WO2020091471A1 - Production de chien transgénique surexprimant la pck1 spécifique du muscle - Google Patents

Production de chien transgénique surexprimant la pck1 spécifique du muscle Download PDF

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WO2020091471A1
WO2020091471A1 PCT/KR2019/014633 KR2019014633W WO2020091471A1 WO 2020091471 A1 WO2020091471 A1 WO 2020091471A1 KR 2019014633 W KR2019014633 W KR 2019014633W WO 2020091471 A1 WO2020091471 A1 WO 2020091471A1
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pck1
muscle
dog
cells
cell
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Korean (ko)
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오현주
김건아
이병천
김민정
라기혜
이석희
김진욱
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서울대학교산학협력단
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Priority to KR1020217011172A priority Critical patent/KR102624831B1/ko
Publication of WO2020091471A1 publication Critical patent/WO2020091471A1/fr

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    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • 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
    • 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
    • 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
    • C12N15/67—General methods for enhancing the expression
    • 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
    • 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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/88—Lyases (4.)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y401/00—Carbon-carbon lyases (4.1)
    • C12Y401/01—Carboxy-lyases (4.1.1)
    • C12Y401/01032—Phosphoenolpyruvate carboxykinase (GTP) (4.1.1.32)
    • 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
    • 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
    • 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
    • C12N2015/8527—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells for producing genetically modified animals, e.g. transgenic for producing animal models, e.g. for tests or diseases

Definitions

  • the present invention relates to a transformed dog and a method for producing the same, and a use thereof for an animal production study having improved energy metabolism and motor performance, and more particularly, to a transgenic dog overexpressing PCK1 and a method for manufacturing the same.
  • PCK1 catalyzes the irreversible stages of grape biosynthesis.
  • PCK1 which is an important factor of grape biosynthesis, and is involved in bioenergy metabolism and vitality improvement, may be a major target gene for animal production research that improves energy metabolism and exercise ability.
  • the present invention aims to provide a medium-large animal with improved energy metabolism and athletic performance and a long life.
  • Another object of the present invention is to provide a method for producing a dog overexpressing PCK1 specifically in the muscle tissue or muscle cell and a muscle specific expression system required for the dog.
  • Another object of the present invention is to provide cells and nuclear transfer embryos transformed with the above muscle specific expression system.
  • Another object of the present invention is to provide various uses of the muscle-specific PCK1 overexpressing dog.
  • the present inventors have developed a technique for transplanting the nucleus of a somatic cell transformed to overexpress PCK1, which is considered to be an important factor in improving the metabolism and vitality of biosynthesis and bioenergy, for the study of animal production with improved energy metabolism and motor performance. By applying it, it was confirmed that a dog specifically overexpressed with PCK1 could be produced and the present invention was completed.
  • the present invention provides a muscle-specific PCK1 overexpressing dog and a method for manufacturing the same.
  • the present invention is an embodiment
  • the alpha-skeletal muscle actin promoter (ACTA promoter) specifically expressed in muscle tissue or muscle cells and the nucleotide sequence encoding PCK1 are operably linked (Knock In, KI).
  • a transgenic dog overexpressing PCK1 is provided.
  • the alpha-skeletal muscle actin promoter (ACTA promoter) specifically expressed in muscle tissue or muscle cells and the nucleotide sequence encoding human-derived PCK1 are operably linked (Knock In, KI) to provide a transgenic dog overexpressing PCK1.
  • the present invention provides a recombinant vector for producing a muscle-specific PCK1 overexpressing animal comprising an ACTA promoter specifically expressed in muscle tissue or muscle cells and a base sequence encoding PCK1.
  • the present invention provides a recombinant vector for producing a muscle-specific PCK1 overexpressing animal comprising an ACTA promoter specifically expressed in muscle tissue or muscle cells and a base sequence encoding human-derived PCK1.
  • the vector may be a vector including one or more transfer factors.
  • the transfer factor may be a piggyBac transfer factor or Sleeping Beauty transfer factor.
  • the metastasis factor may include an inverted terminal repeat sequence (ITR) and / or a direct repeat (DR) sequence at both ends.
  • ITR inverted terminal repeat sequence
  • DR direct repeat
  • the enzyme that mediates the translocation factor may include piggyBac transferase (transposase) or Sleeping Beauty transferase (transposase).
  • the vector may be a piggyBac vector or a Sleeping Beauty vector.
  • the vector may be a plasmid or a viral vector
  • the viral vector may be one or more selected from the group consisting of retrovirus vector, adenovirus vector, adeno-associated virus vector, herpes virus vector, abipox virus vector, and lentivirus vector. It can be a vector.
  • the transformed dog may be provided by somatic cell nuclear transfer (SCNT).
  • SCNT somatic cell nuclear transfer
  • the muscle-specific PCK1 overexpressing dog is characterized by an individual with improved biosynthesis, bioenergy metabolism, vitality, and athletic performance.
  • the present invention provides a method for producing a muscle-specific PCK1 overexpressing dog, characterized in that the nucleus of the nuclear donor cell into which the above-described recombinant vector is introduced is transplanted into a denuclearized egg to produce a mountain.
  • the nuclear donor cell may be a dog embryo cell, a somatic cell or a stem cell.
  • adult fibroblasts or adipocytes can be used.
  • the method for producing a muscle-specific PCK1 overexpressing dog of the present invention may include the following steps as a more specific example.
