EP2303002A2 - Souris transgéniques gpr101 inédites et leurs procédés d'utilisation - Google Patents

Souris transgéniques gpr101 inédites et leurs procédés d'utilisation

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Publication number
EP2303002A2
EP2303002A2 EP09750955A EP09750955A EP2303002A2 EP 2303002 A2 EP2303002 A2 EP 2303002A2 EP 09750955 A EP09750955 A EP 09750955A EP 09750955 A EP09750955 A EP 09750955A EP 2303002 A2 EP2303002 A2 EP 2303002A2
Authority
EP
European Patent Office
Prior art keywords
gprlol
gene
human mammal
disruption
transgenic non
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP09750955A
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German (de)
English (en)
Inventor
Bradford B. Lowell
Harveen Dhillon
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Beth Israel Deaconess Medical Center Inc
Original Assignee
Beth Israel Deaconess Medical Center Inc
Beth Israel Hospital Association
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Filing date
Publication date
Application filed by Beth Israel Deaconess Medical Center Inc, Beth Israel Hospital Association filed Critical Beth Israel Deaconess Medical Center Inc
Publication of EP2303002A2 publication Critical patent/EP2303002A2/fr
Withdrawn legal-status Critical Current

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Classifications

    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K67/00—Rearing or breeding animals, not otherwise provided for; New or modified breeds of animals
    • A01K67/027—New or modified breeds of vertebrates
    • A01K67/0275—Genetically modified vertebrates, e.g. transgenic
    • A01K67/0276—Knock-out vertebrates
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/72—Receptors; Cell surface antigens; Cell surface determinants for hormones
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/8509—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells for producing genetically modified animals, e.g. transgenic
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2217/00—Genetically modified animals
    • A01K2217/07—Animals genetically altered by homologous recombination
    • A01K2217/072—Animals genetically altered by homologous recombination maintaining or altering function, i.e. knock in
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2217/00—Genetically modified animals
    • A01K2217/07—Animals genetically altered by homologous recombination
    • A01K2217/075—Animals genetically altered by homologous recombination inducing loss of function, i.e. knock out
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2227/00—Animals characterised by species
    • A01K2227/10—Mammal
    • A01K2227/105—Murine
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2267/00—Animals characterised by purpose
    • A01K2267/03—Animal model, e.g. for test or diseases
    • A01K2267/035—Animal model for multifactorial diseases
    • A01K2267/0362—Animal model for lipid/glucose metabolism, e.g. obesity, type-2 diabetes

Definitions

  • G protein receptor 101 is an orphan G protein-coupled receptor (GPCR) with no known ligand. GPRlOl is present exclusively in the central nervous system and is abundantly expressed in the hypothalamus and amygdala, specifically in the arcuate nucleus (ARC), ventromedial hypothalamus (VMH), dorsomedial hypothalamus (DMH), posterior hypothalamus (PH), paraventricular nucleus (PVN), medial preoptic area (MPOA), suprachiasmatic (SCN) and anterior hypothalamic area (AHA) of the forebrain regions and the nucleus of the solitary tract (NTS) and lateral parabrachial nucleus (LPB) in the hindbrain regions, areas thought to be involved in metabolic homeostatic function (Nilaweera, K.N.
  • GPCR G protein-coupled receptor
  • GPRlOl G Protein-Coupled Receptor 101 mRNA Expression in the Mouse Brain: Altered Expression in the Posterior Hypothalamus and Amygdala By Energetic Challenges," Journal of Neuroendocrinology, 19: 34-45 (2006), which is incorporated herein by reference in its entirety).
  • GPRlOl is also expressed in areas of the brain involved in regulation of motivated behavior such as the nucleus accumbens in the forebrain and serotonergic nuclei in the midbrain (Bates, et ah, "Characterization of GPRlOl Expression and G-protein Coupling Selectivity, Brain Research, 1087: 1-14 (2006), which is incorporated herein by reference in its entirety)).
  • GPRlOl may have an important role in metabolic homeostasis, it may have an important role in treating and preventing metabolic diseases and disorders.
  • the invention features a novel transgenic mouse model, or cells isolated therefrom, for screening agents that modulate the GPRlOl receptor.
  • Such models can be used to improve diagnosis of diseases relating to energy metabolism as well as identifying and testing pharmaceutical compositions for better treatment and prevention of disease relating to energy metabolism.
