0.1% polyvinylpyrrolidone, 50 mM sodium phosphate buffer at pH 6.5 with 750 mM NaCl, 75 mM sodium citrate at 42° C., (2) hybridization in 50% formamide, 5×SSC (0.75 M NaCl, 0.075 M sodium citrate), 50 mM sodium phosphate (pH 6.8), 0.1% sodium pyrophosphate, 5×Denhardt's solution, sonicated salmon sperm DNA (50 μg/ml), 0.1% SDS, and 10% dextran sulfate at 42° C.; with washes at 42° C. in 0.2×SSC and 0.1% SDS or washes with 0.015 M NaCl, 0.0015 M sodium citrate, 0.1% Na2SO4 at 50° C. or similar procedures employing similar low ionic strength and high temperature washing agents and similar denaturing agents.
The term “substance” means an element, compound, molecule, or a mixture thereof or any other material that could potentially be useful for diagnosing, prognosing, or modulating the amount of adipose tissue on animals, including any drug, chemical entity, or biologic entity.
The term “siRNA” means a polynucleotide that forms a double stranded RNA that reduces or inhibits expression of a gene when the siRNA is expressed in the same cell as the gene. The term encompasses double stranded RNA formed by complementary strands. The siRNA complementary portions that hybridize to form the double stranded molecule typically have substantial or complete identity. Typically, siRNA contains at least about 15-50 nucleotides and the double stranded siRNA contains about 15-50 base pairs, preferably about 20-30 nucleotides and base pairs.
The term “specifically bind” means a special and precise interaction between two molecules which is dependent upon their structure, particularly their molecular side groups. For example, the intercalation of a regulatory protein into the major groove of a DNA molecule, the hydrogen bonding along the backbone between two single stranded nucleic acids, or the binding between an epitope of a protein and an agonist, antagonist, or antibody.
The term “specifically hybridize” means an association between two single stranded polynucleotides of sufficiently complementary sequence to permit such hybridization under predetermined conditions generally used in the art (sometimes termed “substantially complementary”). For example, the term may refer to hybridization of a polynucleotide probe with a substantially complementary sequence contained within a single stranded DNA or RNA molecule according to an aspect of the invention, to the substantial exclusion of hybridization of the polynucleotide probe with single stranded polynucleotides of non-complementary sequence.
The term “variant” means (1) a polynucleotide sequence containing any substitution, variation, modification, replacement, deletion, or addition of one or more nucleotides from or to a polynucleotide sequence and that has the same or substantially the same properties and performs the same or substantially the same function as the original sequence and (2) a polypeptide sequence containing any substitution, variation, modification, replacement, deletion, or addition of one or more amino acids from or to a polypeptide sequence and that has the same or substantially the same properties and performs the same or substantially the same function as the original sequence. The term therefore includes single nucleotide polymorphisms (SNPs) and allelic variants and includes conservative and non-conservative amino acid substitutions in polypeptides. The term also encompasses chemical derivatization of a polynucleotide or polypeptide and substitution of nucleotides or amino acids with nucleotides or amino acids that do not occur naturally, as appropriate.
The invention is not limited to the particular methodology, protocols, and reagents described herein because they may vary. Further, the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of the present invention. As used herein and in the appended claims, the singular forms “a,” “an,” and “the” include plural reference unless the context clearly dictates otherwise, e.g., reference to “a variant” includes a plurality of variants. Further, defined terms include variations of the terms used in the proper grammatical context, e.g., the term “specifically binds” includes “specific binding” and other forms of the term. Similarly, the words “comprise”, “comprises”, and “comprising” are to be interpreted inclusively rather than exclusively.
Unless defined otherwise, all technical and scientific terms and any acronyms used herein have the same meanings as commonly understood by one of ordinary skill in the art in the field of the invention. Although any compositions, methods, articles of manufacture, or other means or materials similar or equivalent to those described herein can be used in the practice of the present invention, the preferred compositions, methods, articles of manufacture, or other means or materials are described herein.
All patents, patent applications, publications, and other references cited or referred to herein are incorporated herein by reference to the extent allowed by law. The discussion of those references is intended merely to summarize the assertions made therein. No admission is made that any such patents, patent applications, publications or references, or any portion thereof, is relevant prior art for the present invention and the right to challenge the accuracy and pertinence of such patents, patent applications, publications, and other references is specifically reserved.
In one aspect, the present invention provides one or more genes or gene segments (“genes” as defined herein) that are differentially expressed in fat animals compared to lean animals. The invention is based upon the discovery of 295 polynucleotides representing 254 genes that are differentially expressed in fat animals compared to lean animals. The genes were identified by comparing the expression of genes in adipose tissue from animals diagnosed as fat with genes in adipose tissue from animals diagnosed as lean using Affymetrix GeneChip® technology. The polynucleotides are shown in the Sequence Listing and referenced in Table 1 as SEQ ID NOs:1-295. Table 1 also shows the Affymetrix Probe Identification Number (herein “APIN”) in Column 2, fold expression (fat/lean) in Column 3, Accession Number of Highest BLAST Hit in Column 4, and Accession Number of Highest BLAST Hit for a Human Sequence in Column 5 (column descriptions are also relevant for Tables 2 and 3). A description of the putative or actual gene function can be obtained from the BLAST database using methods known to skilled artisans. Generally, the putative or actual gene function is determined by (1) identifying the APIN for each gene that had 2 fold or greater gene expression in fat animals compared to lean animals, (2) determining the nucleotide sequence of each such gene by inputting the APIN into the publicly available Affymetrix database that correlates AIPN numbers with sequences, and (3) inputting the nucleotide sequence into the BLAST database provided by the National Institutes of Health and determining the putative or actual gene function from the resulting sequence matches to homologous sequences in the database. Table 4 shows the gene description obtained for the highest blast hit accession number for the corresponding SEQ ID NO and Table 5 shows the gene description for the highest blast hit for a human sequence accession number for the corresponding SEQ ID NO.
The polynucleotides are divided into groups based upon several criteria. First, the polynucleotides are divided into three groups based upon a an analysis of expression that determines the amount of or fold differential gene expression between fat and lean animals. Group 1 corresponds to the polynucleotides identified by SEQ ID NOs:1-295. These polynucleotides are differentially expressed in fat animals compared to lean animals by at least 2 fold. Group 2 corresponds to the polynucleotides identified by SEQ ID NOs:1-70. These polynucleotides are differentially expressed in fat animals compared to lean animals by at least 2.5 fold. Group 3 corresponds to the polynucleotides identified by SEQ ID NOs:1-25. These polynucleotides are differentially expressed in fat animals compared to lean animals by at least 3 fold. Second, the polynucleotides are divided into a group based upon their function. Group 4 corresponds to the polynucleotides identified in Table 2. These polynucleotides are associated with lipid and glucose metabolism pathways in animals. Third, the polynucleotides are divided into a group based upon their relevance. Group 5 corresponds to the polynucleotides identified in Table 3. These polynucleotides were identified as particularly relevant to fat animals compared to lean animals because they were identified by more than one probe when the differential expression analysis was conducted.
The polynucleotides and genes are identified by measuring differences in gene expression from adipose tissue from canines diagnosed as fat with gene expression in adipose tissue from canines diagnosed as lean. Changes in gene expression can be determined by any method known to skilled artisans. Generally, changes in gene expression are determined by measuring transcription (determining the amount of mRNA produced by a gene) or measuring translation (determining the amount of protein produced by a gene). The amount of RNA or protein produced by a gene can be determined using any method known to skilled artisans for quantifying polynucleotides and proteins. Generally, RNA expression is determined using polymerase chain reaction (PCR) (including, without limitation, reverse transcription-PCR (RT-PCR) and quantitative real-time PCR (qPCR)), RNase protection, Northern blotting, and other hybridization methods. The RNA measured is typically in the form of mRNA or reverse transcribed mRNA. Protein or polypeptide expression is determined using various colormetric and spectroscopic assays and methods such as the lowry assay, the biuret assay, fluorescence assays, turbidimetric methods, the bicinchoninic assay, protein chip technology, infrared absorbance, ninhydrin, the bradford assay, and ultraviolet absorbance. In a preferred method, changes in gene expression are determined using Affymetrix Canine-1 and Canine-2 gene chips available for purchase from Affymetrix, Inc. and the instructions for using such chips to determine gene expression.
Generally, differential gene expression in fat animals compared to lean animals is determined by measuring the expression of at least one gene. Preferably, the expression of two or more differentially expressed genes is measured to provide a gene expression pattern or gene expression profile. More preferably, the expression of a plurality of differentially expressed genes is measured to provide additional information for a more significant gene expression pattern or profile.
The polynucleotides, genes, proteins encoded by the polynucleotides and genes, and the complements, homologs, variants, or fragments based upon the sequences are useful in a variety of prognostic and diagnostic assays relating to the amount of adipose tissue on an animal and are useful for screening test substances to determine if the substances are useful for modulating the amount of adipose tissue on an animal. Other uses will be apparent from the description of the invention contained herein.
In another aspect, the invention provides a combination comprising two or more polynucleotides that are differentially expressed in fat animals compared to lean animals or two or more proteins produced by the expression of two or more polynucleotides that are differentially expressed in fat animals compared to lean animals. In one embodiment, the combination comprises two or more polynucleotides or proteins expressed from polynucleotides selected from SEQ ID NOs:1-295. In another, the combination comprises two or more polynucleotides or proteins expressed from polynucleotides selected from SEQ ID NOs:1-70. In another, the combination comprises two or more polynucleotides or proteins expressed from polynucleotides selected from SEQ ID NOs:1-25. In another, the combination comprises two or more polynucleotides or proteins expressed from polynucleotides selected from the SEQ ID NOs identified in Table 2. In a further, the combination comprises two or more polynucleotides or proteins expressed from polynucleotides selected from the SEQ ID NOs identified in Table 3. In another, the combination comprises useful variations of such polynucleotides. Preferably, the combination comprises a plurality of polynucleotides or proteins expressed from polynucleotides, generally about 10, 20, 50, 100, 200, or more polynucleotides or proteins, as appropriate for a particular Group and use. When the combination comprises one or more fragments, the fragments can be of any size that retains the properties and function of the original polynucleotide or protein, preferably from about 30%, 60%, or 90% of the original. The polynucleotides and proteins can be from any animal, preferably canines and felines, most preferable canines.
In another aspect, the invention provides a composition comprising two or more oligonucleotide or polynucleotide probes suitable for detecting the expression of genes differentially expressed in fat animals compared to lean animals. In one embodiment, the probes comprise polynucleotides selected from SEQ ID NOs:1-295. In another, the probes comprise polynucleotides selected from SEQ ID NOs:1-70. In a further, the probes comprise polynucleotides selected from SEQ ID NOs:1-25. In another, the probes comprise polynucleotides selected from the SEQ ID NOs identified in Table 2. In another, the probes comprise polynucleotides selected from the SEQ ID NOs identified in Table 3. In another, the probes comprise useful variations of such polynucleotides. The probes contain a sufficient number of nucleotides to specifically hybridize substantially exclusively with appropriate complementary polynucleotides. Preferably, the probes comprise at least about 10, 15, 20, 25, or 30 nucleotides. In some embodiments, the probes contain more nucleotides and comprise at least about 30, 50, 70, 90 or 100 nucleotides, or more. The probes may comprise full length functional genes of the present invention. Preferably, the composition comprises a plurality of polynucleotide probes suitable for detecting genes differentially expressed in fat animals compared to lean animals, generally about 10, 50, 200, 500, 1000, or 2000, or more probes. The polynucleotide probes are made or obtained using methods known to skilled artisans, e.g., in vitro synthesis from nucleotides, isolation and purification from natural sources, or enzymatic cleavage of the genes of the present invention.