  • the present invention includes all of the transformation vectors, cells, and nuclear transfer eggs required for the production of a muscle-specific PCK1 over-expressing dog, which can be obtained in the process of the method for producing a muscle-specific PCK1 over-expressing dog.
  • the present invention is another specific example,
  • a nuclear transfer embryo of a dog formed by transplanting or fusing nuclei of a dog-derived somatic cell or stem cell transformed with a recombinant vector containing a base sequence encoding the ACTA promoter and PCK1 into a denuclearized egg can also be provided.
  • the muscle-specific PCK1 overexpressing dog of the present invention is a dog with increased muscle mass, muscle strength, muscular endurance, muscle fibers, mitochondrial biosynthesis and activity, energy metabolism and glucose biosynthesis, etc. , It can be used in a variety of ways to improve athletic performance / strength.
  • the present invention provides various uses of the muscle-specific PCK1 overexpressing dog.
  • a method for screening a medicament for improving energy metabolism including the following.
  • a method of screening a medicament for improving athletic performance including the following, is provided.
  • the present invention provides an animal model of muscle-specific PCK1 overexpression that improves energy metabolism, vitality and athletic performance, and various uses thereof.
  • FIG. 1 is a schematic diagram as one embodiment of a recombinant vector for the production of a muscle-specific PCK1 overexpressing dog.
  • red fluorescent protein which is a marker gene of adult fibroblasts and adipose stem cells into which pPB-ACTAp-PCK1-Red / Puro vector is introduced.
  • Figure 5 is a result of confirming the expression pattern of the red fluorescent protein, a marker gene of adipose stem cells into which pPB-ACTAp-PCK1-Red / Puro vector is introduced.
  • N is a negative control
  • S shows a dog cloned with a muscle-specific PCK1 expression.
  • FIG. 8 shows Southern blot results of muscle-specific PCK1 expression transgenic replication beagles, N is a negative control, and S is a muscle specific PCK1 expression transgenic replication dog.
  • Figure 9 shows the results of serum chemistry analysis of muscle-specific PCK1 expression transgenic cloned dogs (PCK1) and controls (Control) at 1, 2, 4, and 6 months of age (ALT, alanine aminotransferase; AST, aspartate aminotransferase; ALP, alkaline phosphatase ; BUN, blood urea nitrogen; CREA, creatinine; TC, total cholesterol; GLU, glucose; ALB, albumin; TP, total protein; TG, triglyceride).
  • ALT alanine aminotransferase
  • AST aspartate aminotransferase
  • ALP alkaline phosphatase
  • BUN blood urea nitrogen
  • CREA creatinine
  • TC total cholesterol
  • GLU glucose
  • ALB albumin
  • TP total protein
  • TG triglyceride
  • 11 is a data showing the spectrum of MRS imaging results of control group 2.
  • 13 is a data showing the spectrum of MRS imaging results of control group 4.
  • 15 is a data showing the spectrum of MRS results of a muscle-specific PCK1 overexpressed cloned dog.
  • FIG. 16 is chest radiographic data of a muscle-specific PCK1 overexpressed cloned dog.
  • 17 is abdominal radiographic data of a muscle-specific PCK1 overexpressed cloned dog.
  • Figure 18 is the results of skull radiographs of cloned dogs overexpressing muscle-specific PCK1.
  • 19 is lumbar radiographic data of a muscle-specific PCK1 overexpressing cloned dog.
  • 20 is a pelvic radiograph of a muscle-specific PCK1 overexpressing cloned dog.
  • FIG. 21 is a CT image of a muscle-specific PCK1 overexpressing cloned dog and is visualized by visualizing the normality of the occipital bone and cervical spine.
  • Muscle specific expression system is a generic term for a system that allows any gene or protein to be expressed in muscle tissue or cells comprising muscle tissue.
  • the muscle-specific expression system is a regulatory element that can specifically regulate the expression of any gene or protein in muscle tissue or cells constituting muscle tissue, for example, a promoter or enhancer.
  • the expression system may include a vector system including a nucleic acid sequence encoding an arbitrary gene, a viral system, etc., but also includes any system capable of expressing any gene or protein in a cell. .
  • Vector or "expression vector” is a plasmid known in the art capable of inserting a nucleic acid encoding a structural gene and expressing the nucleic acid in a host cell, a vector containing a transposable element, a viral vector Or other medium. Preferably, it may be a vector containing a transfer factor or a viral vector.
  • a "recombinant vector” refers to a gene construct comprising essential regulatory elements operably linked to express a gene insert as a vector capable of expressing a target protein or target RNA in a suitable host cell.
  • control sequence or "regulatory element” is meant a DNA sequence that regulates the expression of nucleic acid sequences operably linked in a particular host cell.
  • regulatory sequences include any operator sequence to regulate transcription, a sequence encoding a suitable mRNA ribosomal binding site, and a sequence that regulates the termination of transcription and translation.
  • the regulatory factor may be a promoter, an enhancer, or the like.
  • “Promoter” refers to a DNA sequence that can regulate the transcription of a specific nucleotide sequence into mRNA when linked to a specific sequence. Typically, the promoter is not applicable in all cases, but is located at 5 '(i.e., upstream) of the desired nucleotide sequence to be transcribed into mRNA, and a site to which the RNA polymerase and other transcription factors for initiating transcription specifically bind. to provide.