  • the invention provides GPRlOl knock-out transgenic non-human animals, or cells isolated therefrom, and their use as a model for disease relating to energy metabolism such as obesity, metabolic syndrome, dyslipidemia, insulin resistance syndrome, type 2 diabetes, anorexia nervosa and cachexia.
  • the invention provides transgenic non-human animals, or cells isolated therefrom, wherein the GPRlOl gene is constitutively active and their use as a model for disease relating to energy metabolism such as obesity, metabolic syndrome, dyslipidemia, insulin resistance syndrome, diabetes, particularly type 2 diabetes, anorexia nervosa and cachexia.
  • the invention relates to a transgenic knock-out non-human mammal whose genome comprises a disruption in the endogenous GPRlOl gene.
  • said disruption has been introduced into the genome by homologous recombination with a DNA targeting construct in an embryonic stem cell.
  • the disruption of the GPRlOl gene results in an inability of said transgenic non-human mammal to produce detectable levels GPRl 01.
  • the mammal is a mouse.
  • the invention further relates to an isolated cell from a transgenic knock-out non-human mammal whose genome comprises a disruption in the endogenous GPRlOl gene.
  • the invention further relates to a method of producing a knock-out non-human mammal whose genome comprises a disruption in the endogenous GPRlOl gene.
  • the invention further relates to a method for screening a candidate agent for the ability to modulate body weight and food intake in a knock-out non-human mammal:
  • the invention further relates to a method for screening a candidate agent for the ability modulate body weight and food intake in a knock-out non-human mammal:
  • the invention relates to a transgenic non-human mammal whose genome comprises a constitutively active endogenous GPRlOl gene.
  • the constitutively active GPRlOl gene has been introduced into the genome by a single amino acid substitution.
  • amino acid substitution has been introduced into the genome by homologous recombination with a DNA targeting construct in an embryonic stem cell.
  • the mammal is a mouse.
  • the invention further relates to an isolated cell from a transgenic non-human mammal whose genome comprises a constitutively active endogenous GPRlOl gene.
  • the invention further relates to a method of producing a transgenic non-human mammal whose genome comprises a constitutively active endogenous GPRlOl gene.
  • the invention further relates to a method for screening a candidate agent for the ability to modulate body weight and food intake in a transgenic non-human mammal whose genome comprises a constitutively active endogenous GPRlOl gene:
  • ((aa)) providing a transgenic non-human mammal whose genome comprises a constitutively active endogenous GPRlOl gene
  • FIG. 1 shows a three primer multiplex polymerase chain reaction (PCR) strategy to genotype mice.
  • FIG. 2 is a graph showing body weight in GPRlOl knock-out mice (GPRlOl-KO) and wild-type littermate controls (WT). The Y axis represents body weight in grams (g) and the X axis represents age in weeks (wks).
  • FIG. 3 A is a bar graph of food intake in grams (g) during a twenty-four hour period in GPRlOl knock-out mice (GPRlOl-KO) and wild-type littermate controls (WT).
  • FIG. 3B is a graph showing cumulative food intake in GPRlOl knock-out mice (GPRlOl-KO) and wild-type littermate controls (WT).
  • the Y axis represents food intake in grams (g) and the X axis represents time in minutes.
  • FIG. 4 is a graph showing alterations in food consumption for wild-type controls (WT).
  • the Y axis represents food intake in grams (g) and the X axis represents time in minutes.
  • FIG. 5 is a graph showing alterations in food consumption for GPRlOl knock-out mice (GPRlOl KO).
  • the Y axis represents food intake in grams (g) and the X axis represents time in minutes.
  • FIG. 6 is a graph showing body weight of GPRlOl knock-out mice (GPRlOlKO) and wild-type littermate controls (WT) on a chow diet.
  • the Y axis represents body weight in grams (g) and the X axis is age in weeks (wks).
  • FIG. 7 is a graph showing body weight of GPRlOl knock-out mice (GPRlOlKO) and wild-type littermate controls (WT) on a high fat diet.
  • the Y axis represents body weight in grams (g) and the X axis is age in weeks (wks).
  • FIG. 8 is a bar graph of body composition (fat mass and lean mass in grams) for mice
  • the invention relates to transgenic non-human GPRlOl knock-out animals, and methods of using the animals for the development of drugs for the treatment or prevention of diseases related to energy metabolism, such as obesity, metabolic syndrome, dyslipidemia, insulin resistance syndrome, diabetes, particularly type 2 diabetes, anorexia nervosa and cachexia.