In another aspect, the invention provides a device suitable for detecting the expression of a plurality of genes differentially expressed in fat animals compared to lean animals. The device comprises a substrate having a plurality of the oligonucleotide or polynucleotide probes of the present invention affixed to the substrate at known locations. The device is essentially an immobilized version of the oligonucleotide or polynucleotide probes described herein. The device is useful for rapid and specific detection of genes and polynucleotides and their expression patterns and profiles. Typically, such probes are linked to a substrate or similar solid support and a sample containing one or more polynucleotides (e.g., a gene, a PCR product, a ligase chain reaction (LCR) product, a DNA sequence that has been synthesized using amplification techniques, or a mixture thereof) is exposed to the probes such that the sample polynucleotide(s) can hybridize to the probes. Either the probes, the sample polynucleotide(s), or both, are labeled, typically with a fluorophore or other tag such as streptavidin, and detected using methods known to skilled artisans. If the sample polynucleotide(s) is labeled, hybridization may be detected by detecting bound fluorescence. If the probes are labeled, hybridization is typically detected by label quenching. If both the probe and the sample polynucleotide(s) are labeled, hybridization is typically detected by monitoring a color shift resulting from proximity of the two bound labels. A variety of labeling strategies and labels are known to skilled artisans, particularly for fluorescent labels. Preferably, the probes are immobilized on substrates suitable for forming an array (known by several names including DNA microarray, gene chip, biochip, DNA chip, and gene array) comparable to those known in the art.
In another aspect, the invention provides a composition comprising two or more peptide or polypeptide probes suitable for detecting the expression of genes differentially expressed in fat animals compared to lean animals. In one embodiment, the probes comprise peptides or polypeptides that specifically bind to proteins produced by the expression of one or more polynucleotides comprising sequences selected from SEQ ID NOs:1-295. In another, the probes comprise peptides or polypeptides that specifically bind to proteins produced by expression of one or more polynucleotides comprising sequences selected from SEQ ID NOs:1-70. In another the probes comprise peptides or polypeptides that specifically bind to proteins produced by expression of one or more polynucleotides selected from SEQ ID NOs:1-25. In a further the probes comprise peptides or polypeptides that specifically bind to proteins produced by expression of one or more polynucleotides selected from the SEQ ID NOs identified in Table 2. In another, the probes comprise peptides or polypeptides that specifically bind to proteins produced by expression of one or more polynucleotides selected from the SEQ ID NOs identified in Table 3. In another, the probes comprise peptides or polypeptides that specifically bind to proteins produced by expression of one or more useful variations of such polypeptides. The probes contain a sufficient number of amino acids to specifically bind to the appropriate polypeptides. Preferably, the probes comprise at least about 4, 10, 20, 40, or 80 amino acids. In some embodiments, the probes contain more amino acids and comprise at least about 100 or more amino acids. The probes may comprise full length functional proteins derived from the expression of full length functional genes identified by the present invention. Preferably, the invention provides a plurality of polypeptide probes suitable for detecting genes differentially expressed in fat animals compared to lean animals, more preferably a collection of about 10, 50, 100, 500, or 1000 or more of such probes. In one embodiment, the probes are antibodies, preferably monoclonal antibodies.
The polypeptide probes may be made according to conventional methods, e.g., using the nucleotide sequence data provided for polynucleotides of the present invention and methods known in the art. Such methods include, but are not limited to, isolating polypeptide directly from cells, isolating or synthesizing DNA or RNA encoding the polypeptides and using the DNA or RNA to produce recombinant products, synthesizing the polypeptides chemically from individual amino acids, and producing polypeptide fragments by chemical cleavage of existing polypeptides.
In another aspect, the invention provides a device suitable for detecting the expression of a plurality of genes differentially expressed in fat animals compared to lean animals. The device comprises a substrate having a plurality of the peptide or polypeptide probes of the present invention affixed to the substrate at known locations. The device is essentially an immobilized version of the peptide or polypeptide probes described herein. The device is useful for the rapid and specific detection of proteins and their expression patterns. Typically, such probes are linked to a substrate and a sample containing one or more proteins is exposed to the probes such that the sample proteins can hybridize to the probes. Either the probes, the sample proteins, or both, are labeled and detected, typically with a fluorophore or other agent known to skilled artisans. Generally, the same methods and instrumentation used for reading polynucleotide microarrays is applicable to protein arrays. Preferably, the probes are immobilized on a substrate suitable for forming an array.
Methods for determining the amount or concentration of protein in a sample are known to skilled artisans. Such methods include radioimmunoassays, competitive-binding assays, Western blot analysis, and ELISA assays. For methods that use antibodies, polyclonal and monoclonal antibodies are suitable. Such antibodies may be immunologically specific for a protein, protein epitope, or protein fragment.
Some embodiments of the invention utilize antibodies for the detection and quantification of proteins produced by expression of the polynucleotides of the present invention. Although proteins may be detected by immunoprecipitation, affinity separation, Western blot analysis, protein arrays, and the like, a preferred method utilizes ELISA technology wherein the antibody is immobilized on a solid support and a target protein or peptide is exposed to the immobilized antibody. Either the probe, or the target, or both, can be labeled using known methods.
In some embodiments, expression patterns or profiles of a plurality of genes differentially expressed in fat animals compared to lean animals are observed utilizing an array of probes for detecting polynucleotides or polypeptides. In one embodiment, arrays of oligonucleotide or polynucleotide probes may be utilized, whereas another embodiment may utilize arrays of antibodies or other proteins that specifically bind to the differentially expressed gene products of the present invention. Such arrays may be commercially available or they may be custom made using methods known to skilled artisans, e.g., in-situ synthesis on a solid support or attachment of pre-synthesized probes to a solid support via micro-printing techniques. In various embodiments, arrays of polynucleotides or polypeptides probes are custom made to specifically detect transcripts or proteins produced by the differentially expressed genes of the present invention.
In one embodiment, arrays of polynucleotide or polypeptide probes are custom made to specifically detect transcripts or proteins produced by two or more polynucleotides or genes identified in Table 2. These probes are designed to detect genes associated with lipid and glucose metabolism pathways in animals. In another embodiment, arrays of polynucleotide or polypeptide probes are custom made to specifically detect transcripts or proteins produced by two or more polynucleotides or genes identified in Table 3. These probes are designed to detect genes that are particularly relevant to fat animals compared to lean animals.
In a further aspect, the invention provides a method for detecting the differential expression of one or more genes differentially expressed in fat animals compared to lean animals in a sample. The method comprises (a) hybridizing a combination comprising a plurality of polynucleotide probes that are differentially expressed in fat animals compared to lean animals with polynucleotides in the sample to form one or more hybridization complexes; (b) optionally, hybridizing a combination comprising a plurality of polynucleotide probes that are differentially expressed in fat animals compared to lean animals with polynucleotides in a standard to form one or more hybridization complexes; (c) detecting the hybridization complexes from the sample and, optionally, the standard from step (b); and (d) comparing the hybridization complexes from the sample with the hybridization complexes from a standard, wherein a difference in the amount of hybridization complexes between the standard and sample indicate differential expression of genes differentially expressed in fat animals compared to lean animals in the sample. In various embodiments, the plurality of polynucleotide probes are selected from SEQ ID NOs:1-295 with difference of 2 fold or more, SEQ ID NOs:1-70 with difference of 2.5 fold or more, SEQ ID NOs:1-25 with difference of 3 fold or more, polynucleotides identified in Table 2 with difference of 2 fold or more, polynucleotides identified in Table 3 with difference of 2 fold or more, and useful variations of such polynucleotides with the appropriate fold for the Group. These polynucleotides are used to prepare probes that hybridize with sample polynucleotides to form hybridization complexes that are detected and compared with those of the standard. In some embodiments, the sample polynucleotides are amplified prior to hybridization. In some embodiments, the probes are bound to a substrate, preferably in an array.
Step (b) and part of step (c) are optional and are used if a relatively contemporaneous comparison of two or more test systems is to be conducted. However, in a preferred embodiment, the standard used for comparison is based upon data previously obtained using the method.
These probes are exposed to a sample to form hybridization complexes that are detected and compared with those of a standard. The differences between the hybridization complexes from the sample and standard indicate differential expression of polynucleotides and therefore genes differentially expressed in fat animals compared to lean animals in the sample. In a preferred embodiment, probes are made to specifically detect polynucleotides or fragments thereof produced by one or more of the genes or gene fragments identified by the present invention. Methods for detecting hybridization complexes are known to skilled artisans.
In one embodiment, the method further comprises exposing the animal or sample to a test substance before hybridization. Then, the comparison is indicative of whether the test substance altered the expression of genes differentially expressed in fat animals compared to lean animals, particularly fat-associated genes, in the sample.
In another aspect, the invention provides a method for detecting the differential expression of genes differentially expressed in fat animals compared to lean animals in a sample. The method comprises (a) reacting a combination comprising a plurality of polypeptide probes with proteins in the sample under conditions that allow specific binding between the probes and the proteins to occur, wherein the proteins bound by the probes are differentially expressed in a fat animal compared to a lean animal; (b) optionally, reacting a combination comprising a plurality of polypeptide probes with proteins in a standard under conditions that allow specific binding between the probes and the proteins to occur, wherein the proteins bound by the probes are differentially expressed in a fat animal compared to a lean animal; (c) detecting specific binding in the sample and, optionally, the standard from step (b); and (d) comparing the specific binding in the sample with that of a standard, wherein differences between the specific binding in the standard and the sample indicate differential expression of genes differentially expressed in fat animals compared to lean animals in the sample.
In various embodiments, the plurality of polypeptide probes are probes that specifically bind to proteins produced by expression of one or more polynucleotides selected from SEQ ID NOs:1-295 with difference of 2 fold or more, SEQ ID NOs:1-70 with difference of 2.5 fold or more, SEQ ID NOs:1-25 with difference of 3 fold or more, polynucleotides identified in Table 2 with difference of 2 fold or more, polynucleotides identified in Table 3 with difference of 2 fold or more, and useful variations of such polynucleotides with the appropriate fold for the Group. These polynucleotides are used to prepare probes that specifically bind to proteins that are detected and compared with those of the standard. In some embodiments, the probes are bound to a substrate, preferably in an array. In one embodiment the probes are antibodies.
Step (b) and part of step (c) are optional and are used if a relatively contemporaneous comparison of two or more test systems is to be conducted. However, in a preferred embodiment, the standard used for comparison is based upon data previously obtained using the method.
These probes are exposed to a sample to form specific binding that is detected and compared with those of a standard. The differences between the specific binding from the sample and standard indicate differential expression of proteins and therefore genes differentially expressed in fat animals compared to lean animals, particularly fat-associated genes, in the sample. In a preferred embodiment, probes are made to specifically detect proteins or fragments thereof produced by one or more of the genes or gene fragments identified by the present invention.
In one embodiment, the method further comprises exposing the animal or sample to a test substance before reacting the polypeptides with the proteins. Then, the comparison is indicative of whether the test substance altered the expression of genes differentially expressed in fat animals compared to lean animals, particularly fat-associated genes, in the sample.
In another aspect, the method for detecting the expression of genes differentially expressed in fat animals compared to lean animals in a sample is used to monitor an animal's progress when attempting to modulate the amount of adipose tissue on the animal in response to an adipose tissue modulation program. The method is performed at intervals, preferably set intervals, during the modulation program and the animal's progress monitored by comparing the results of the method at two or more points during the modulation program. A change in expression of one or more of the genes differentially expressed in fat animals compared to lean animals, particularly fat-associated genes, or in the pattern of gene expression, or the tack of any change, resulting from the comparison indicates the effectiveness of the modulation program. For example, an adipose tissue modulation program designed to reduce the amount of adipose tissue on an animal could be monitored and shown to be effective if the amount of gene expression for genes differentially expressed in fat animals compared to lean animals, particularly fat-associated genes, declines over time in response to the stimulus in the program. Similarly, a program to increase adipose tissue in a lean or overly lean animal should increase the expression profile for such genes. The modulation program can be any plan to modulate the amount of adipose tissue on the animal such as a diet, exercise, drug, or other similar program.
In a further aspect, the invention provides a method for measuring the effect of a test substance on the expression profile of one or more genes differentially expressed in fat animals compared to lean animals and a method for screening a test substance to determine if it is likely to be useful for modulating the amount of adipose tissue on an animal. The methods comprise (a) determining a first expression profile by measuring the transcription or translation products of two or more polynucleotides selected from SEQ ID NOs:1-295 or useful variations thereof in a test system in the absence of the test substance; (b) determining a second expression profile by measuring the transcription or translation products of two or more polynucleotides selected from SEQ ID NOs:1-295 or useful variations thereof in a test system in the presence of the test substance; and (c) comparing the first expression profile to the second expression profile.
A change in the second expression profile compared to the first expression profile of 2 fold or more indicates that the test substance effects the expression of genes differentially expressed in fat animals compared to lean animals and that the test substance is likely to be useful for modulating the amount of adipose tissue on an animal. In a preferred embodiment, the genes differentially expressed in fat animals compared to lean animals are fat-associated genes and the change is a 2 fold or more change in expression of at least two genes between the first expression profile to the second expression profile. The invention also provides the substances identified using the method.