  • the promoter of the present invention is a constitutive promoter.
  • the term “constitutive” as used in connection with a promoter means that the promoter is capable of directing the transcription of a nucleic acid sequence to which it is operatively linked without stimulation (eg heat shock, chemicals, etc.).
  • the promoter of the present invention may preferably be an alpha-skeletal muscle actin promoter (ACTA promoter).
  • ACTA promoter can be regulated such that specific sequences linked to the promoter are muscle-specific.
  • the ACTA promoter may be from a dog.
  • “Operably linked” refers to a functional linkage between a nucleic acid expression control sequence and a nucleic acid sequence encoding a desired protein or RNA to perform a general function.
  • a promoter and a nucleic acid sequence encoding a protein or RNA are operably linked to influence the expression of the encoding nucleic acid sequence.
  • Operational linkage with recombinant vectors can be made using genetic recombination techniques well known in the art.
  • Transformation means changing the genetic properties of an organism by DNA given from outside.
  • various methods known in the art for example, microinjection, electroporation, particle bombardment, and sperm-mediated gene transfer, virus infection It can be appropriately selected and applied from techniques using a viral infection, direct muscle injection, insulator, and transposon.
  • the expression vector containing human PCK1 can be transformed into a dog fetal fibroblast through a virus infection method.
  • Animal “or” experimental animal “means any mammalian animal other than humans.
  • the animal includes animals of all ages, including embryos, fetuses, newborns, and adults.
  • Eg commercial sources such as laboratory animals or other animals, rabbits, rodents (eg mice, rats, hamsters, gerbils and guinea pigs), cattle, sheep, pigs, goats, Horses, dogs, cats, birds (eg, chickens, turkeys, ducks, geese), primates (eg, chimpanzees, monkeys, rhesus monkeys), but is not limited to the most preferred animals are dogs.
  • “Overexpression” means that any gene or protein is expressed above a normal level. In the present invention, it means that the expression of the PCK1 protein is expressed above the expression level of normal cells, and specifically, it may be expressed specifically above the normal level in muscle tissue or cells constituting muscle tissue.
  • the overexpressed transformant may be a transformed cell, transformed tissue or transformed animal in which the PCK1 protein is expressed at a normal level or higher. For example, it may be an overexpressed transformant produced by exposing the cell, tissue, or animal to a substance containing a muscle-specific expression system or introducing the substance.
  • Nuclear transplant refers to a genetic manipulation technique that artificially binds other cells or nuclei to a denuclearized egg to have the same trait. "Nuclear transplant” refers to an egg in which nuclear donor cells have been introduced or fused.
  • “Replication” is a genetic manipulation technique to create a new individual with the same gene set as one individual.
  • the dog's somatic cells, embryonic cells, embryo-derived cells and / or adult-derived cells are substantially identical to the nuclear DNA sequences of other cells. Refers to having a nuclear DNA sequence.
  • the present invention uses the technique of cloning a dog using nuclear transfer technology.
  • the somatic cell nuclear transfer technology is a technology capable of producing offspring without passing through meiosis and haploid chromosome-bearing germ cells, which are generally performed in the reproductive process. It is a method of producing and transplanting the fertilized egg in vivo to generate a new individual.
  • Nuclear donor cell refers to a cell or nucleus of a cell that delivers the nucleus to a nuclear receptor, a nuclear recipient.
  • the "ovum” preferably refers to a mature egg that has reached the middle of the second meiosis.
  • dog cells or stem cells may be used as the nuclear donor cells.
  • “Obesity resistance” refers to a state in which the incidence of obesity is suppressed or reduced by suppressing or inhibiting factors and environments that may cause obesity by increasing or activating mechanisms or factors that inhibit obesity, such as fatty acid oxidation and energy consumption.
  • “About” means 30, 25, 20, 25, 10, 9, 8, 7, 6, 5, 4 for reference amount, level, value, number, frequency, percent, dimension, size, amount, weight or length , Amount, level, value, number, frequency, percent, dimension, size, amount, weight or length, varying by 3, 2 or 1%.
  • the present invention relates to a transgenic dog with improved nucleotide sequence encoding the ACTA promoter and PCK1 (Knock in, KI) and improved energy metabolism and exercise ability, a method for manufacturing the same, and use thereof.
  • nucleotide sequence encoding a dog-derived ACTA promoter and PCK1 is KI, and thus relates to a transformed dog having improved energy metabolism and athletic ability, a method for manufacturing the same, and use thereof.
  • genes involved in energy metabolism or growth may be over-expressed to improve the motor performance of the medium-large animal. More specifically, the gene may be PCK.
  • PCK Phosphoenolpyruvate carboxykinase
  • PCK1 Cytosolic isoform
  • PCK2 mitochondrial isoform
  • PCK1 corresponds to an enzyme that converts oxaloacetate (OAA) to phosphoenolpyruvate (PEP) in the cytoplasm during gluconeogenesis and glyceroneogenesis.
  • One aspect of the invention may be a transgenic dog overexpressing muscle tissue or muscle cell-specific PCK1. Any cell of the transgenic dog may have a foreign nucleotide sequence encoding PCK1 in addition to the intrinsic nucleotide sequence encoding PCK1. It may further include.
  • the muscle tissue or muscle cells of the transgenic dog may further include a foreign nucleotide sequence encoding PCK1 in addition to an intrinsic base sequence encoding PCK1.