  • the invention also relates to transgenic non-human animals whose GPRlOl gene is constitutively active, and methods of using the animals for the development of drugs for the treatment or prevention of diseases related to energy metabolism, such as obesity, metabolic syndrome, dyslipidemia, insulin resistance syndrome, diabetes, particularly type 2 diabetes, anorexia nervosa and cachexia.
  • modulation of the amount or activity of the GPRlOl gene may be beneficial in the treatment of such energy metabolism diseases.
  • Metabolic disease include diseases related to energy metabolism, such as, but not limited to, obesity, metabolic syndrome, dyslipidemia, insulin resistance syndrome, diabetes, particularly type 2 diabetes, anorexia nervosa and cachexia.
  • transgenic non-human animal includes the founder transgenic non-human animals and progeny of the founders, as well as cells, cell lines and tissues from such animals in which one or more of the cells of the animal includes one or more transgenes.
  • Transgenic non-human animals can be farm animals such as pigs, goats, sheep, cows, horses, and rabbits, rodents such as rats, guinea pigs, and mice, and non-human primates such as baboons, monkeys, and chimpanzees. Transgenic mice are particularly useful.
  • a transgene is exogenous DNA which is integrated into the genome of a cell from which a transgenic animal develops and which remains in the genome of the mature animal, thereby directing the expression of an encoded gene product in one or more cell types or tissues of the transgenic animal.
  • a "knock-out" of a gene means an alteration or disruption in the sequence of the gene that results in a decrease of function of the target gene, preferably such that target gene expression is undetectable or insignificant.
  • a knock-out of an endogenous gene means that function of the gene has been substantially decreased so that expression is not detectable or only present at insignificant levels.
  • the terms “disruption” and “alteration” connote a partial or complete reduction in the expression and/or function of the GPRlOl gene. Alteration or disruption of the GPRlOl gene can be accomplished by a variety of methods known to those of skill in the art. For example, gene targeting using homologous recombination, mutagenesis (e.g., point mutation) and antisense technology can be used to disrupt a GPRlOl gene.
  • homologous recombination refers to a type of homologous recombination which occurs as a consequence of the introduction of a targeting construct (e.g., vector) into a mammalian cell (e.g., an ES cell) which is designed to locate and recombine with a corresponding portion of the nucleic acid sequence of the genomic locus targeted for alteration (e.g., disruption) thereby introducing an exogenous recombinant nucleic acid sequence capable of conferring a planned alteration to the endogenous gene.
  • a targeting construct e.g., vector
  • a mammalian cell e.g., an ES cell
  • homologous recombination is a process (e.g., method) by which a particular DNA sequence can by replaced by an exogenous genetically engineered sequence.
  • regions of the targeting vector which have been genetically engineered to be homologous (e.g., complimentary) to the endogenous nucleotide sequence of the gene which is targeted for disruption line up or recombine with each other such that the nucleotide sequence of the targeting vector is incorporated into (e.g, integrates with) the corresponding position of the endogenous gene.
  • a "construct" is meant a recombinant nucleic acid, generally recombinant
  • the term "genotype" refers to the genetic makeup of an animal with respect to the GPRlOl chromosomal locus. More specifically, the term genotype refers to the status of the animal's GPRlOl alleles. GPRlOl is located on the X chromosome. Since female mice have two X chromosomes, mice can be of the following three genotypes: wildtype mice
  • This X chromosome inactivation is random cell to cell and is often referred to as "lyonization".
  • roughly half the cells in the organism will express no GPRlOl (because the X chromosome bearing the wild-type allele ( ⁇ GPR101+ ) has been inactivated) while the other half of the cells in the organism will express normal levels of GPRlOl (because the X chromosome bearing the "null” allele ( ⁇ GPR10 '- nu11 ) was inactivated, and hence the X chromosome bearing the wild-type allele ( ⁇ GPR101+ ) remains active).
  • mice have one Y chromosome (which lacks a GPRlOl gene) and one X chromosome (which has the GPRlOl gene).
  • mice can only be of one or another genotype: wild-type (Y, ⁇ GPR101+ ) m j ce which have normal amounts of GPRlOl in every cell and knockout (Y, ⁇ GPR101 - nu11 ) m i ce that lack any GPRlOl in every cell.