In one embodiment, the polynucleotides are selected from SEQ ID NOs:1-70 or useful variations thereof and the change is 2.5 fold or higher. In another, the polynucleotides are selected from SEQ ID NOs:1-25 or useful variations thereof and the change is 3 fold or higher. In a further, the polynucleotides are identified in Table 2 or Table 3, or useful variations thereof, and the change is 2 fold or higher.
In one embodiment, the test system is an in vitro test system such as a tissue culture, cell extract, or cell line. In another, the test system is an in vivo test system, i.e., an animal such as a canine. In other embodiments, the test system is an ex vivo tissue system or an in silico system.
Test substances can be any substance that may have an effect on polynucleotides or genes differentially expressed in fat animals compared to lean animals, particularly fat-associated genes. Test substances include, but are not limited to, amino acids; proteins, peptides, polypeptides, nucleic acids, oligonucleotides, polynucleotides, small molecules, macromolecules, vitamins, minerals, simple sugars; complex sugars; polysaccharides; carbohydrates; medium-chain triglycerides (MCTs), triacylglycerides (TAGs); n-3 (omega-3) fatty acids including DHA, EPA, ALA; n-6 (omega-6) fatty acids including LA, γ-linolenic acid (GLA) and ARA; SA, conjugated linoleic acid (CLA); choline sources such as lecithin; fat-soluble vitamins including vitamin A and precursors thereof such as carotenoids (e.g., β-carotene), vitamin D sources such as vitamin D2 (ergocalciferol) and vitamin D3 (cholecalciferol), vitamin E sources such as tocopherols (e.g., α-tocopherol) and tocotrienols, and vitamin K sources such as vitamin K1 (phylloquinone) and vitamin K2 (menadione); water-soluble vitamins including B vitamins such as riboflavin, niacin (including nicotinamide and nicotinic acid), pyridoxine, pantothenic acid, folic acid, biotin and cobalamin; and vitamin C (ascorbic acid); antioxidants, including some of the vitamins listed above, especially vitamins E and C; also bioflavonoids such as catechin, quercetin and theaflavin; quinones such as ubiquinone; carotenoids such as lycopene and lycoxanthin; resveratrol; and α-lipoic acid, L-carnitine; D-limonene; glucosamine; S-adenosylmethionine; and chitosan. In a preferred embodiment test substances are nutrients that may be added to food or consumed as a supplement. Examples include, but are not limited to, fatty acids such as omega-3 fatty acids (e.g., DHA and EPA) and omega-6 fatty acids (erg., ARA), carnitine, methionine, vitamin C, vitamin E, and vitamin D.
In a preferred embodiment, the substances useful for affecting the expression of genes differentially expressed in fat animals compared to lean animals, particularly fat-associated genes, may be identified using methods discloses in co-pending U.S. Provisional Patent Application No. 60/657,980, filed Mar. 2, 2005, and any subsequent US or foreign patent application that claims priority thereto.
In a further aspect, the invention provides a method for formulating a prognosis that an animal is likely to become fat or developing a diagnosis that an animal is fat. The method comprises determining if one or more polynucleotides selected from SEQ ID NOs:1-295 or useful variations thereof or one or more polypeptides that specifically bind to proteins produced by expression of one or more polynucleotides selected from SEQ ID NOs:1-295 or useful variations thereof are differentially expressed in the animal compared to one or more lean animals. The animal is determined to be likely to become fat or determined to be fat if the comparison indicates that the polynucleotides are differentially expressed in the animal compared to the lean animals by a fold of 2 or more.
In various embodiments, the prognosis or diagnosis is based upon the polynucleotides selected from SEQ ID NOs:1-70, SEQ ID NOs:1-25, the sequences identified in Table 2, the sequences identified in Table 3, or useful variations of such polypeptides.
The expression profile for lean animals used in the comparison can be obtained from one or more lean animals contemporaneously with the expression profile for the animal being tested of from a database of lean animal expression profiles. Preferably, a database of expression profiles for lean animals accumulated over time is available for use as a reference.
Determining if the polynucleotides or polypeptides are differentially expressed can be accomplished by detecting the polynucleotides or polypeptides using methods known to skilled artisans some of which are described herein.
In another aspect, the invention provides a method for manipulating the genome or the expression of the genome of an animal, particularly a non-human animal. The method comprises disrupting the expression of one or more genes differentially expressed in fat animals compared to lean animals, preferably using oligonucleotides or polynucleotides constructed using polynucleotides selected from SEQ ID NOs:1-295 or useful variations thereof.
Methods of manipulating the genome are known to those of skilled in the art. Such methods include the production of transgenic and knockout animals and the disruption of transcription or translation. In one embodiment, one or more polynucleotides selected from SEQ ID NOs:1-295 or useful variations thereof are used to prepare a construct useful to disrupt or “knock out” the corresponding endogenous gene in an animal. This method produces an animal having a null mutation for that gene locus. In other embodiments, the animals exhibit a reduction or complete elimination of the expression of one or more genes differentially expressed in fat animals compared to lean animals, particularly fat-associated genes. The invention also provides an animal produced using the method. In various embodiments, the genome is manipulated using the one or more polynucleotides selected from SEQ ID NOs:1-70, SEQ ID NOs:1-25, the sequences identified in Table 2, the sequences identified in Table 3, or useful variations of such sequences. The transgenic animals are preferably mammals, e.g., rodents such as mice and rats, but may be other mammal such as felines and canines.
Methods of manipulating the expression of genome are known to those of skilled in the art. Such methods include the use of antisense or siRNA molecules and using such molecules to disrupt the translation or transcription of the genome. In one embodiment, one or more polynucleotides selected from SEQ ID NOs:1-295 or useful variations thereof are used to prepare antisense and similar DNA binding molecules that are useful for disrupting transcription or to prepare short (small) interfering RNAs (siRNA) useful for functionally disrupting translation. Briefly, gene expression is inhibited by antisense molecules through binding to DNA and preventing transcription and a siRNA through RNA interference (RNAi) or post-transcriptional gene silencing (PTGS). siRNA molecules target homologous mRNA molecules for destruction by cleaving the mRNA molecule within the region spanned by the siRNA molecule, Accordingly, siRNAs capable of targeting and cleaving a mRNA transcribed from a fat-associated gene is used to decrease or eliminate expression of one or more of such genes. In other embodiments, antisense molecules capable of binding to DNA and siRNAs capable of targeting and cleaving mRNA transcribed from one or more polynucleotides or genes selected from Group 2, Group 3, Group 4, or Group 5 polynucleotides or genes may be used to decrease or eliminate expression of one or more of these genes. In preferred embodiments, siRNAs are constructed from the transcripts of polynucleotides selected from SEQ ID NOs:1-295 or useful variations thereof.
In another aspect, the invention provides a composition suitable for manipulating the genome of an animal. The composition comprises one or more substances that interfere with the expression of one or more genes differentially expressed in fat animals compared to lean animals, particularly fat-associated genes. Preferably, substances comprise oligonucleotides or polynucleotides that bind to one or more of the genes or their transcription products and interferes with their replication, transcription, or translation, most preferably oligonucleotides or polynucleotides constructed using polynucleotides selected from SEQ ID NOs:1-295 or useful variations thereof. In various embodiments, the substances comprise antisense molecules or siRNAs.
In another aspect, the invention provides a method for modulating the expression of one or more genes differentially expressed in fat animals compared to lean animals, particularly fat-associated genes, or modulating the amount of adipose tissue on an animal comprising administering to the animal a gene expression or tissue modulating amount of a composition comprising one or more of DHA, EPA, EPA and DHA, ALA, LA, ARA, and SA. In preferred embodiments the composition comprises, in milligrams per kilogram of body weight per day (mg/kg/day), DHA in amounts of from about 1 to about 30, preferably from about 3 to about 15; EPA in amounts of from about 1 to about 30, preferably from about 3 to about 15; EPA/DHA Combo (1.5:1 ratio) in amounts of from about 412 to about 30/45, preferably from about 9/6 to about 18/12; ALA in amounts of from about 10 to about 100, preferably from about 30 to about 60; LA in amounts of from about 30 to about 600, preferably from about 60 to about 300; ARA in amounts of from about 5 to about 50, preferably from about 15 to about 30; SA in amounts of from about 3 to about 60, preferably from about 6 to about 30; and CLA (as a control) in amounts of from about 6 to about 120, preferably from about 12 to about 60. The composition can be administered to the animal in any manner or form suitable for the composition. Preferably, the composition is administered to the animal orally in the form of a food composition or a supplement. The food composition may be of any form, e.g., a nutritionally balanced food composition known in the art such as dry foods, semi-moist foods, and wet foods for animals, particularly companion animals such as feline and canine animals. Supplements include dosage forms such as tablets, capsules, and similar forms. In a further aspect, the composition is administered in combination with one or more drugs or other substances that modulate the amount of adipose tissue on an animal. The drugs or substances include, but are not limited to, substances that suppress appetite, increase metabolism, or interfere with the absorption of specific nutrients, particularly from food. Examples include, but are not limited to, orlistat (blocks fat breakdown and absorption), anorexigenics such as dexedrine (suppresses appetite), anorectics such as fenfluramine and phentermine, and sibutramine, and phenylpropanolamine.
In another aspect, the invention provides a composition suitable for modulating the expression of one or more genes differentially expressed in fat animals compared to lean animals, particularly fat-associated genes, or modulating the amount of adipose tissue on an animal. The composition comprises a gene expression or tissue modulating amount of one or more of DHA, EPA, EPA and DHA, ALA, LA, ARA, and SA. In various embodiments, the composition comprises, in mg/kg/day, DHA in amounts sufficient to administer to an animal from about 1 to about 30; EPA in amounts sufficient to administer to an animal from about 1 to about 30; EPA/DHA Combo (1.5:1 ratio) in amounts sufficient to administer to an animal from about 4/2 to about 30/45; ALA in amounts sufficient to administer to an animal from about 10 to about 100; LA in amounts sufficient to administer to an animal from about 30 to about 600; ARA in amounts sufficient to administer to an animal from about 5 to about 50; SA in amounts sufficient to administer to an animal from about 3 to about 60; and CLA (as a control) in amounts sufficient to administer to an animal from about 6 to about 120. Such substances are useful for modulating the amount of adipose tissue on an animal, Preferably, the substances affect the expression of a plurality of such genes. In one embodiment, the composition further comprises one or more drugs or other substances that modulate the amount of adipose tissue on an animal.
In another aspect, the invention provides a method for selecting an animal for inclusion in one or more groups or subgroups. The method comprises determining the expression profile of the animal for (a) polynucleotides selected from SEQ ID NOs:1-295 or useful variations thereof or (b) polypeptides each of which specifically binds to proteins produced by expression of one or more polynucleotides selected from SEQ ID NOs:1-295 or useful variations thereof and assigning the animal to a group based upon the expression profile. The groups can be any useful groups, preferably those involved in a research experiment, trial, clinical trial, or other similar category. For example, the groups can be groups involved in a research experiment or clinical trial that requires a one or more control groups and one or more treatment groups. In one embodiment, the control group comprises lean animals and the treatment group comprises fat animals, or vice versa in another. The expression profile for a plurality of animals can be determined and the animals assigned to the control group or treatment group based upon the results of the profile, i.e., animals with a differential expression of 2 fold or more compared to a standard are assigned to the fat group and animals with a differential expression of 2 fold or less compared to a standard are assigned to the lean group. The method is particularly useful for assigning animals to a clinical trial when testing potential drugs or other substances for their ability to reduce the amount of adipose tissue on the animal.
In another aspect, the invention provides a computer system suitable for manipulating data relating to one or more genes differentially expressed in fat animals compared to lean animals, particularly fat-associated genes. The system comprises a database containing information identifying the expression level of one or more polynucleotides selected from SEQ ID NOs:1-295 or useful variations thereof and/or polypeptides that specifically bind to proteins produced by the expression of one or more polynucleotides selected from SEQ ID NOs:1-295 or useful variations thereof in lean animals and/or fat animals and a user interface to interact with the database, particularly to input, manipulate, and review the information for different animals or categories or animals, e.g., lean or fat animals. In one embodiment, the database further contains information identifying the activity level of one or more polypeptides encoded by one or more polynucleotides selected from SEQ ID NOs:1-295 or useful variations thereof. In another, the database further comprises sequence information for one or more of the polynucleotides selected from SEQ ID NOs:1-295 or useful variations thereof. In other embodiments, the database contains additional information describing the putative description of the genes in one or more animal species. The computer system is any electronic device capable of containing and manipulating the data and interacting with a user., e.g., a typical computer or an analytical instrument designed to facilitate using the present invention and outputting the results relating to the status of an animal.