  • the muscle tissue or muscle cells of the transgenic dog may include two or more base sequences encoding PCK1.
  • the PCK1 expression level of any cell of the transgenic dog may be higher than that of any cell of the wild-type dog.
  • the PCK1 expression level of the muscle tissue or muscle cell of the transgenic dog may be higher than that of the wild type dog muscle tissue or muscle cell.
  • the PCK1 expression level of the muscle tissue or muscle cells of the transformed dog may be significantly higher than that of the wild-type dog muscle tissue or muscle cells.
  • the transformed dog's blood lipase concentration value may have a value of about 0.5 times, about 0.4 times, about 0.3 times, about 0.2 times, or about 0.1 times or less of the wild-type dog's blood lipace concentration value. Can be.
  • a recombinant vector comprising an ACTA promoter derived from a dog and a base sequence encoding PCK1 may be provided to prepare the transformed cell.
  • PCK1 The mechanism of PCK1 in the body is as follows.
  • Fatty acid produces acetyl CoA through bate oxidation, and then acetyl CoA enters the TCA cycle to produce ATP.
  • triglyceride is produced from PEP and fatty acid and glycerol are produced from triglyceride, they are used for energy production through the TCA cycle.
  • the present invention aims to prepare a transgenic animal having an increased expression level of PCK1 by artificially inserting a nucleotide sequence encoding PCK1 into the genome of a specific cell.
  • nucleotide sequence encoding PCK1 derived from a dog or a nucleotide sequence encoding a substance functionally equivalent to a PCK1 gene derived from a dog may be used.
  • the present invention can use a nucleotide sequence encoding a human-derived PCK1 or a substance functionally equivalent to a human-derived PCK1 gene.
  • the present invention provides a recombinant vector comprising a promoter and a base sequence encoding any material.
  • the present invention provides a recombinant vector that includes a promoter and a base sequence encoding any substance and is capable of modifying the base sequence of any cell.
  • the promoter and the base sequence may be operably linked to each other.
  • the promoter and the base sequence may not be operably linked to each other.
  • a recombinant vector comprising an ACTA promoter and a base sequence encoding PCK1 is provided.
  • a recombinant vector comprising an ACTA promoter from any animal and a base sequence encoding PCK1 is provided.
  • a recombinant vector comprising an ACTA promoter derived from a dog and a base sequence encoding PCK1 is provided.
  • the ACTA promoter and the PCK1 gene may be operably linked.
  • the ACTA promoter and PCK1 gene may not be operably linked.
  • the vector may be a vector further comprising one or more transfer factors.
  • the transfer factor may be a piggyBac transfer factor or Sleeping Beauty transfer factor.
  • the enzyme that mediates the translocation factor may include piggyBac translocation enzyme (transposase) or Sleeping Beauty translocation enzyme (transposase).
  • inverted terminal repeat sequences (ITR) and / or direct repeat (DR) may be further included at both ends of the metastasis factor.
  • the vector may be a piggyBac vector or Sleeping Beauty vector.
  • a recombinant vector including an ACTA promoter, a base sequence encoding PCK1, a transfer factor, a repeating sequence (ITR and / or DR), and a selectable marker may be provided.
  • a recombinant vector comprising an ACTA promoter, a nucleotide sequence encoding PCK1, a transfer factor, a repeating sequence (ITR and / or DR), and a selectable marker, arranged in the same order as shown in FIG. 1. have.
  • the recombinant vector may be used for knocking in a nucleotide sequence encoding a promoter and any substance into the genome of any cell (Knock in, KI).
  • the knock-in means that it is introduced into the genome of the host so that a specific foreign gene can be expressed.
  • the recombinant vector may be used for knocking in a nucleotide sequence encoding a promoter and any substance in the genome of muscle tissue or muscle cells.
  • the base sequence encoding PCK1 can be appropriately used by those skilled in the art from sequences having a base sequence encoding non-limiting PCK1 known in the art.
  • the base sequence encoding the PCK1 can be prepared by genetic recombination methods known in the art. Examples include PCR amplification to amplify nucleic acids from genomes, chemical synthesis or cDNA sequence production techniques, Fmoc techniques, and the like.
  • nucleotide sequence may have a nucleotide sequence encoding each functional equivalent of PCK1.
  • the functional equivalent is about 70%, about 71%, about 72%, about 73%, about 74%, about 75%, about 76% of the amino acid sequence of the wildtype as a result of amino acid addition, substitution or deletion. , About 77%, about 78%, about 79%, about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or about 100% or more homology
  • the "equivalent physiological activity” refers to the activity of regulating mitochondrial phyto-biosynthesis, bioenergy metabolism, glucose homeostasis, or athletic performance.
  • deletion or substitution of amino acids may be preferably located in a region not related to the physiological activity of the polypeptide of the present invention.
  • deletion or substitution of amino acids may be preferably located in a region related to the physiological activity of the polypeptide of the present invention.
  • the muscle specific PCK1 expressing recombinant vector of the present invention is a plasmid, a vector containing a transfer factor, a viral vector or a plasmid known in the art capable of expressing a nucleic acid specifically encoding a PCK1 gene in muscle tissue or cells constituting muscle tissue.
  • it may preferably be a vector containing a transfer factor or a viral vector.
  • the viral vector is not limited thereto, and may be a retrovirus vector, an adenovirus vector, a herpes virus vector, an abipoxvirus vector, or a lentivirus vector.