  • wild-type Y, ⁇ GPR101+
  • knockout Y, ⁇ GPR101 - nu11
  • m i ce that lack any GPRlOl in every cell.
  • the GPRlOl knock-out mammal of the present invention can manifest a particular phenotype.
  • the term "phenotype” refers to the resulting biochemical or physiological consequences attributed to a particular genotype.
  • the GPRlOl knock-out mammal has altered metabolic homeostasis.
  • “Knock-out” transgenics can be transgenic animals having a heterozygous knock-out of a gene or a homozygous knock-out of a gene. “Knock-outs” also include conditional knock-outs, where alteration of the target gene can occur upon, for example, exposure of the animal to a substance that promotes target gene alteration, introduction of an enzyme that promotes recombination at the target gene site (e.g., Cre in the Cre-lox system), or other method for directing the target gene alteration postnatally.
  • an enzyme that promotes recombination at the target gene site e.g., Cre in the Cre-lox system
  • Recombineering is a homologous recombination-based, highly efficient genetic engineering system that can be used to introduce mutations in a target sequence that is part of a vector, such as a BAC.
  • Methods of recombineering are known to those skilled in the art (for example see Zhang et at, Nature Biotech. 18: 1314-7, 2000; Zhang et at, Nature Genetics 20: 123-8 (1998; and Datsenko and Wanner, Proc. Natl. Acad. Sci. USA, 97: 6640-5 (2000). Reviews of recombineering can be found in Court, et al., Annu. Rev. Genet.
  • constitutively active receptor shall mean a receptor stabilized in an active state by means other than through binding of the receptor to its ligand or a chemical equivalent thereof.
  • a constitutively active receptor may be endogenous or non-endogenous.
  • Constutively activated receptor shall mean an endogenous receptor that has been modified so as to be constitutively active or to be more constitutively active.
  • ES cell refers to pluripotent embryonic stem cells and to such pluripotent cells in the very early stages of embryonic development, including but not limited to cells in the blastocyst stage of development.
  • Site specific mutagenesis or "site directed mutagenesis” is a production of a specific predetermined change in a DNA sequence. Methods for site specific mutagenesis can be found in Sambrook et al., Molecular Cloning: A Laboratory Manual, CSH Press 1989, pp.
  • a transgenic animal is produced by the integration of a given transgene into the genome in a manner that permits the expression of the transgene.
  • Methods for producing transgenic animals are generally described by Wagner and Hoppe (U.S. Pat. No. 4,873,191); Brinster et al, Proc. Natl. Acad. Sci. USA 82: 4438-4442 (1985); and in
  • a gene flanked by genomic sequences is transferred by microinjection into a fertilized egg.
  • the microinjected eggs are implanted into a host female, and the progeny are screened for the expression of the transgene.
  • Transgenic animals may be produced from the fertilized eggs from a number of animals including, but not limited to reptiles, amphibians, birds, mammals, and fish.
  • the transgenic animals may be obtained by utilizing embryonic stem (ES) cells for the generation of the transgenes.
  • the transgene is introduced into embryonic stem cells and the transfected stem cells are utilized to form an embryo.
  • ES cells are obtained by culturing pre-implantation embryos in vitro under appropriate conditions (Evans et al, Nature 292: 154- 156 (1981); Bradley et al., Nature 309: 255-256 (1984); Gossler et al., Proc. Acad. Sci. USA 83: 9065-9069 (1986); and Robertson et al, Nature 323: 445-448 (1986), which are incorporated herein by reference in their entirety).
  • the offspring may be analyzed for the integration of the transgene by isolating genomic DNA from tail tissue and the fragment coding for the gene identified by conventional DNA-hybridization techniques (Southern, J. MoI. Biol. 98: 503-517 (1975), which is incorporated herein by reference in its entirety)).
  • One aspect of the invention pertains to isolating cells or cell lines from the non-human transgenic animals of the invention and growing the cells in culture.
  • Another aspect of the invention pertains to host cells into which a recombinant expression vector of the invention has been introduced, containing sequences which allow it to homologously recombine into a specific site of the host cell's genome, or sequences that allow it to randomly or semi-randomly recombine into the host cell's genome. It is understood that such cells refer not only to the particular subject cell but to the progeny or potential progeny of such a cell. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term as used herein.