In another aspect, the invention provides a method for using a computer system or the present invention to present information identifying the expression profile of one or more genes differentially expressed in fat animals compared to lean animals, particularly fat-associated genes. The method comprises comparing the expression level of two or more polynucleotides or proteins expressed from polynucleotides selected from SEQ ID NOs:1-295 form a sample to the expression profile of the polynucleotides or proteins in the computer system.
In a further aspect, the present invention provides kits suitable for determining the differential expression of one or more genes differentially expressed in fat animals compared to lean animals, particularly fat-associated genes, in a test system. The kits comprise in separate containers in a single package or in separate containers in a virtual package, as appropriate for the use and kit component, two or more probes suitable for detecting the expression of genes differentially expressed in fat animals compared to lean animals, the probes comprising (a) polynucleotides selected from SEQ ID NOs:1-295 or useful variations thereof or (b) polypeptides that specifically bind to proteins produced by the expression of one or more polynucleotides selected from SEQ ID NOs:1-295 or useful variations thereof and at least one of (1) instructions for how to use the probes of the present invention; (2) reagents and equipment necessary to use the probes, (3) a composition suitable for modulating the expression of one or more genes differentially expressed in fat animals compared to lean animals; (4) a composition suitable for disrupting the expression of one or more genes differentially expressed in fat animals compared to lean animals; (5) a food composition suitable for modulating the amount of adipose tissue on an animal; and (6) one or more drugs or other substances that that modulate the amount of adipose tissue on an animal. In one preferred embodiment, the probes are bound to a substrate, preferably in an array.
When the kit comprises a virtual package, the kit is limited to instructions in a virtual environment in combination with one or more physical kit components. In one embodiment, the kit contains probes and/or other physical components and the instructions for using the probes and other components are available via the internet. The kit may contain additional items such as a device for mixing samples, probes, and reagents and device for using the kit, e.g., test tubes or mixing utensils.
In another aspect, the present invention provides a means for communicating information about or instructions for one or more of (1) using the polynucleotides of the present invention for detecting the expression of genes differentially expressed in fat animals compared to lean animals in a sample, (2) using the polynucleotides of the present invention for measuring the effect of a test substance on the expression of one or more genes differentially expressed in fat animals compared to lean animals, (3) using the polynucleotides of the present invention for screening a test substance to determine if it is likely to be useful for modulating the amount of adipose tissue on an animal, (4) using the polynucleotides of the present invention for formulating a prognosis that an animal is likely to become fat or developing a diagnosis that an animal is fat, (5) using the polynucleotides of the present invention for manipulating the genome of a non-human animal or the expression of the genome of an animal, (6) using the polynucleotides of the present invention for modulating the expression of one or more genes differentially expressed in fat animals compared to lean animals, particularly fat-associated genes, or modulating the amount of adipose tissue on an animal, (7) using the polynucleotides of the present invention for selecting an animal for inclusion in one or more groups (8) using the polynucleotides of the present invention for using computer system to manipulate data relating to genes differentially expressed in fat animals compared to lean animals, particularly fat-associated genes, (9) administering substances of the present invention to an animal, alone or in combination with the other elements of the present invention, (10) using the substances of the present invention for modulating the amount of adipose tissue on an animal, (11) using the computer system of the present invention, (12) using the kits of the present invention, and (13) instructions for using the methods and compositions of the present invention with one or more drugs or other substances that that modulate the amount of adipose tissue on an animal. The means comprises a document, digital storage media, optical storage media, audio presentation, or visual display containing the information or instructions. In certain embodiments, the communication means is a displayed web site, visual display, kiosk, brochure, product label, package insert, advertisement, handout, public announcement, audiotape, videotape, DVD, CD-ROM, computer readable chip, computer readable card, computer readable disk, computer memory, or combination thereof containing such information or instructions. Useful information includes one or more of (1) methods for promoting the health and wellness of animals and (2) contact information for the animal's caregivers to use if they have a question about the invention and its use. Useful instructions include techniques for using the probes, instructions for performing a gene expression assay, and administration amounts and frequency for the substances. The communication means is useful for instructing on the benefits of using the present invention.
Disclosed herein are typical preferred embodiments of the invention and, although specific terms are employed, they are used in a generic and descriptive sense only and not for purposes of limitation as many modifications and variation of the invention are possible in light of the teachings contained herein. The invention can be further illustrated by the following examples, although it will be understood that these examples are included merely for purposes of illustration and are not intended to limit the scope of the invention unless otherwise specifically indicated.
Isolation of Ribonucleic Acid (RNA) from Tissue
Tissue samples that have been collected, frozen in liquid nitrogen, and thawed are homogenized and processed using a TRIzol® RNA extraction method to produce good quality RNA which is then subjected to further genomic analysis.
Materials: ice, liquid nitrogen, frozen canine or feline tissue, TRIzol® lysis reagent, chloroform minimum 99%, isopropyl alcohol, 70% ethanol (prepared with ethanol, absolute and deionized, RNase-free water), RNase Zap®, deionized water, RNA Storage Solution®, from Ambion.
Equipment: Ultra-Turrax T25 Power Homogenizer, Beckman Coulter Allegra 25R Centrifuge, Eppendorf Centrifuge, forceps, scalpel, hard cutting surface, i.e. cutting board, 1.5 mL DNase and RNase free/sterile microcentrifuge tubes, 50 mL DNase and RNase free/sterile disposable polypropylene tubes, P1000, P200, P20, P10 and P2 Rainin Pipetman pipettes, filter pipette tips for P1000, P200, P20, P10 and P2 pipettes, DNase and RNase free/sterile, and lint free wipes.
Preparations: Prepare 50 mL polypropylene tubes with 4 mL TRIzol® (one tube for each tissue selected for RNA isolation).
Tissue Homogenization: Fill a container capable of holding liquid nitrogen with 3-4 scoops of liquid nitrogen. Place a piece of frozen tissue immediately into the aforementioned container (the tissue should be about the size of a pea) and place the tissue into the appropriate labeled 50 mL polypropylene tube (that already contains 4 mL TRIzol®). Immediately begin homogenization using the Ultra-Turrax T25 Power Homogenizer. Homogenize on the highest setting (6) for 10-15 seconds. Cool the sample on ice for another 10-15 seconds and then repeat. Continue until the tissue is fully homogenized and the solution is cloudy. Upon complete homogenization, cap the 50 mL tube and return to the ice. Incubate the homogenized tissues at room temperature for 5 minutes before proceeding with the isolation procedure.
RNA Isolation: The procedures given in the Invitrogen instructions provided with the TRIzol® reagent are generally followed. Separate the homogenized sample into four 1 mL aliquots in four 1.5 mL microcentrifuge tubes. Add 200 uL of chloroform to each 1 mL aliquot. Cap the tubes, vortex for 15 seconds and then shake up and down. The result should be a pink milky liquid. Incubate the tubes at room temperature for 2-3 minutes. Centrifuge the tubes for 15 minutes at 14,000 rpm and 4° C. Transfer the aqueous phase (top layer) to a sterile 1.5 mL microcentrifuge tube. The typical volume of the aqueous phase which should be transferred to the new tube is about 500 uL. Be sure not to transfer any of the intermediate or lower phase. Precipitate the RNA from solution by adding 500 uL of Isopropyl Alcohol to each microcentrifuge tube containing the aqueous layer. Shake the tubes up and down for at least 20 seconds. Incubate the samples at room temperature for 10 minutes. Centrifuge the samples for 10 minutes, 14,000 rpm at 4° C. Remove the supernatant carefully by aspirating off the liquid being sure not to lose the pellet. Add 1 mL of 70% ethanol to wash the pellet. Dislodge the pellet by flicking the tube (or tapping the tube on the bench top) and shake to mix. Centrifuge for 5 minutes, 8,200 rpm at 4° C. Remove the supernatant carefully by aspirating off the liquid being sure not to lose the pellet. Using a lint free wipe carefully soak up excess ethanol to make sure the pellet is dry. Resuspend each pellet into 30 uL of RNA Storage Solution. Mix gently by pipetting until the RNA goes back into solution and then store at −80° C. It may be necessary to vortex the sample for a few seconds at a low speed to facilitate the resuspension of the RNA. If this is necessary spin down the samples, using the microcentrifuge, prior to freezing.
RNA Cleaning: The procedures given in the RNeasy® Mini Handbook are followed.
RNA Isolation from Cells Cultured in OptiCell Chambers Using the RNeasy Mini Kit.
Cells cultured from mammalian cell lines are used to isolate good quality RNA which is then used for future downstream genomic analysis. All work related to the culturing of the cells is to be done under strict aseptic conditions.
Reagents: 10×PBS, deionized H2O, absolute ethanol, RNA Storage Solution, β-Mercaptoethanol, RNase Zap®, Buffer RLT, and Buffer RW1 and Buffer RPE (provided in the RNeasy Mini Kit)
Equipment/Materials: RNeasy Mini Kit, QIAshredder spin columns, OptiCell knife, 20 mL sterile syringe, OptiCell tips, Cell scraper, P1000 Pipetman pipette, Rainin, P200 Pipetman pipette, Rainin, 100-100 uL filtered pipette tips, 1-200 uL filtered pipette tips, sterile transfer pipettes, 55 mL sterile solution basin, 1.5 mL sterile microcentrifuge tubes, and Eppendorf Microcentrifuge.
Solutions: Buffer RLT (stock provided in RNeasy Mini Kit); —Add 100 uL of β-Mercaptoethanol per 10 mL of Buffer RLT prior to beginning protocol. 70% Ethanol: Make 50 mL of 70% ethanol by adding 35 mL absolute ethanol to 15 mL deionized, RNase-free water. 1×PBS: RNase-free water. Filter the solution using a 0.22 um filter.
Procedure: Removing Cells from the OptiCell Chamber (proceed one OptiCell at a time). Check the cells under a microscope to ensure that the cells are alive before isolating RNA. Remove and discard the cell culture medium. Using the OptiCell knife cut away the top membrane exposing the cells on the lower membrane. Wash the membrane to which the cells are attached three times with 1×PBS. Pipette 600 uL of the Buffer RLT solution (containing β-Mercaptoethanol) onto the center of the membrane to which the cells are attached. Using the cell scraper, gently spread the Buffer RLT over the entire surface of the membrane, and then collect the liquid in one corner. Pipette off the entire volume of Buffer RLT and place into a QIAshredder spin column.
RNA Isolation: Centrifuge the QIAshredder spin columns at 14,000 rpm for 2 minutes. Discard the spin column but keep the collection tube and its contents. Add 600 uL of 70% ethanol to the collection tube and mix well by pipetting (the total volume now 1.2 mL). Transfer 600 uL of the cell lysate to an RNeasy mini column and centrifuge for 15 seconds at 14,000 rpm. Discard the flow through but keep the collection tube and the spin column. Transfer the remaining volume of cell lysate (˜600 uL) to the spin column and repeat the centrifugation. Discard the flow through but keep the collection tube and the spin column. Add 700 uL Buffer RW1 to the spin column. Centrifuge for 15 seconds at 14,000 rpm to wash the column. Discard the flow through and the collection tube. Transfer the spin column to a new 2 mL collection tube and add 500 uL Buffer RPE to the column. Centrifuge for 15 seconds at 14,000 rpm. Discard the flow through, keep the collection tube/column. Add another 500 uL Buffer RPE to the column. Centrifuge for 2 minutes at 14,000 rpm. Transfer the spin column to a 1.5 mL collection tube. Add 30 uL of RNA Storage Solution directly to the silica gel membrane and centrifuge for 1 minute at 14,000 rpm to elute the RNA. Store the final RNA at −70° C.
Using the Agilent 2100 Bioanalyzer and the RNA 6000 Nano Assay, analyze RNA isolated from cultured mammalian cells, lymphocytes or tissues for quality.
Reagents: RNA 6000 Nano gel matrix, RNA 6000 Nano dye concentrate, RNA 6000 Nano Marker, (all of the above reagents are contained in the RNA 6000 Nano Assay kit, Agilent), RNA 6000 ladder, RNase Zap, and RNase-free water, from Ambion.