  • the recombinant vector may further include a selection marker.
  • the selection marker may be an antibiotic resistance gene such as a kanamycin resistance gene or a neomycin resistance gene, or a fluorescent protein such as a green fluorescent protein or a red fluorescent protein, but is not limited thereto.
  • a tag sequence for protein separation purification or identification may be further included in the vector of the present invention.
  • the tag sequence may be GFP, mRuby, GST (Glutathione S-transferase) -tag, HA, His-tag, Myc-tag, or T7-tag, but the tag sequence of the present invention is limited by the examples It does not work.
  • the tag sequence can be used to confirm the presence or absence of expression and the amount of expression using GFP or mRuby.
  • Transformed cells can be provided using the recombinant vector disclosed in the specification,
  • a transformed dog can be provided using the transformed cells.
  • a transformed dog in which a phytosynthetic reaction is activated in the muscle itself can be provided.
  • the production of glucose in the muscle itself can be increased to provide a transformed dog with improved energy metabolism and athletic performance.
  • the present invention provides a transformed cell with a knock-in sequence encoding a promoter and a specific substance.
  • the present invention provides a transformed cell having a gene sequence encoding the ACTA promoter and PCK1.
  • the base sequence encoding the ACTA promoter and PCK1 may be knocked through a known method obvious to those skilled in the art.
  • the transformed cells can be produced by introducing the recombinant vector into nuclear donor cells.
  • the present invention provides a method for producing the transformed cell and the cell into which the recombinant vector has been introduced.
  • the recombinant vector can be introduced into nuclear donor cells by any method apparent to those skilled in the art.
  • the nuclear donor cell may be a dog embryonic cell, a somatic cell or a stem cell.
  • the nuclear donor cells include, but are not limited to, blastocytes, epithelial cells, fibroblasts, neurons, keratinocytes, hematopoietic cells, melanocytes, chondrocytes, macrophages, monocytes, muscle cells, B lymphocytes, T lymphocytes, embryonic stem cells, embryonic germ cells, embryo-derived cells, placental cells and embryonic cells.
  • adult stem cells derived from tissues of various origins for example, fat, uterus, bone marrow, muscle, placenta, cord blood, or skin (epithelial) may be stem cells.
  • the nuclear donor cell may be a non-human host embryo, and is generally a 2-cell stage, 4-cell stage, 8-cell stage, 16-cell stage, 32-cell stage, 64-cell stage, loss Embryos, or embryos comprising blastocysts.
  • the nuclear donor cells may be embryo-derived cells, adult fibroblasts, and adipose stem cells.
  • the nuclear donor cells may be adipose stem cells or adult fibroblasts of dogs.
  • the characteristics of the cells have the advantage of being able to obtain a large number of cells at the time of initial separation, cell culture is relatively easy, and it is easy to culture and manipulate in vitro.
  • the embryonic cells, somatic cells or stem cells provided as nuclear donor cells can be obtained from a method for preparing a surgical specimen or biopsy specimen using conventional methods known in the art.
  • Recombinant vectors according to the invention can be introduced into cells by methods known in the art.
  • transient transfection for example, but not limited to, transient transfection, microinjection, transduction, cell fusion, calcium phosphate precipitation, liposome-mediated transfection, DEAE dextran -DEAEDextran-mediated transfection, polybrene-mediated transfection, electroporation, gene gun and other known methods for introducing nucleic acids into cells It can be introduced into cells for the production of transgenic animals by methods.
  • a specific gene can be KI in the genome of a transformed cell using the recombinant vector. Certain regions in the cell genome can be used to overexpress PCK1 specifically in muscle tissue or muscle cells.
  • the transgene can be inserted into a safe harbor site in the genome of liver cells.
  • the safe harbor site is a specific region in the genome that does not cause serious side effects, such as cancer, even when a foreign gene is inserted, and the foreign gene inserted in the specific region has a high level of permanent and safe expression. It is possible.
  • the transformed dog in order to produce the transformed dog, it is possible to provide a transformed cell in which the nucleotide sequence encoding the ACTA promoter derived from the dog and PCK1 is KI.
  • the transformed cell may further include a foreign nucleotide sequence encoding PCK1 in addition to the intrinsic nucleotide sequence encoding PCK1.
  • the transformed cell may further include a foreign nucleotide sequence encoding PCK1 in addition to the intrinsic nucleotide sequence encoding PCK1.
  • the transformed cell may include two or more base sequences encoding PCK1.
  • the transformed dog can be used to produce the transformed dog.
  • transformed cells may be proliferated or cultured according to methods known in the art.
  • the transformed cells can be grown or cultured in the medium.
  • the medium can be any medium that can be prepared for incubation of animal cells, mammalian cells, or in vitro with appropriate components necessary for animal cell growth, such as anabolic carbon, nitrogen and / or micronutrients. Can be used.
  • the medium is any basic medium suitable for animal cell growth, as a non-limiting example, Minimal Essential Medium (MEM), Dulbecco modified Eagle Medium (DMEM), Roswell Park Memorial Institute Medium (RPMI), Keratinocyte Serum Free (K-SFM) Medium), ⁇ -MEM medium (GIBCO), K-SFM medium, DMEM medium (Welgene), MCDB 131 medium (Welgene), IMEM medium (GIBCO), DMEM / F12 medium, PCM medium, M199 / F12 (mixture) ( GIBCO), and MSC extended media (Chemicon).