  • vectors refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked.
  • plasmid refers to a circular double stranded DNA loop into which additional DNA segments can be ligated.
  • viral vector Another type of vector is a viral vector, wherein additional DNA segments can be ligated into the viral genome.
  • Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors).
  • vectors e.g., non-episomal mammalian vectors
  • Other vectors are integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome.
  • certain vectors are capable of directing the expression of genes to which they are operatively linked.
  • Such vectors are referred to herein as "expression vectors.”
  • expression vectors of utility in recombinant DNA techniques are often in the form of plasmids.
  • plasmid and vector can be used interchangeably as the plasmid is the most commonly used form of vector.
  • the invention is intended to include such other forms of expression vectors, such as viral vectors (e.g., replication defective retroviruses, adenoviruses and adeno-associated viruses), which serve equivalent functions.
  • the recombinant expression vectors of the invention comprise a nucleic acid of the invention in a form suitable for expression of the nucleic acid in a host cell, which means that the recombinant expression vectors include one or more regulatory sequences, selected on the basis of the host cells to be used for expression, which is operably linked to the nucleic acid sequence to be expressed.
  • regulatory sequences are described, for example, in Goeddel, Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, Calif. (1990), which is incorporated herein by reference in its entirety.
  • Regulatory sequences include those which direct constitutive expression of a nucleotide sequence in many types of host cells and those which direct expression of the nucleotide sequence only in certain host cells.
  • the design of the expression vector can depend on such factors as the choice of the host cell to be transformed, the level of expression of protein desired, etc.
  • the expression vectors of the invention can be introduced into host cells to thereby produce proteins or peptides, including fusion proteins or peptides, encoded by nucleic acids as described herein.
  • the recombinant expression vectors of the invention can be designed for expression of the target gene in prokaryotic or eukaryotic cells.
  • the target gene or fragments can be expressed in bacterial cells such as E. coli, insect cells (using baculovirus expression vectors), yeast cells or mammalian cells. Suitable host cells are discussed further in Goeddel, Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, Calif.
  • the recombinant expression vector can be transcribed and translated in vitro.
  • the target gene can also be expressed in mammalian cells using a mammalian expression vector.
  • mammalian expression vectors include pCDM8 (Seed, B., Nature, 329: 840 (1987) and pMT2PC (Kaufman et al, EMBO J, 6: 187 (1987)).
  • the expression vector's control functions are often provided by viral regulatory elements.
  • commonly used promoters are derived from polyoma, adenovirus 2, cytomegalovirus and Simian virus 40.
  • suitable expression systems for both prokaryotic and eukaryotic cells see chapters 16 and 17 of Sambrook, J., Fritsh, E. F., and Maniatis, T. Molecular Cloning: A Laboratory Manual. 2nd, ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N. Y., 1989.
  • a host cell can be any prokaryotic or eukaryotic cell.
  • host cells can be bacterial cells such as E. coli, insect cells, yeast, Xenopus cells, or mammalian cells (such as Chinese hamster ovary cells (CHO), African green monkey kidney cells (COS), or fetal human cells (293T)). Other suitable host cells are known to those skilled in the art.
  • a host cell is derived from the transgenic non-human animals described herein.
  • Vector DNA can be introduced into prokaryotic or eukaryotic cells via conventional transformation or transfection techniques.
  • transformation and transfection are intended to refer to a variety of art-recognized techniques for introducing foreign nucleic acid (e.g., DNA) into a host cell, including calcium phosphate or calcium chloride co-precipitation, DEAE-dextran-mediated transfection, lipofection, or electroporation. Suitable methods for transforming or transfecting host cells can be found in Sambrook, et al. (Molecular Cloning: A Laboratory Manual. 2nd, ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N. Y., 1989), and other laboratory manuals.
  • modulators i.e., candidate or test compounds or agents (e.g., peptides, cyclic peptides, peptidomimetics, small molecules, small organic molecules, or other drugs) which effect (i.e., modulate, inhibit, reduce, prevent or reverse) diseases related to energy metabolism, such as obesity, metabolic syndrome, dyslipidemia, insulin resistance syndrome, type 2 diabetes, anorexia nervosa and cachexia.
  • modulating or modulator” or modulate refers to agonizing or antagonizing the GPRlOl receptor.