Equipment/Other Materials: Agilent Chip Priming Station, Agilent, RNA 6000 chip, Agilent, electrode cleaners, P2, P10, P200, and P1000 Rainin Pipetman pipettes, sterile, DNase/RNase free filtered pipette tips, 1.5 mL microcentrifuge tubes, sterile, vortex, IKA vortex mixer, microcentrifuge, and heating block.
Procedure: The procedure is given in the Reagent Kit Guide, RNA 6000 Nano Assay, Edition November 2003, by Agilent Technologies. The procedures are followed as given in the Guide, with the following modifications: Preparing the Gel, pg. 17-rather than separating the filtered gel into aliquots of 65 uL each, keep the stock filtered gel in the original microcentrifuge tube and aliquot the 65 uL as needed. Loading the RNA 6000 Nano Marker, pg. 22—add 1 uL of RNase-free water (instead of RNA 6000 Nano Marker) to each sample well that will not contain sample. Not only will this conserve the amount of Marker used but also serves as a negative control to see that none of the reagents are contaminated, including the RNase-free water. Loading the Ladder and Samples, pg. 23—heat denature the samples and RNA 6000 Ladder for an additional 30 seconds (total of 2.5 minutes) at 71° C. Starting the Chip Run, pg. 26-choose the “Eukaryote Total RNA Nano” option from the assay menu.
Gene expression is analyzed using Affymetrix Canine 1 and Canine 2 GeneChip® Arrays are available commercially from Affymetrix, Inc., Santa Clara, Calif. 95051. Total RNA is reverse transcribed into cDNA. The cDNA is used to generate cRNA which is fragmented and used as probes for GeneChip hybridization. The gene chip is washed and the hybridization signal is measured with an Affymetrix laser scanner. The hybridization data is then validated and normalized for further analysis.
Materials: Affymetrix provides most of the reagents and kit. Other reagents listed in the Affymetrix Manual but not supplied in the kit may be obtained separately (refer to GeneChip Expression Analysis Technical Manual (701021 Rev.4) for details), RNase Zap® and deionized water.
Equipment: Eppendorf microcentrifuge, 1.5 mL DNase and RNase free/sterile microcentrifuge tubes, 50 mL DNase and RNase free/sterile disposable polypropylene tubes, P1000, P200, P205 P10 and P2 Rainin Pipetman pipettes, Filter pipette tips for P1000, P200, P20, P10 and P2 pipettes, DNase and RNase free/sterile, and Peltier Thermal Cycler PTC-200.
Procedure: follow all procedures exactly as described in GeneChip Expression Analysis Technical Manual (Affymetrix Copyright 1999-2003). Use 5 microgram of total RNA for the first strand cDNA synthesis. Use either Peltier Thermal Cycler PTC-200 or heat block for temperature control on reactions and probe denaturing. The quality control is performed using RNA NanoDrop chips with BioAnalyer 2100. Use 100 Format (Midi Array) for the canine genechip.
Adipose tissue samples are obtained from 16 (3 lean and 13 fat) canine animals diagnosed as either “fat” or “lean” using conventional methods. The “fatness” or “leanness” of an animal is determined based on measurements by DEXA using conventional methods or based on a 5 point body condition scoring system. For example, an animal is considered lean if it has a body condition score of 2 or 2.5 and/or a DEXA total body fat percentage of 27% or less. An animal is considered to be fat if it has a body condition score of 4 or higher and a total body fat percentage of 30% or higher. All tissue samples are snap frozen in liquid nitrogen immediately after removal from the animal.
The tissues are analyzed using Affymetrix “Canine-2” canine gene chip according to conventional methods in order to determine which genes, if any, are differentially expressed in fat animals compared to lean animals. Data from the fat and lean samples are compared and analyzed using the GeneSpring and R-Bioconductor software. For any given gene to be assigned a “present” call, it had to exhibit a 2-fold change in expression level to be considered for further scrutiny. Furthermore, genes that are present in only one condition and are either “absent” or “marginal” in the other group are also selected for further scrutiny. Results are provided in the tables below:
Homo sapiens thymopoietin (TMPO), transcript variant 3, mRNA
Homo sapiens zinc finger protein 227 (ZNF227), mRNA
Homo sapiens, clone IMAGE: 5171802, mRNA
Caenorhabditis elegans BMP receptor Associated protein family member (bra-1)
Homo sapiens mRNA for Acetyl-CoA carboxylase 2 (ACACB gene)
Mus musculus Murr1 and U2af1-rs1 genes, partial and complete cds
Campylobacter jejuni 81-176 (pflA) gene, complete cds, orf1 and orf2, partial cds
Plasmodium yoelii yoelii str. 17XNL hypothetical protein (PY04060) mRNA, partial
Homo sapiens zinc finger protein 233 (ZNF233), mRNA
Homo sapiens zinc finger protein 233 (ZNF233), mRNA
Homo sapiens bcl6 gene, 5′ flanking region
Homo sapiens G protein-coupled receptor 51 (GPR51), mRNA
C. familiaris mRNA for orphan nuclear receptor dNGFI-B protein
L. japonicus mRNA for small GTP-binding protein, RAB7C
Canis familiaris chemokine (C-C motif) ligand 2 (CCL2), mRNA
Canis familiaris inducible T-cell co-stimulator (ICOS) mRNA, complete cds
Nicotiana tabacum mRNA for cyclin D3.1 protein (CycD3.1)
Canis familiaris chemokine (C-C motif) ligand 8 (CCL8), mRNA
Oryza sativa (japonica cultivar-group) chromosome 11 clone B1356E08,
Homo sapiens transmembrane 4 L six family member 18, mRNA (cDNA clone
Schizosaccharomyces pombe 972h-isoleucine-tRNA ligase (SPBC8D2.06),
Homo sapiens acetyl-Coenzyme A carboxylase beta, mRNA (cDNA clone
Canis familiaris podoplanin (PDPN), mRNA
Homo sapiens cDNA FLJ13037 fis, clone NT2RP3001268, highly similar to Homo
sapiens zinc finger protein ZNF228 (ZNF228) mRNA
Homo sapiens serine/threonine protein kinase Kp78 splice variant CTAK75a
Canis familiaris IgA heavy chain constant region gene, partial cds
Homo sapiens cyclin-dependent kinase inhibitor mRNA, partial cds
Canis familiaris ucp2 mRNA for uncoupling protein 2, complete cds
Canis familiaris podoplanin (PDPN), mRNA
Canis familiaris immunoglobulin gamma heavy chain C mRNA, complete cds
Homo sapiens mRNA for Acetyl-CoA carboxylase 2 (ACACB gene)
Homo sapiens acetyl-Coenzyme A carboxylase beta (ACACB), mRNA
Nitella japonica chromoplast atpB gene for ATP synthase beta subunit, partial
Homo sapiens solute carrier family 26, member 7 (SLC26A7), transcript variant 1,
Homo sapiens leucine rich repeat containing 17 (LRRC17), transcript variant 1,
Homo sapiens sortilin-related receptor, L(DLR class) A repeats-containing
Canis familiaris dystrophin (DMD) mRNA, 5′ untranslated region, alternatively
Pisum sativum ribosomal protein L34 homolog (RPL34) mRNA, complete cds
Canis familiaris serum amyloid A protein (SAA) mRNA, partial cds
Homo sapiens BTB (POZ) domain containing 11 (BTBD11), transcript variant 3,
Homo sapiens lamin B1 (LMNB1), mRNA
Macaca fascicularis testis cDNA clone: QtsA-13105, similar to human armadillo
Homo sapiens KIAA0040 (KIAA0040), mRNA
Homo sapiens thyroid hormone receptor, beta (erythroblastic leukemia viral (verb-
Plasmodium yoelii yoelii str. 17XNL hypothetical protein (PY01308) mRNA, partial
Homo sapiens glucose transporter 14 short isoform mRNA, complete cds;
Ustilago maydis 521 hypothetical protein (UM05082.1), mRNA
Canis familiaris mRNA for putative secreted frizzled related protein 2 (sfrp2 gene)
Homo sapiens BAC clone RP11-216H12 from 4, complete sequence
C. familiaris MHC class Ib gene (DLA-79) gene, complete CDS
Homo sapiens claudin 4, mRNA (cDNA clone MGC: 1778 IMAGE: 3349211),
Homo sapiens ELOVL family member 6, elongation of long chain fatty acids
Homo sapiens genomic DNA, chromosome 18 clone: RP11-883A18, complete
Canis familiaris T cell receptor beta chain hcvb3 (hcvb3) mRNA, partial cds
Canis familiaris dihydrodiol dehydrogenase (dimeric) (DHDH), mRNA
Canis familiaris glucose-6-phosphatase mRNA, complete cds
Tursiops truncatus IgM heavy chain mRNA, complete cds
Homo sapiens serum/glucocorticoid regulated kinase 2, mRNA (cDNA clone
Xenopus laevis ubiquitously transcribed tetratricopeptide repeat gene, Y-linked,
Canis familiaris nitric oxide synthase 2A (inducible, hepatocytes) (NOS2A),
Canis familiaris triadin isoform 3 mRNA, complete cds
Homo sapiens neuropilin 2 (NRP2) gene, complete cds, alternatively spliced
Petunia integrifolia subsp. inflata S2 self-incompatibility ribonuclease (S2-RNase)
Homo sapiens mRNA; cDNA DKFZp686G0638 (from clone DKFZp686G0638)
Homo sapiens B-box and SPRY domain containing (BSPRY), mRNA
Oryza sativa (japonica cultivar-group) genomic DNA, chromosome 1, complete
Homo sapiens tetratricopeptide repeat domain 25, mRNA (cDNA clone
Homo sapiens mRNA for laminin alpha 2 subunit precursor variant protein
Homo sapiens lipin 1 (LPIN1), mRNA
Bos taurus similar to Asporin precursor (Periodontal ligament associated protein
Aspergillus nidulans FGSC A4 hypothetical protein (AN4185.2), mRNA
Sus scrofa epidermal growth factor precursor (EGF) mRNA, complete cds
Canis familiaris IgA heavy chain constant region gene, partial cds
Canis familiaris mRNA for metallothionein-II, complete cds
Macaca fascicularis mRNA, clone QnpA-12979: similar to Homo sapiens
Homo sapiens leucyl/cystinyl aminopeptidase (LNPEP), transcript variant 2,
Homo sapiens adenosine monophosphate deaminase (isoform E) (AMPD3),
Hordeum vulgare subsp. vulgare cultivar Morex inducer of CBF expression 2
Pongo pygmaeus mRNA; cDNA DKFZp469L0319 (from clone DKFZp469L0319)
Homo sapiens cDNA clone MGC: 51010 IMAGE: 5270267, complete cds
Drosophila melanogaster CG18211-PA (betaTry) mRNA, complete cds
Homo sapiens protein upregulated in metastatic prostate cancer mRNA,
Bos taurus homeodomain only protein, mRNA (cDNA clone MGC: 127764
Leishmania major strain Friedlin hypothetical protein (LMJ_1048) mRNA, partial
Homo sapiens alanine-glyoxylate aminotransferase 2-like 1, mRNA (cDNA clone
Canis familiaris IgA heavy chain constant region gene, partial cds
Felis catus CD8 antigen, beta polypeptide (CD8B), mRNA
Homo sapiens mRNA for alanine:glyoxylate aminotransferase 2 homolog 1,
Canis familiaris metallothionein 1X (MT1X), mRNA
Canis familiaris metallothionein 1X (MT1X), mRNA
Homo sapiens WNK lysine deficient protein kinase 3 (WNK3), transcript variant 2,
Canis familiaris IgA heavy chain constant region gene, partial cds
Xenopus laevis MGC83953 protein, mRNA (cDNA clone MGC: 83953
Pongo pygmaeus C6 gene for complement component 6, partial cds