  • MEM Minimal Essential Medium
  • DMEM Dulbecco modified Eagle Medium
  • RPMI Roswell Park Memorial Institute Medium
  • K-SFM Keratinocyte Serum Free
  • ⁇ -MEM medium ⁇ -MEM medium
  • K-SFM medium K-SFM medium
  • DMEM medium Welgene
  • MCDB 131 medium Welgene
  • IMEM medium DM
  • a person having ordinary skill in the art can select or combine a suitable medium and appropriately culture it by a known method. In addition, it can be cultivated while adjusting conditions such as a suitable culture environment, time, and temperature based on common knowledge in this field.
  • a transformed dog can be produced using the transformed cells.
  • Somatic cell nuclear transfer (SCNT) can be used as the method.
  • the nucleus of the transformed cell is transplanted into the fertilized egg of an animal, and the fertilized egg implanted with the nucleus is conceived to produce a transformed individual encoding a promoter and a specific sequence encoding a specific substance. Implanted in the fertilized egg of the nucleus, and implanted with the nuclear implanted embryo, it is possible to produce a transformed individual encoding the ACTA promoter and the PCK1 encoding base sequence.
  • the present invention relates to a transgenic dog with improved nucleotide sequence encoding the ACTA promoter and PCK1 (Knock in, KI) and improved energy metabolism and exercise ability, a method for manufacturing the same, and use thereof.
  • nucleotide sequence encoding a dog-derived ACTA promoter and PCK1 is KI, and thus relates to a transformed dog having improved energy metabolism and athletic ability, a method for manufacturing the same, and use thereof.
  • nucleotide sequence encoding a dog-derived ACTA promoter and a human-derived PCK1 is KI, and thus relates to a transformed dog having improved energy metabolism and exercise ability, a method for manufacturing the same, and a use thereof.
  • Any of the cells of the transgenic dog may further include a foreign nucleotide sequence encoding PCK1 in addition to the intrinsic nucleotide sequence encoding PCK1.
  • the muscle tissue or muscle cells of the transgenic dog may further include a foreign nucleotide sequence encoding PCK1 in addition to an intrinsic base sequence encoding PCK1.
  • the muscle tissue or muscle cells of the transgenic dog may include two or more base sequences encoding PCK1.
  • the PCK1 expression level of any cell of the transgenic dog may be higher than that of any cell of the wild-type dog.
  • the PCK1 expression level of the muscle tissue or muscle cell of the transgenic dog may be higher than that of the wild type dog muscle tissue or muscle cell.
  • the amount of PCK1 expression in the muscle tissue or muscle cells of the transgenic dog is about 1 fold, about 1.1 fold, about 1.2 fold, and about 1.3 fold higher than that of the wild type dog muscle tissue or muscle cells.
  • the amount of PCK1 expression in muscle tissue or muscle cells of the transformed dog may be higher than that of wild-type dog muscle tissue or muscle cells.
  • the amount of PCK1 expression in muscle tissue or muscle cells of the transgenic dog may be significantly higher than that of wild-type dog muscle tissue or muscle cells.
  • the transformed dog's blood lipase concentration value may have a value of about 0.5 times, about 0.4 times, about 0.3 times, about 0.2 times, or about 0.1 times or less of the wild-type dog's blood lipace concentration value. Can be.
  • the transformed dog's blood lipase concentration value may have a value of about 2 times, about 3 times, about 4 times, about 5 times, or about 6 times or more of the wild-type dog's blood lipace concentration value. have.
  • the transformed dog's blood lipase concentration value may have a value of 0.25 times or less or 4 times or more of the wild-type dog's blood lipace concentration value.
  • the transformed cells can be used to produce the transformed dog.
  • the transformed cells may be implanted under suitable conditions to induce pregnancy.
  • transformed cells can be induced to conceive through somatic cell nuclear transfer (SCNT).
  • SCNT somatic cell nuclear transfer
  • the present invention a transformed animal of the present invention by a somatic cell nuclear transfer method (somatic cell nuclear transfer, SCNT) using a transformed cell line encoding the base sequence encoding the ACTA promoter and PCK1 derived from dogs Can produce.
  • SCNT somatic cell nuclear transfer
  • the transformed animal may be a dog overexpressing PCK1.
  • the transformed animal may be a dog overexpressing PCK1 in muscle tissue or muscle cells.
  • each step can be used with reference to a conventional method for producing cloned animals using somatic cell nuclear transfer technology known in the art.
  • a method for manufacturing a nuclear transfer embryo of a dog formed by transplanting the nucleus of the transformed cell into a denuclearized egg and a nuclear transfer embryo prepared thereby may be provided.
  • the nuclear transfer embryo is implanted into the fallopian tubes of the surrogate mother to produce an acid-producing ACTA promoter and PCK1 encoding a sequence of PCK1 overexpressing transgenic dogs or a method of producing the ACTA promoter and PCK1 produced thereby It provides a dog over-expressing PCK1, characterized in that the nucleotide sequence is encoded.
  • a method for producing the transgenic animal can be provided.
  • Nuclease may be used for the transformation.
  • the method exposes the embryo or cell to a recombinant vector (eg, a recombinant vector containing PCK1),
  • the nuclease may specifically bind to a target chromosome site in an embryo or cell, thereby causing a nucleotide sequence change in the cell chromosome.