  • transgenic non-human animals of the invention can be used to identify a compound or composition effective for the treatment or prevention of diseases related to energy metabolism.
  • Compounds or compositions can be identified by administering a test compound or composition to a transgenic non-human animal of the invention or by contacting the test compound or composition with an organ, a tissue (e.g., skeletal muscle) or cells (e.g., neuronal cells or muscle cells) derived from the transgenic non-human animal. Effects of the test compound or composition on the energy metabolism on the transgenic non-human animal, organ, tissues or cells are evaluated. For example, a candidate agent can be assessed in the transgenic non-human animals. Test compounds or compositions that alter energy metabolism can be effective for the treatment or prevention of diseases related to energy metabolism.
  • test compounds or compositions identified as described herein in an appropriate animal model as described herein.
  • test compounds or compositions identified as described herein can be used in an animal model (e.g., a transgenic GPRlOl knock-out non-human animal) to determine the efficacy, toxicity, or side effects of treatment with such test compounds or compositions.
  • animal model e.g., a transgenic GPRlOl knock-out non-human animal
  • test compounds or compositions identified as described herein can be used in an animal model to determine the mechanism of action of such test compounds or compositions.
  • Test compounds can be formulated into pharmaceutical compositions by admixture with pharmaceutically acceptable non-toxic excipients or carriers and administered to transgenic non- human animals of the invention by any route of administration.
  • parenteral routes such as subcutaneous, intramuscular, intravascular, intradermal, intranasal, inhalation, intrathecal, or intraperitoneal administration
  • enteral routes such as sublingual, oral, or rectal administration
  • a test compound or composition can be tested for physiologic actions on GPRlOl receptors by, for example, administering the test compound or composition to wildtype mice and GPRlOl knock-out mice. If the test compound or composition causes an effect in wildtype mice, but not in GPRlOl knock-out mice, then the test compound or composition produces the effect by changing the activity GPRlOl . If the test compound or composition causes loss of body weight, loss of body fat, and/or decreased food intake in wildtype mice, but not in GPRlOl knock-out mice, then the test compound or composition is likely to be an activator of GPRlOl .
  • test compound or composition causes an increase in body weight, an increase in body fat, and/or an increase in food intake, then test compound or composition is likely to be an inhibitor of GPRlOl . If the test compound or composition produces an effect in both wildtype mice and GPRlOl knock-out mice, then the test compound or composition is not producing its effects by changing GRPlOl activity.
  • test compound or composition can be tested in GPRlOl knock-out mice. For example, if effects seen in wildtype mice are also seen in GPRlOl knock-out mice, then the test compound or composition is not specific for GPRlOl .
  • the GPRlOl knock-out mice can be used to determine whether toxicity caused by a test compound or composition is caused by altering GPRlOl activity (on-target toxicity) or some other mechanism (off-target toxicity). For example, if a test compound or composition causes a toxic or undesirable effect in wildtype mice but not in GPRlOl knock-out mice, then the toxic effect is a byproduct of altering GPRlOl activity (an on-target effect). If the test compound or composition causes a toxic or undesirable effect in both wildtype mice and GPRlOl knock-out mice, then the toxic effect of the test compound or composition is not related to any changes in the activity of GPRlOl (an off-target effect).
  • the GPRlOl constitutively active mice may be particularly useful for testing, for example, a test compound or composition that might decrease the activity of GPR 101.
  • a test compound or composition might be useful for stimulating food intake in patients with anorexia nervosa or cachexia. Since the receptor is activated in these mice, they should be sensitive to potential inhibitors of GPRlOl activity.
  • a replacement targeting construct was prepared using a bacterial artificial chromosome (BAC) genomic clone containing -100 kb upstream and -40 kb downstream sequence of GPRlOl .
  • the BAC clone was engineered such that a 2216 bp portion of GPRlOl sequence (NCBI accession number NC 000086) spanning the coding exon, including the start codon, was removed and replaced with an Ires-Cre-Frt-Kanamycin-Frt (Ires-Cre FKF) cassette.
  • the genomic DNA sequence for GPRlOl is in NC_000086 and is from position 54756894 to 54749845 on the assembly.