Plasmodium yoelii yoelii str. 17XNL hypothetical protein (PY02022) mRNA, partial
Pongo pygmaeus mRNA; cDNA DKFZp470P1633 (from clone DKFZp470P1633)
Canis familiaris podoplanin (PDPN), mRNA
Pongo pygmaeus mRNA; cDNA DKFZp468I0813 (from clone DKFZp468I0813)
Homo sapiens zinc finger, DHHC-type containing 17 (ZDHHC17), mRNA
Bos taurus mRNA for VSGP/F-spondin, complete cds
Sus scrofa estrogen sulfotransferase (STE), mRNA
Homo sapiens Kallmann syndrome 1 sequence (KAL1), mRNA
Homo sapiens Kruppel-like factor 9 (KLF9), mRNA
Mus musculus expressed sequence AI854703 (AI854703), mRNA
Homo sapiens WAP four-disulfide core domain 1 (WFDC1), mRNA
Homo sapiens armadillo repeat containing 9, mRNA (cDNA clone MGC: 74894
Homo sapiens jun dimerization protein gene, partial cds; cfos gene, complete
Homo sapiens EGR1 gene for early growth response protein 1
Homo sapiens early growth response 1, mRNA (cDNA clone MGC: 88036
Homo sapiens 12 BAC RP11-181C3 (Roswell Park Cancer Institute Human BAC
Homo sapiens mRNA; cDNA DKFZp686J04124 (from clone DKFZp686J04124)
Homo sapiens zinc finger protein 227 (ZNF227), mRNA
Homo sapiens solute carrier family 7 (cationic amino acid transporter, y+ system),
Homo sapiens PAC clone RP5-1003N18 from 14q24.3, complete sequence
Homo sapiens serum/glucocorticoid regulated kinase 2 mRNA, complete cds
Homo sapiens BAC clone RP11-198M19 from 2, complete sequence
Homo sapiens FK506 binding protein 5 (FKBP5), mRNA
Homo sapiens pyruvate dehydrogenase kinase, isozyme 4, mRNA (cDNA clone
Homo sapiens BAC clone RP11-617I14 from 4, complete sequence
Homo sapiens chromosome 3 clone RP11-6B7, complete sequence
Homo sapiens acetyl-Coenzyme A carboxylase beta (ACACB), mRNA
Homo sapiens 12 BAC RP11-451H11 (Roswell Park Cancer Institute Human
Homo sapiens BAC clone RP11-398G12 from 2, complete sequence
Homo sapiens suprabasin (SBSN), mRNA
Homo sapiens 3 BAC RP11-1D19 (Roswell Park Cancer Institute Human BAC
Homo sapiens cDNA FLJ38032 fis, clone CTONG2013352, moderately similar to
Homo sapiens zinc finger protein 233 (ZNF233), mRNA
sapiens (human)
Homo sapiens 3 BAC RP11-211G3 (Roswell Park Cancer Institute Human BAC
Homo sapiens transmembrane protein with EGF-like and two follistatin-like
Homo sapiens nuclear receptor subfamily 4, group A, member 1, transcript
Homo sapiens cDNA FLJ39913 fis, clone SPLEN2018643, highly similar to
Homo sapiens mRNA for activation-inducible lymphocyte immunomediatory
Homo sapiens cDNA clone IMAGE: 5175186, containing frame-shift errors
Homo sapiens ELOVL family member 6, elongation of long chain fatty acids
Homo sapiens BAC clone RP11-384E2 from 4, complete sequence
Homo sapiens chromosome 14 open reading frame 119, mRNA (cDNA clone
Homo sapiens chemokine (C-C motif) ligand 8 (CCL8), mRNA
Homo sapiens transmembrane 4 L six family member 18 (TM4SF18), mRNA
Homo sapiens clone 25081 tropomodulin mRNA sequence
Homo sapiens acetyl-Coenzyme A carboxylase beta (ACACB), mRNA
Homo sapiens podoplanin (PDPN), transcript variant 3, mRNA
Homo sapiens zinc finger protein 228 (ZNF228), mRNA
Homo sapiens cDNA clone MGC: 88772 IMAGE: 4765168, complete cds
Homo sapiens glutamate-cysteine ligase, catalytic subunit (GCLC), mRNA
Homo sapiens gene for p16/CDKN2A, complete cds
Homo sapiens ATPase, H+ transporting, lysosomal 42 kDa, V1 subunit C isoform
Homo sapiens interferon regulatory factor 4, mRNA (cDNA clone MGC: 23069
Homo sapiens ARV1 homolog (yeast) (ARV1), mRNA
Homo sapiens podoplanin (PDPN), transcript variant 1, mRNA
Homo sapiens glutamate-cysteine ligase, catalytic subunit (GCLC), mRNA
Homo sapiens clone DNA44205 NLGN4 (UNQ365) mRNA, complete cds
Homo sapiens BAC clone RP11-197H3 from 2, complete sequence
Homo sapiens carnitine palmitoyltransferase 1A (liver) (CPT1A), nuclear gene
Homo sapiens hypoxia inducible factor 3, alpha subunit, mRNA (cDNA clone
Homo sapiens claudin 10 (CLDN10), transcript variant 1, mRNA
Homo sapiens acetyl-Coenzyme A carboxylase beta (ACACB), mRNA
Homo sapiens 12q24 BAC RGPI11-443D10 (Roswell Park Cancer Institute
Homo sapiens carnitine palmitoyltransferase 1A (liver), transcript variant 2,
Homo sapiens FK506 binding protein 5, mRNA (cDNA clone MGC: 34489
Homo sapiens NOVH protein mRNA, complete cds
Homo sapiens partial CPT1A gene for carnitine O-palmitoyltransferase 1,
Homo sapiens chromosome 19, cosmid R31341, complete sequence
Homo sapiens solute carrier family 26, member 7 (SLC26A7), transcript variant 1,
Mus musculus 17 days embryo stomach cDNA, RIKEN full-length enriched
Homo sapiens jun B proto-oncogene (JUNB), mRNA
Homo sapiens chromosome 8, clone RP11-734H6, complete sequence
Homo sapiens mRNA for Cdc7-related kinase, complete cds
Homo sapiens leucine rich repeat containing 17 (LRRC17), transcript variant 1,
Homo sapiens sortilin-related receptor, L(DLR class) A repeats-containing
Homo sapiens chromosome 8, clone CTD-3071K10, complete sequence
Macaca fascicularis brain cDNA, clone: QccE-21970, similar to human aldolase
Homo sapiens chromosome 17, clone 193h18, complete sequence
Homo sapiens BAC clone RP11-1191J2 from 4, complete sequence
Homo sapiens full open reading frame cDNA clone RZPDo834A0126D for gene
Homo sapiens diphtheria toxin receptor (heparin-binding epidermal growth factor-
Homo sapiens BTB (POZ) domain containing 11 (BTBD11), transcript variant 3,
Homo sapiens regulator of G-protein signalling 1, mRNA (cDNA clone MGC: 9198
Homo sapiens nudE nuclear distribution gene E homolog 1 (A. nidulans), mRNA
Homo sapiens sortilin-related receptor, L(DLR class) A repeats-containing
Homo sapiens chromosome 5 clone RP11-1152B5, complete sequence
Homo sapiens serum-inducible kinase mRNA, complete cds
Homo sapiens mRNA; cDNA DKFZp313N1532 (from clone DKFZp313N1532)
Homo sapiens leucine rich repeat containing 39 (LRRC39), mRNA
Homo sapiens lamin B1 (LMNB1), mRNA
Homo sapiens reelin (RELN), transcript variant 2, mRNA
Homo sapiens cDNA FLJ41271 fis, clone BRAMY2036396
Macaca fascicularis testis cDNA clone: QtsA-13105, similar to human armadillo
Homo sapiens chromosome 17, clone CTD-3193K9, complete sequence
sapiens (human)
Homo sapiens CC chemokine ligand 4L2f (CCL4L) mRNA, CCL4L-2 allele,
Homo sapiens thyroid hormone receptor, beta (erythroblastic leukemia viral (verb-
Homo sapiens 3 BAC RP11-211G3 (Roswell Park Cancer Institute Human BAC
Homo sapiens 12 BAC RP4-605O3 (Roswell Park Cancer Institute Human BAC
Homo sapiens polo-like kinase 2 (Drosophila) (PLK2), mRNA
Homo sapiens cDNA FLJ36603 fis, clone TRACH2015180, highly similar to
Homo sapiens BAC clone RP11-216H12 from 4, complete sequence
Homo sapiens HLA-C gene for MHC class I antigen, CW*15021 allele, exons 1-8
Homo sapiens L-pipecolic acid oxidase (LPIPOX) mRNA, complete cds
Homo sapiens mRNA; cDNA DKFZp686C24224 (from clone DKFZp686C24224)
Homo sapiens claudin 4, mRNA (cDNA clone MGC: 1778 IMAGE: 3349211),
Homo sapiens cDNA: FLJ23378 fis, clone HEP16248
Homo sapiens gp250 precursor, mRNA, complete cds
Homo sapiens fem-1 homolog b (C. elegans), mRNA (cDNA clone MGC: 19792
Homo sapiens aminoacylase 1-like 2, mRNA (cDNA clone IMAGE: 5262663),
Homo sapiens hypothetical protein FLJ20920, mRNA (cDNA clone MGC: 19867
Homo sapiens genomic DNA, chromosome 18 clone: RP11-883A18, complete
Homo sapiens hypothetical protein LOC392636, mRNA (cDNA clone
Homo sapiens partial BV03S1J2.2 gene for T-cell receptor beta, variable region
Homo sapiens ligand effect modulator-6 (LEM6) mRNA, complete cds
Homo sapiens chromosome 16 clone CTA-237H1, complete sequence
Homo sapiens phosphatidylinositol-4-phosphate 5-kinase, type I, beta (PIP5K1B),
Homo sapiens dihydrodiol dehydrogenase (dimeric) (DHDH), mRNA
Homo sapiens BAC clone RP11-433O3 from 4, complete sequence
sapiens (human)
Homo sapiens creatine kinase, mitochondrial 1B (CKMT1B), nuclear gene
Homo sapiens cDNA FLJ45560 fis, clone BRTHA3003417
Homo sapiens chromosome 15, clone RP11-253M7, complete sequence
Homo sapiens C1q-C mRNA, complete cds
Homo sapiens ARV1 homolog (yeast) (ARV1), mRNA
Homo sapiens mRNA; cDNA DKFZp781J0852 (from clone DKFZp781J0852)
Homo sapiens full open reading frame cDNA clone RZPDo834F0920D for gene
Homo sapiens complement component 1, q subcomponent, alpha polypeptide,
Homo sapiens cDNA FLJ16122 fis, clone BLADE2008995
Homo sapiens chromosome 20 open reading frame 155 (C20orf155), mRNA
Homo sapiens neuropilin 2 (NRP2) gene, complete cds, alternatively spliced
Homo sapiens chromosome 4 clone RP11-603B8, complete sequence
Homo sapiens protein phosphatase 2C, magnesium-dependent, catalytic subunit,
sapiens (human)
Homo sapiens mRNA; cDNA DKFZp779B086 (from clone DKFZp779B086)
sapiens (human)
Homo sapiens sulfotransferase family, cytosolic, 1B, member 1 (SULT1B1),
Homo sapiens cDNA FLJ30638 fis, clone CTONG2002721, weakly similar to
Homo sapiens cytochrome P450, family 26, subfamily B, polypeptide 1
Homo sapiens BAC clone RP11-678H22 from 4, complete sequence
Homo sapiens chromosome 11, clone RP13-25N22, complete sequence
Homo sapiens cystatin 9-like (mouse), mRNA (cDNA clone MGC: 34724
Homo sapiens dystonin (DST), transcript variant 1eA, mRNA
Homo sapiens arrestin domain containing 2, transcript variant 1, mRNA (cDNA
Homo sapiens cDNA FLJ14958 fis, clone PLACE4000052, highly similar to Homo
sapiens ATP cassette binding transporter 1 (ABC1) mRNA
Homo sapiens zinc finger and BTB domain containing 16, transcript variant 2,
Homo sapiens chromosome 9 open reading frame 72, mRNA (cDNA clone
Homo sapiens mitogen-activated protein kinase kinase kinase 15 (MAP3K15),
Homo sapiens laryngeal carcinoma related protein 1 mRNA, complete cds
Homo sapiens tetratricopeptide repeat domain 25, mRNA (cDNA clone
Homo sapiens mRNA for laminin alpha 2 subunit precursor variant protein
Homo sapiens protein phosphatase 2C, magnesium-dependent, catalytic subunit,
Homo sapiens BAC clone RP11-484O9 from 2, complete sequence
Homo sapiens asporin (LRR class 1) (ASPN), mRNA
Homo sapiens clone DNA34392 ASPN (UNQ215) mRNA, complete cds
Homo sapiens solute carrier family 27 (fatty acid transporter), member 6
Homo sapiens chromosome 3 clone RP11-189A1, complete sequence
Homo sapiens dehydrogenase/reductase (SDR family) member 9 (DHRS9),
Homo sapiens truncated epidermal growth factor (beta-urogastrone) (EGF) gene,
Homo sapiens cDNA clone MGC: 86772 IMAGE: 4765168, complete cds
Homo sapiens PAC clone RP1-85D24 from Y, complete sequence