  • One embodiment of the present invention provides the use of a dog in which the PCK1 gene is overexpressed, a muscle-specific PCK1 protein is overexpressed.
  • the PCK1 gene knocked-out transgenic dog with the PCK1 gene knocked-out according to the present invention can be produced through crossing, and later, the external gene can be delivered.
  • the dog characterized in that the PCK1 gene of the present invention is knocked in and that the knocked-in PCK1 gene is specifically expressed as a protein in muscle. It can be useful as an animal model having improved bioenergy metabolism and motor ability by expression and / or an animal model resistant to diseases such as metabolic diseases, such as diabetes.
  • the muscle-specific PCK1 overexpressing dog of the present invention is used as a quarantine dog requiring improved motor performance or is a mechanism research involved in inducing or treating metabolic diseases, the role of PCK1, muscle mass, muscle strength, muscle fibers, mitochondrial biosynthesis, Various applications such as models for studying changes in energy metabolism will be possible.
  • the present invention may include various uses of muscle-specific PCK1 overexpressing dogs in the above method from another viewpoint.
  • the animal model according to the present invention can be used as a method of screening a medicament for improving energy metabolism.
  • the screening method of the present invention is a method of the present invention.
  • the muscle tissue of the dog may be analyzed by comparison with a control group not administered with the candidate substance.
  • energy metabolism may refer to mitochondrial biosynthesis, mitochondrial activity, and vinegar biosynthesis.
  • the candidate substance may be any one selected from the group consisting of peptides, proteins, non-peptidic compounds, synthetic compounds, fermentation products, cell extracts, plant extracts, animal tissue extracts, and plasma. However, it is not limited thereto.
  • the compound may be a novel compound, or may be a well-known compound. These candidate substances can form and do salts.
  • the method for administering the candidate substance may be, for example, oral administration, intravenous injection, subcutaneous administration, intradermal administration or intraperitoneal administration. It can be appropriately selected according to the symptoms of the target animal and the properties of the candidate substance. In addition, the dosage of the candidate substance can be appropriately selected according to the administration method or the properties of the candidate substance.
  • the animal model according to the present invention can be used as a method of screening a medicament for improving athletic performance.
  • the screening method of the present invention is a method of the present invention.
  • the tissue of the dog may be analyzed by comparison with a control group in which the candidate substance is not administered.
  • the athletic performance may be muscle mass, muscle strength, and endurance compared to the control group.
  • the candidate substance may be any one or more selected from the group consisting of peptides, proteins, non-peptidic compounds, synthetic compounds, fermentation products, cell extracts, plant extracts, animal tissue extracts, and plasma. However, it is not limited thereto.
  • the compound may be a novel compound, or a well-known compound. These candidate substances may form salts.
  • the method for administering the candidate substance as described above may be, for example, oral administration, intravenous injection, subcutaneous administration, intradermal administration, or intraperitoneal administration, and may be appropriately selected according to the symptoms of the target animal and the properties of the candidate substance.
  • the dosage of the candidate substance can be appropriately selected according to the administration method or the properties of the candidate substance.
  • obesity generally means that the fat tissue in the body is excessive, and the energy consumed as food does not balance the energy consumed by physical activity and the like, and thus means a phenomenon in which excess energy is accumulated as body fat.
  • Abnormal increase in body fat due to energy imbalance over a long period of time can lead to various metabolic diseases such as diabetes, hyperlipidemia, heart disease, stroke, arteriosclerosis, fatty liver, and adult disease.
  • Obesity may be caused by an increase in the size (hypertrophy) or increase in the number of fat cells in the body (hyperplasia).
  • Obesity is caused by abnormal hypertrophy of the subcutaneous tissue caused by the accumulation of excess energy into the body when an imbalance of metabolic processes occurs due to endocrine factors, genetic factors, and social and environmental factors.
  • Fat tissue enlargement is a phenomenon in which the size of fat cells increases (fat cell enlargement) or increases in number (fat cell hyperplasia), which also affects the stagnation of the local venous-lymph system, resulting in vascular tissue disease of the dermis-subcutaneous tissue. Can also cause
  • Triglycerides which are excessively accumulated in obese patients, can be stored in the liver or muscles as well as in adipose tissue, leading to insulin resistance. Therefore, excessively stored consumption of triglycerides can be the prevention and treatment of underlying obesity and metabolic diseases.
  • the recombinant vector can be used to treat or alleviate the obesity and metabolic disease.
  • Obesity and metabolic diseases may be treated or alleviated when administered to a specific region of the living body using the recombinant vector.
  • the amount of adipose tissue or adipocytes may be reduced.
  • the obesity or metabolic disorders include, but are not limited to, metabolic syndrome, hypertriglyceridemia, high-density lipidemia, low-density lipidemia, angina pectoris, myocardial infarction, hypogonadism, sleep apnea, premenstrual syndrome, stress urinary incontinence Urinary incontinence, hyperactivity disorder, chronic fatigue syndrome, osteoarthritis, weight-related cancer, orthostatic hypotension, pulmonary hypertension, menstrual disorder, diabetes, hypertension, impaired glucose tolerance, coronary thrombosis, somnolence, depression, anxiety, psychosis, Reproductive disorders such as delayed movement disorder, drug addiction, substance abuse, cognitive impairment, Alzheimer's disease, cerebral ischemia, obsessive-compulsive behavior, panic attack, social phobia, bulimia, atherosclerosis, gallbladder disease such as cholelithiasis, anorexia, polycystic ovary disease Skin infections such as infection, varicose veins
  • the dog for the muscle-specific PCK1 overexpression model of the present invention artificially increases the activity of PCK1 as the PCK1 protein is overexpressed in muscle tissue, thereby inducing an increase in glucose production by activating the phytosynthetic reaction in the muscle itself, and also energy Since it induces an improvement in metabolism and athletic performance, energy metabolism and athletic performance can be provided as an animal model with improved obesity resistance.