  • the primers used to engineer the deletion were: (SEQ ID NO: 1) HD73 Forward 5'- TCC TCT GCA AGG CAC TAA CCC TAG CCA CAT GTT TCT CTC GTC CTC AAT CTA GTG ATG TAA TTC CGC CCC TCT CCC T-3' and (SEQ ID NO: 2) HD74 Reverse 5'-CCT AGC TCC TCA TTT CAG GCT TGC CCT TTT CTG GAT CCC TTT TGA AGA CCT AAA CAA AAT ATT AAC GCT TAC A-3.
  • a 1 1.4 kb portion of BAC containing the deletion was inserted into pCR-Blunt containing Zeocin. This targeting plasmid was then linearized and electroporated into embryonic stem cells. Targeted clones were identified by PCR analysis using primers spanning the 3' as well as 5' end of the targeted locus (FIG. 1). Cells expanded from targeted ES clones were injected into C57BL6 blastocysts and germline transmitting chimeric animals were obtained and then mated with FLPe-recombinase mice.
  • a targeting construct to generate a constitutively activated form of GPRlOl with A397 ⁇ K397 was created using standard techniques involving recombineering.
  • a galK positive and counterselection scheme was used to make the point mutation in the GPRlOl BAC (Warming, S. et ah, "Simple and Highly Efficient BAC Recombineering Using galK Selection," Nucleic Acids Research, 33: 1-12 (2005), which is incorporated herein by reference in its entirety).
  • the modified BAC was then inserted into a plasmid and injected into blastocysts for generation of a mouse harboring a point mutation.
  • GPRlOl knock-out mice at eight weeks of age also exhibit alterations in overall patterns of food consumption as shown in FIG. 5.
  • the GPRlOl knockout mice consumed larger meals during the dark phase.
  • GPRlOl knock-out mice have an increase in body weight and food intake, and, therefore, the GPRlOl knock-out mice can be useful in identifying agonists and antagonists of GPRlOl, which can be used to treat metabolic diseases.
  • GPRlOlKO mice fed a chow diet weigh more than wildtype controls (FIG. 6).

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Abstract

La présente invention concerne des animaux transgéniques non humains, ou des cellules isolées à partir de tels animaux, dont le génome comprend le gène GPR101 mis en sommeil, ainsi que des animaux transgéniques non humains, ou des cellules isolées à partir de tels animaux, dont le génome comprend le gène GPR101 constitutionnellement actif. L'invention concerne également des procédés d'utilisation de ces animaux transgéniques non humains en tant que modèles en vue de l'amélioration du traitement, de la prévention ou du diagnostic de maladies associées au métabolisme énergétique, dont l'obésité, le syndrome métabolique, la dyslipidémie, le syndrome de résistance à l'insuline, le diabète de type 2, l'anorexie nerveuse et la cachexie.
EP09750955A 2008-05-19 2009-05-18 Souris transgéniques gpr101 inédites et leurs procédés d'utilisation Withdrawn EP2303002A2 (fr)

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EP2024746A2 (fr) 2006-05-31 2009-02-18 Arena Pharmaceuticals, Inc. Utilisation du récepteur gpr101 dans des procédés d'identification de modulateurs de la sécrétion de peptides actifs biologiquement dérivés de la proopiomélanocortine (pomc) hypothalamique, utiles dans le traitement de troubles liés aux peptides actifs biologiqu
US20110173706A1 (en) * 2008-05-19 2011-07-14 Beth Israel Deaconess Medical Center, Inc. Novel gpr101 transgenic mice and methods of use thereof
EP2918166A1 (fr) 2014-03-10 2015-09-16 Westfälische Wilhelms-Universität Münster Souris double knock-out pour TTP/MRP14 modèle pour le psoriasis

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US4873191A (en) 1981-06-12 1989-10-10 Ohio University Genetic transformation of zygotes
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US7202394B1 (en) 1999-10-05 2007-04-10 Japan Science And Technology Corporation Animal with the mass expression of human gene and test method by using the animal
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US20060123502A1 (en) 2004-12-03 2006-06-08 Murphy Andrew J Assay methods for identifying RE2-like antagonists, methods of use, and non-human transgenic animals
EP2024746A2 (fr) 2006-05-31 2009-02-18 Arena Pharmaceuticals, Inc. Utilisation du récepteur gpr101 dans des procédés d'identification de modulateurs de la sécrétion de peptides actifs biologiquement dérivés de la proopiomélanocortine (pomc) hypothalamique, utiles dans le traitement de troubles liés aux peptides actifs biologiqu

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