Homo sapiens complement component 1, q subcomponent, beta polypeptide,
Homo sapiens chromosome 3 clone RP11-944L7, complete sequence
Homo sapiens SH3 domain protein D19 (SH3D19), mRNA
Homo sapiens mRNA; cDNA DKFZp686E15208 (from clone DKFZp686E15208)
Macaca fascicularis mRNA, clone QnpA-12979: similar to Homo sapiens
Homo sapiens chromosome 11, clone RP11-68C8, complete sequence
Homo sapiens tocopherol (alpha) transfer protein (ataxia (Friedreich-like) with
Homo sapiens cDNA FLJ32190 fis, clone PLACE6002102
Homo sapiens CCBL1 gene, last two exons
Homo sapiens cysteine conjugate-beta lyase; cytoplasmic (glutamine
Homo sapiens triggering receptor expressed on myeloid cells 2 (TREM2), mRNA
Homo sapiens chromosome 21 open reading frame 24 isoform 7 (C21orf24)
Homo sapiens mRNA; cDNA DKFZp779B086 (from clone DKFZp779B086)
Homo sapiens BAC clone RP11-642E20 from 4, complete sequence
Homo sapiens leucine rich repeat containing 47 (LRRC47), mRNA
Homo sapiens translation initiation factor 2 (MTIF2) gene, exons 6 through 9;
Homo sapiens insulin receptor, mRNA (cDNA clone IMAGE: 4823710), partial cds
Homo sapiens ring finger protein 150 (RNF150), mRNA
Homo sapiens chromosome 5 clone CTC-361G14, complete sequence
Homo sapiens matrix metallopeptidase 2 (gelatinase A, 72 kDa gelatinase, 72 kDa
Homo sapiens Zic family member 3 heterotaxy 1 (odd-paired homolog,
Drosophila) (ZIC3), mRNA
Homo sapiens BAC clone RP11-395A12 from 2, complete sequence
Homo sapiens genomic DNA, chromosome 18 clone: RP11-815J4, complete
Homo sapiens protein upregulated in metastatic prostate cancer mRNA,
Homo sapiens Kruppel-like factor 5 (intestinal), mRNA (cDNA clone MGC: 52153
Homo sapiens (human)
Homo sapiens interferon-related developmental regulator 1 (IFRD1), transcript
Homo sapiens (human)
Homo sapiens homeodomain-only protein, mRNA (cDNA clone MGC: 20820
Homo sapiens coagulation factor II receptor-like 2 (F2RL2) gene, complete cds
Homo sapiens cysteine conjugate-beta lyase; cytoplasmic (glutamine
Homo sapiens alpha-2-macroglobulin, mRNA (cDNA clone MGC: 47683
Homo sapiens phospholipid transfer protein, transcript variant 1, mRNA (cDNA
Homo sapiens WD repeat domain 66 (WDR66), mRNA
Homo sapiens mRNA for alanine:glyoxylate aminotransferase 2 homolog 1,
Homo sapiens immunoglobulin alpha 2m(1) heavy chain constant region gene,
Homo sapiens BAC clone RP11-348G16 from 2, complete sequence
Homo sapiens erbb2 interacting protein (ERBB2IP), transcript variant 2, mRNA
Homo sapiens mRNA for alanine:glyoxylate aminotransferase 2 homolog 1,
Homo sapiens phospholipase C, delta 4 (PLCD4), mRNA
Homo sapiens cDNA FLJ39109 fis, clone NTONG2005137, highly similar to
Homo sapiens mRNA; cDNA DKFZp586M2121 (from clone DKFZp586M2121)
Homo sapiens HIF-3A mRNA for hypoxia-inducible factor-3 alpha, complete cds
Homo sapiens BAC clone CTB-118E13 from 7, complete sequence
Homo sapiens mRNA; cDNA DKFZp313I2220 (from clone DKFZp313I2220);
Homo sapiens WNK lysine deficient protein kinase 3 (WNK3), transcript variant 1,
Homo sapiens immunoglobulin alpha 2m(1) heavy chain constant region gene,
Macaca fascicularis testis cDNA clone: QtsA-11169, similar to human hypothetical
Macaca fascicularis brain cDNA, clone: QflA-10289, similar to human TU3A
Homo sapiens centrosome and spindle pole associated protein 1 (CSPP1),
Homo sapiens osteomodulin (OMD), mRNA
Homo sapiens 12 BAC RP11-424C20 (Roswell Park Cancer Institute Human
Homo sapiens family with sequence similarity 59, member A (FAM59A), mRNA
Homo sapiens ps20 WAP-type four-disulfide core domain protein mRNA,
Homo sapiens chromosome 5 clone CTC-428I11, complete sequence
Homo sapiens chromosome 5 clone CTC-229P9, complete sequence
Homo sapiens chromosome 8, clone RP13-895A16, complete sequence
Homo sapiens lung type-I cell membrane-associated protein hT1a-2 (hT1a-2)
Homo sapiens ATPase, H+ transporting, lysosomal 42 kDa, V1 subunit C isoform
Homo sapiens aminopeptidase puromycin sensitive (NPEPPS), mRNA
Homo sapiens chromosome 5 clone CTC-575N7, complete sequence
Homo sapiens BAC clone RP11-308K2 from 4, complete sequence
Homo sapiens chromosome 5 clone RP11-270H9, complete sequence
Homo sapiens coiled-coil domain containing 18, mRNA (cDNA clone
Homo sapiens, clone RP11-44B13, complete sequence
Macaca fascicularis testis cDNA clone: QtsA-14119, similar to human lipin 1
Bos taurus phytanoyl-CoA hydroxylase [human: Refsum disease], mRNA (cDNA
Homo sapiens chromosome 6 open reading frame 81 (C6orf81), mRNA
Homo sapiens coagulation factor II receptor-like 2 (F2RL2) gene, complete cds
Homo sapiens WD repeat domain 66, mRNA (cDNA clone MGC: 33630
Macaca fascicularis testis cDNA, clone: QtsA-18294, similar to human interferon-
sapiens (human)
Homo sapiens cysteine conjugate-beta lyase; cytoplasmic (glutamine
Homo sapiens chromosome 3 open reading frame 14, mRNA (cDNA clone
Homo sapiens chromosome 15 clone CTD-2270N23 map 15q21, complete
Homo sapiens mRNA; cDNA DKFZp667H2312 (from clone DKFZp667H2312)
Homo sapiens fibronectin type III domain containing 1 (FNDC1), mRNA
Homo sapiens chromosome 16 clone RP11-486L19, complete sequence
Homo sapiens BAC clone RP11-178D14 from 2, complete sequence
Homo sapiens PAC clone RP5-839O24 from 7, complete sequence
Homo sapiens 3 BAC RP11-364F11 (Roswell Park Cancer Institute Human BAC
Affymetrix canine gene chips Canine-1 and Canine-2 are used to determine the effect of various test substances or ingredients such as MCTs; TAGs; ALA; EPA; DHA; linoleic acid; stearic acid (SA), conjugated linoleic acid (CLA), GLA; arachidonic acid; lecithin; vitamin A, vitamin D, vitamin E, vitamin K, riboflavin, niacin, pyridoxine, pantothenic acid, folic acid, biotin vitamin C, catechin, quercetin, theaflavin; ubiquinone; lycopene, lycoxanthin; resveratrol; α-lipoic acid; L-carnitine; D-limonene; glucosamine; S-adenosylmethionine; chitosan, various materials containing one or more of these compounds, and various combination thereof on gene expression in four canine cell lines and appropriate controls. Each ingredient is tested in two concentrations as illustrated for selected sample ingredients shown in Table 6. The solvent at the higher of the two concentrations is used as a control. Four canine cell lines are used: CCL34 (kidney), CRL1430 (thymus), CCL183 (bone) (obtained from The American Tissue Culture Collection) and CTAC (thyroid) (See, Measurement of NK Activity in Effector Cells Purified from Canine Peripheral Lymphocytes, Veterinary Immunology and Immunopathology, 35 (1993) 239-251). A cell line treated with an ingredient at a specific concentration is referred to as “treatment” and an untreated sample is referred to as “control.” The words “genes” and “probes” are used synonymously in this method. Gene expression is measured for the treatment cell lines and controls using the instructions provided with the Affymetrix chips.
The gene expression data is determined to be either “up” or “down”-regulated for any given treatment. The decision on whether a gene is “up” or “down” is based on the fold change, which is calculated as treatment intensity/control intensity for each individual probe. The fold change is considered down-regulated if its value is <1/1.5 (for across all 4 cell lines analysis) or <½ (for within cell lines analysis) and is up-regulated if it is >1.5 (for across all 4 cell lines analysis) or >2 (for within cell lines analysis). Also, a probe is considered significant for further scrutiny if it is called as present in only one of the conditions being compared (treatment or control) and is “absent” or “marginal” in the other and the fold change is significant according to the software used. Probes that appear to be regulated in opposite directions in the two treatments are excluded from further analysis.
The raw data is analyzed using GeneSpring version 7.0 (GS) software (Agilent Corporation) and validated using the R-Bioconductor (RB) freeware. Both software packages are used to compute probe intensities from the CEL files generated by the Affymetrix Instrument. The Present/Absent/Marginal calls per probe and P-values are computed using the R-Bioconductor and GeneSpring software separately.
Two schemes are used for data analysis. First; “across cell lines” and “within individual cell lines.” In the first scheme, genes are selected for scoring provided they are found to be significant and common across all cell-lines. The “across cell lines” yields the highest confidence data with minimum noise and may provide the best possible clues as to which genes are affected by individual ingredients. In the second scheme, only those genes that show a significant fold change in the two treatments according to both software packages within an individual cell lines are scored. A sample of the data obtained from these experiments is shown in Table 7. Table 7 shows the correlation between treatment substance (Column 1), Probe (data link) (Column 2), Direction (Column 3), Best BLAST Annotation (determined statistically) (Column 4), and Human Accession Number (Column 5). The information for all ingredients tested is stored in a database for reference.
Based upon the physiological condition of the canines (a diagnosis as fat) and a comparison of the information from the Tables1-7, i.e, noting genes that are influenced by a test substance or ingredient and are also differentially expressed in fat canines compared to lean canines, a nutritional formula useful for selecting and preparing a food composition for fat canines would be believed to contain one or more of the following ingredients in the following amounts (in vivo amounts in milligrams per kilogram of body weight per day (mg/kg/day) are based upon extrapolation from amounts used in vitro, for example: DHA—from about 1 to about 30; EPA—from about 1 to about 30; EPA/DHA Combo (1.5:1 ratio)—from about 412 to about 30/45; ALA—from about 10 to about 100; LA—from about 30 to about 600; ARA—from about 5 to about 50; and SA—from about 3 to about 60. Based upon these data, a food composition and related diet containing one or more of these ingredients can be prepared and used to regulate the genes that are differentially expressed in fat animals compared to lean animals. Such regulation will cause the modulation of the amount of adipose tissue on the animal and, therefore, in one embodiment, promote a shift to a desirable or normal (more lean) status and promote better health and wellness of the animal.