  • the activity of PCK1 is artificially increased, thereby activating the phytosynthetic reaction in the muscle itself, leading to an increase in the production of glucose, and also energy Since it induces the improvement of metabolism and motor performance, it can be provided as an animal model for energy metabolism / motor performance research accordingly.
  • the present invention provides a muscle-specific PCK1 overexpressing dog and various uses thereof, thereby increasing the production of glucose by activating the phytosynthetic reaction by muscle-specific overexpressing PCK1, obesity using improved energy metabolism and improved dogs It can be used in various ways such as metabolic disease-related research (such as obesity resistance research) and energy metabolism / motor enhancement research.
  • the PCK1 gene is expressed in various tissues such as liver, kidney, adipocyte, small intestine, lung, muscle and brain. Since PCK1 is expressed in a wide range of tissues, it is important to select a promoter capable of increasing expression limited to a specific tissue in order to reduce side effects.
  • the muscle-specific promoter ⁇ -skeletal muscle actin promoter (2,372 bp) and human-derived PCK1 gene (1,896 bp) were synthesized, and then muscle specific PiggyBac vector cloning, which introduced the PCK1 gene, was synthesized under the control of an appropriate promoter.
  • the mRuby gene was used (Fig. 1).
  • the base sequence of the ACTA promoter is SEQ ID NO: 1
  • the base sequence of PCK1 is SEQ ID NO: 2
  • the base sequence of the vector constructed in this experiment is SEQ ID NO: 3
  • 5 'PB termunal repeat, and 3' PB termunal repeat are SEQ ID NO: 4, respectively.
  • 5 'PB termunal repeat is SEQ ID NO: 4
  • adipose stem cells are more suitable than adult fibroblasts as a cell line to be used for the production of transgenic cloned dogs.
  • a cell line was constructed and subjected to molecular bioassay, and the cell line confirmed the metastasis and expression of the vector was used for somatic cell nuclear transfer. (Fig. 4, Fig. 5).
  • PCK1 overexpressing transformed individuals showed the following Phenotype due to the increase in Glyceroneogenesis. It was confirmed that PCK1 production in muscle, activity in cage, exercise capacity, mitochondrial number in muscle, and feed intake increased. As the number of mitochondria in the muscle increased, it was confirmed that the over-produced triglyceride could be consumed, so that the lactate concentration in the tissue was observed low in the control group.
  • Example 5 Result of MRS imaging of femoral muscle of transgenic cloned dog
  • MRS magnetic resonance spectrography
  • Control group and PCK1 expressing dog MRS results Control 1 Control 2 Control 3 Control 4 Control 5 PCK1 Ala: Cr 0.590654 2.440945 2.852113 2.53463 1.61 2.28 Asp: Cr 3.439252 2.952756 1.725352 0.765811 1.61 1.87 Cr: Cr One One One One One One One One One One One One One One One One One One One One One One One One One One One One One One One One One One One One One GABA: Cr 0 0 0 0 0 0 0 GPC: Cr 0.842991 0.850394 0.739437 0.583113 0.63 0.24 Glc: Cr 0.623364 0.373228 0.493662 0.551045 0.872 0.264 Gln: Cr 0.86729 0.714173 0.264085 1.000515 0.84 0.141 Glth: Cr 0.324299 0.338583 0.371127 0.251672 0 0.356 Glu: Cr 0.373832 0.565354 0.760563 3.064577 0.731
  • Example 6 Evaluation of anatomical normality using X-ray in muscle-specific PCK1 overexpressed cloned dog
  • Example 7 Evaluation of normality using echocardiography in a cloned dog overexpressing muscle-specific PCK1
  • control group 4 control group 5
  • PCK1 overexpressing cloned dogs were all considered to be normal.
  • Example 8 Assessment of anatomical normality using CT in muscle-specific PCK1 overexpressing cloned dogs
  • CT scans were performed to evaluate the anatomical normality of cloned dogs overexpressing muscle-specific PCK1. Hepatobiliary system malformations and occipital-vertebral malformations were judged not to occur in both control 4, control 5, and PCK1 overexpressing cloned dogs (FIG. 21).

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Abstract

L'invention concerne des chiens transgéniques surexprimant PCK1 d'une manière spécifique au muscle, un procédé de production de ceux-ci et une utilisation de ceux-ci. En particulier, la présente invention concerne une utilisation de chiens transformés de manière à surexprimer la protéine PCK1 en particulier dans les tissus musculaires en tant que modèle animal pour étudier la masse musculaire, la force musculaire, la fibre musculaire, la biosynthèse mitochondriale, des variations du métabolisme énergétique et similaires.
PCT/KR2019/014633 2018-10-31 2019-10-31 Production de chien transgénique surexprimant la pck1 spécifique du muscle WO2020091471A1 (fr)

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