Canis familiaris type I
Mus musculus RIKEN cDNA
Homo sapiens 12 BAC RP11-
Canis familiaris urate oxidase
Homo sapiens mRNA; cDNA
Momo sapiens toll-interleukin 1
Homo sapiens hypoxia-inducible
Mus musculus chromosome 14
Homo sapiens interleukin 8
Homo sapiens clone DNA22780
Homo sapiens prodynorphin
Homo sapiens lymphocyte adaptor
Homo sapiens cDNA: FLJ21199
Homo sapiens, clone
Homo sapiens 12 BAC RP11-
Homo sapiens hypothetical protein
Canis familiaris clone RP81-
Mus musculus mRNA for
Homo sapiens aryl hydrocarbon
Canis familiaris clone RP81-
Homo sapiens pyruvate
Homo sapiens, Similar to secreted
Sus scrofa carnitine
Homo sapiens methyl CpG binding
Homo sapiens RAB37, member
Homo sapiens hypoxia-inducible
Homo sapiens chromosome 15
Homo sapiens integrin-linked
Homo sapiens clone DNA22780
Homo sapiens 12 BAC RP11-
Homo sapiens Kruppel-like factor
Homo sapiens RNA binding motif
Sus scrofa peptidyl-prolyl cis-trans
Homo sapiens chromosome 16
Homo sapiens chromosome 8,
Bos taurus mRNA for sodium
Yarrowia lipolytica CLIB99,
H. sapiens mRNA for skeletal
Homo sapiens chromosome 16
Homo sapiens, clone
Mus musculus BAC clone RP23-
Homo sapiens S164 gene, partial
Homo sapiens cDNA clone
Haemonchus contortus
Homo sapiens cDNA FLJ33460 fis,
Homo sapiens mRNA; cDNA
Homo sapiens mRNA; cDNA
Canis familiaris secreted B7-1
Homo sapiens pyruvate
Mus musculus mbt domain
Homo sapiens cDNA FLJ38323 fis,
Canis familiaris L-type Ca channel
Canis familiaris fibroblast growth
Bos taurus clone IMAGE: 7961516
Sus scrofa carnitine
Homo sapiens mRNA for UDP-
Homo sapiens chromosome 11,
Bos taurus mRNA for transcription
Homo sapiens RAD51-like 1 (S. cerevisiae)
Hirudo medicinalis intermediate
Homo sapiens mRNA for
Homo sapiens clone alpha1 mRNA
Homo sapiens solute carrier family
Homo sapiens clone DNA22780
Homo sapiens Kruppel-like factor
Felis catus clone RP86-117J4,
Felis catus growth arrest and DNA
Homo sapiens cytochrome P450,
Homo sapiens serine palmitoyl
Homo sapiens chromosome 8,
Homo sapiens mRNA; cDNA
Homo sapiens mRNA; cDNA
Canis familiaris clone RP81-
Canis Familiaris, clone XX-25A1,
Homo sapiens lactamase, beta 2,
Homo sapiens BAC clone RP11-
Homo sapiens cDNA FLJ33134 fis,
Homo sapiens mRNA; cDNA
Homo sapiens cDNA FLJ13648 fis,
Homo sapiens chromosome 16
Homo sapiens pyruvate
Homo sapiens mRNA; cDNA
Mus musculus mRNA for NFI-B
Canis familiaris type I collagen pre-
Hirudo medicinalis intermediate
Homo sapiens fibroblast growth
Homo sapiens hypothetical protein
Homo sapiens mRNA for cAMP
Homo sapiens mRNA for
Homo sapiens clone DNA22780
Canis familiaris natural resistance
Homo sapiens Kruppel-like factor
Canis familiaris clone RP81-
Homo sapiens chromosome 17,
Homo sapiens cDNA FLJ34120 fis,
Pongo pygmaeus mRNA; cDNA
Homo sapiens, clone
Drosophila melanogaster
Mustela vison tyrosine
Homo sapiens chromosome 5
Homo sapiens pyruvate
Canis familiaris Na+-dependent
Homo sapiens mRNA; cDNA
Homo sapiens calsyntenin 2,
Mus musculus microtubule
Homo sapiens downregulated in
Homo sapiens hypoxia-inducible
Homo sapiens clone DNA22780
Homo sapiens BAC clone CTB-
Homo sapiens RGM domain
Homo sapiens HMGIC fusion
Bos taurus mRNA for sodium
Homo sapiens CrkRS mRNA,
Mus musculus piccolo (presynaptic
Homo sapiens mRNA; cDNA
Homo sapiens pyruvate
Rattus norvegicus nuclear receptor
Canis familiaris cyclin-dependent
Sus scrofa carnitine
Homo sapiens chromosome 11,
Homo sapiens hypoxia-inducible
Homo sapiens mRNA; cDNA
Homo sapiens BAC clone RP11-
Homo sapiens clone DNA22780
Homo sapiens clone DNA77624
Homo sapiens cDNA clone
Homo sapiens hypothetical protein
Homo sapiens Kruppel-like factor
Gallus gallus mRNA for
Homo sapiens chromosome 8,
Homo sapiens mRNA; cDNA
Mustela vison NADH
Oryza sativa (japonica cultivar-
Homo sapiens CASK interacting
Homo sapiens cDNA clone
Homo sapiens, clone
Homo sapiens TRIAD1 type I
Mus musculus expressed
Homo sapiens BAC clone RP11-
Homo sapiens genomic DNA,
Rattus norvegicus chromosome
Homo sapiens cDNA clone
Bos taurus mRNA for similar to
Homo sapiens chromosome 17,
Homo sapiens pyruvate
Homo sapiens Kruppel-like factor
Homo sapiens G protein-coupled
Canis familiaris organic anion
Homo sapiens fatty acid
Homo sapiens lamin B1 (LMNB1),
Lotus corniculatus var. japonicus
Homo sapiens mRNA for dual
Homo sapiens 12 BAC RP11-
Mus musculus SNF8, ESCRT-II
Homo sapiens hypoxia-inducible
Homo sapiens cyclophilin-related
Plasmodium yoelii yoelii str. 17XNL
Canis familiaris isolate cOR5D23
Nicotiana benthamiana clone 6-
Homo sapiens amyotrophic lateral
Homo sapiens Kazal type serine
Homo sapiens mRNA for TSC-22
Homo sapiens hypothetical
Homo sapiens cDNA clone
Homo sapiens solute carrier family
Homo sapiens BAC clone RP11-
Danio rerio POU domain, class 4,
Homo sapiens mRNA; cDNA
Aspergillus nidulans FGSC A4
Arabidopsis thaliana clone
Mus musculus ubiquitin-like 4,
Mus musculus piwi-like 4
Homo sapiens CDC14 cell division
Canis familiaris gonadotropin-
Mus musculus nephrin NPHS1
Xenopus laevis MGC80410
Homo sapiens glutathione
Homo sapiens fibroblast growth
Mus musculus RIKEN cDNA
Canis familiaris forssman
Arabidopsis thaliana At1g50920
Homo sapiens gene for LIM-
Homo sapiens olfactory receptor,
Nicotiana benthamiana clone 6-
purpuratus similar to
Mus musculus solute carrier family
In order to simplify clinical and scientific analyses and eliminate the need for using solid tissue samples that have to be biopsied from live animals, blood samples from fat and lean dogs may be obtained and used to develop a “class predictor” that can be used to differentiate between fat and lean animals Class prediction is a form of pattern recognition that involves the use of supervised learning algorithms familiar to one of skill in the art (e.g., Weighted Voting, Class Neighbors, K-Nearest Neighbors and Support Vector Machines) to define a group of genes or gene products that can recognize and differentiate between two groups or classes of animals Developing class predictors generally involves the following steps:
In our studies with fat and lean animals, Affymetrix Canine-2 GeneChips are used according to methods provided hereinabove to measure the gene expression levels in blood samples taken from animals that are conventionally identified as clinically fat (28 animals with a body condition score of 4 or 5) or lean (12 animals with a body condition score of 2 or 2.5). The GeneChip data is then used to train an algorithm (Support Vector Machines) that is included in the software program GeneSpring (version 7.2, Agilent Technologies) to generate the class predictor. Accordingly, data indicate 65 probes that exhibit differential expression levels between the fat and lean samples with a “p” value of 0.01 (after the application of a false discovery rate correction) (see Table 8). RMA normalized data provided in Table 9 indicates the intensity of the fold change in expression in a fat animal versus lean animal such that a value greater than one indicates that the gene is upregulated in a fat animal, a value of one indicates no change in expression in a fat versus lean animal and a value of less than one indicates that the expression of the gene is greater in a lean animal than a fat animal. Thus, it is contemplated herein that these probes and the genes and gene products that they represent can potentially be used as class predictors to identify fat and lean animals using blood samples without the need to use adipose tissue samples.
Homo sapiens elk1 oncogene; complete cds
Pongo pygmaeus mRNA; cDNA DKFZp468H0312 (from
Canis familiaris angiotensin II type 2 receptor mRNA;
Canis familiaris Sec61 beta subunit (Sec61b); mRNA
Magnaporthe grisea 70-15 hypothetical protein
Homo sapiens mRNA; cDNA DKFZp761M0111 (from
Homo sapiens (human)
C. familiaris mRNA for TRAM-protein
Canis familiaris carboxypeptidase B1 (tissue) (CPB1);
Mus musculus olfactory receptor MOR232-2 gene;
Homo sapiens protocadherin 15 (PCDH15); mRNA
Macaca fascicularis brain cDNA; clone: QflA-12135; similar
Canis familiaris non-metastatic cells 2; protein (NM23B)
Homo sapiens mRNA for KIAA1045 protein; partial cds
The data obtained from in vitro ingredient screens discussed above indicate that some ingredients that are high in long chain fatty acids (see Table 7) may have the potential to affect the expression of genes involved in fat metabolism in a way that would promote leanness of the animal as a whole. This is determined by analyzing data obtained from adipose tissue and from the ingredient assays discussed above using conventional computer algorithm analyses. Code for algorithms useful in this regard are familiar to one of skill in the art and may be developed without undue experimentation. An example of such code is provided below:
To confirm that the inclusion of linolenic acid or EPA/DHA (1.5:1) in diets fed to dogs does affect weight loss in dogs, three high protein diets containing either no added long chain fatty acids (Diet A) or added linolenic acid (approximately 1% based on 100% dry matter basis, Diet B) or EPA/DHA (1.5-1, approximately 0.30%:0.20%) (Diet C) were developed for comparison to a high fiber diet that is known to induce weight loss in dogs. In the study, 45 clinically fat dogs are all first fed a nutritionally complete control diet for 30 days prior to the start of the test. After the initial 30 days, the dogs are randomized into 4 groups. Three of the four groups receive one of the test diets and one group is given the high fiber diet as a control for a set period of time, e.g., 4 months. Results indicate that the three experimental foods (Diets A, B and C) have substantially higher digestibility than the higher fiber food. Results also indicate that approximately 38% of the dogs consuming the food containing EPA/DHA reach their weight loss goal at 90 days. Interestingly, dogs consuming the EPA/DHA food also maintain lean muscle mass and bone mineral content. The results also indicate that, at least at the clinical level, diets containing EP/DHA may be as effective as high fiber diets in affecting weight loss.
In order to validate the class predictor probe set and to test its ability to predict fatness or leanness in animals) the class predictor probe set (described in Example 3 above) is applied to gene expression data obtained from the 45 animals participating in the experiment above (expression data not shown). The class predictor analysis confirms that 41 of the 45 animals (approximately 90%) designated “fat” at the beginning of the test are in fact fat (the discrepancy may be due to the subjective nature of the conventional body condition scoring system that is currently used in the clinic). Interestingly, after 14 days of feeding the four diets described above, the class predictor analysis indicates that all animals, regardless of diet, display a “lean” gene expression profile. At the end of the study, it appears that all the animals on the control high fiber diet reflect a “fat” gene expression profile, approximately 25% of the animals on test Diets A and B reflect a biochemically “lean” gene expression profile and approximately 40% of the animals fed on Diet C containing EPA/DHA exhibit a biochemically “lean” gene expression profile (see Table 10).
Based on the results of the weight loss experiment discussed above, it is hypothesized that animals fed a diet containing EPA/DHA will not only lose weight but also will maintain the loss for a longer period of time compared to animals fed the other test and control high fiber diets.
In order to characterize the effects of Diets A, B, and C and the high fiber diet on weight loss maintenance, one could perform, for example, the following type of experiment:
Fat animals may be fed the four different diets (as described in Example 4) until they reach an optimum level of “leanness”. They may then be randomized and divided into subgroups that either continue to be fed the same test diet that they were fed previously or are switched to a maintenance diet that is nutritionally balanced but is not designed to induce or maintain weight loss and does not include appreciable amounts of linolenic acid or EPA/DHA, for example.
The animals may then be observed for a set period of time, e.g., up to 3 months, with their weights recorded daily, their body condition scores determined weekly and their percentage body fat determined on a monthly basis using conventional DEXA technologies.
This application claims benefit of U.S. Provisional No. 60/778,567 filed Mar. 2, 2006 and U.S. Provisional application No. 60/824,318 filed Sep. 1, 2006, PCT/US07/05438, filed Mar. 2, 2007, which are both hereby incorporated by reference for all purposes.
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/US07/05438 | 3/2/2007 | WO | 00 | 11/13/2008 |
| Number | Date | Country | |
|---|---|---|---|
| 60778567 | Mar 2006 | US | |
| 60824318 | Sep 2006 | US |
