WO2020091301A1 - Inflammation-forming transgenic animal model through selective overexpression of lipocalin-2 and method for manufacturing same - Google Patents

Inflammation-forming transgenic animal model through selective overexpression of lipocalin-2 and method for manufacturing same Download PDF

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WO2020091301A1
WO2020091301A1 PCT/KR2019/014021 KR2019014021W WO2020091301A1 WO 2020091301 A1 WO2020091301 A1 WO 2020091301A1 KR 2019014021 W KR2019014021 W KR 2019014021W WO 2020091301 A1 WO2020091301 A1 WO 2020091301A1
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lcn2
animal model
inflammatory
brain
lipocalin
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PCT/KR2019/014021
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French (fr)
Korean (ko)
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박재용
권오성
황은미
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고려대학교 산학협력단
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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
    • 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
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • 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
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing

Definitions

  • the present invention relates to an inflammatory animal model transforming inflammation through selective overexpression of Lipocalin-2 (LCN2), and more specifically, a targeting vector comprising sequences that induce selective overexpression of LCN2.
  • a targeting vector comprising sequences that induce selective overexpression of LCN2.
  • Lipocalin-2 (LCN2) is also known as oncogene 24p3 or neutrophil gelatinase-associated lipocalin (NGAL).
  • LCN2 The role of LCN2 in the inflammatory situation has not been elucidated yet, and research on brain inflammation is incomplete.
  • Inflammation is a type of repair mechanism or defensive response that attempts to repair and repair the damaged area when an invasion that causes a substrate change in a living body or tissue is applied, and when stimulation is applied, inflammatory components such as inflammatory cytokines increase locally And inflammation. When inflammation occurs, symptoms such as redness, fever, swelling, pain, and dysfunction appear. Degenerative lesions, circulation Disorders and excessive proliferation of tissues.
  • the causes of inflammation include various causes such as physical damage, damage caused by chemical substances, and infection of microorganisms such as bacteria and viruses.
  • brain inflammation is also called encephalitis.
  • our head bone contains the brain, which is protected from the hard skull of the head, is supplied with nutrients in the blood, and is called the brain membrane, which serves to discharge waste products from the brain. It is surrounded by a thin film, and the case where the brain itself becomes inflamed is called encephalitis.
  • Encephalitis is a disease in which the brain parenchyma (intraventricular masses) is destroyed by an inflammatory reaction caused by bacteria or viruses.
  • Speaking of encephalitis it mainly shows symptoms of brain tissue.
  • the most typical encephalitis is Japanese encephalitis.
  • there are many cases of encephalitis caused by bacteria and viruses and the most common is herpetic encephalitis.
  • LCN2 animal models lacking the gene have been reported, but there is no animal model overexpressing the gene, so the necessity of production was required.
  • LCN2 is expected to have different functions in important cells in the brain, such as neurons, astrocytes, and microglial cells, and the development of a technical tool for this is urgently needed.
  • the object of the present invention is a CAG (Cytomegalovirus early enhancer / chicken beta action) promoter; A first loxP (locus of X-over P1) site; Stop codon sites; A second loxP site; And it provides a lipocalin-2 (Lipocalin-2; LCN2) gene targeting vector for producing an inflammatory animal model comprising a DNA sequence consisting of LCN2.
  • CAG Cytomegalovirus early enhancer / chicken beta action
  • an object of the present invention is to provide an animal cell for producing an inflammatory animal model in which the LCN2 gene target vector has been transformed.
  • an object of the present invention is to provide an inflammatory LCN2 chimeric animal model prepared by injecting animal cells into surrogate mothers.
  • an object of the present invention is to provide an inflammatory LCN2 animal model prepared by infecting a virus expressing the Cre protein in a chimeric animal model or crossing it with an astrocyte specific Cre protein producing animal model.
  • the object of the present invention (a) preparing a target vector transformed animal cells; And (b) preparing a chimeric animal model by injecting the animal cell of step (a) into a surrogate mother.
  • the object of the present invention is (a) treating the candidate substance to the subject of the inflammatory LCN2 animal model; (b) measuring the expression level of LCN2 in the subject; And (c) to provide a screening method of an inflammatory treatment material comprising the step of selecting a candidate substance for reducing the expression level of LCN2 as an inflammatory treatment substance, compared to the non-treatment group of candidate substances.
  • the object of the present invention (a) inflammatory lipocalin-2 (Lipocalin-2; LCN2) treating a candidate substance in a subject of an animal model; (b) observing a change in the shape of astrocytes and microglia in the subject; And (c) to provide a screening method of an inflammatory treatment material comprising the step of selecting a candidate substance for reducing the change in the form of astrocytes and microglia compared to the untreated group of candidate substances as an inflammatory treatment substance.
  • inflammatory lipocalin-2 Lipocalin-2; LCN2
  • the present invention is a CAG (Cytomegalovirus early enhancer / chicken beta action) promoter (SEQ ID NO: 2); A first loxP (locus of X-over P1) site (SEQ ID NO: 3); Stop codon sites; A second loxP site; And LCN2 (SEQ ID NO: 1) provides a lipocalin-2 (Lipocalin-2; LCN2) gene targeting vector for producing an inflammatory animal model comprising a DNA sequence.
  • CAG Cytomegalovirus early enhancer / chicken beta action promoter
  • p2A porcine teschovirus-1 2A
  • SEQ ID NO: 4 enhanced green fluorescent protein
  • EGFP enhanced green fluorescent protein
  • WPRE woodchuck hepatitis virus transcription factor after transcription
  • SEQ ID NO: 7 a DNA sequence consisting of SV40 polyA signal
  • the present invention provides an animal cell for producing an inflammatory animal model in which the LCN2 gene target vector has been transformed.
  • the present invention provides an inflammatory LCN2 chimeric animal model prepared by injecting animal cells into surrogate mothers.
  • the present invention provides an inflammatory LCN2 animal model prepared by infecting a virus expressing Cre protein with a chimeric animal model or crossing it with an astrocyte specific Cre protein producing animal model.
  • the present invention comprises the steps of: (a) preparing an animal cell transformed with the target vector; And (b) preparing a chimeric animal model by injecting the animal cell of step (a) into a surrogate mother.
  • the manufacturing method may include (c) infecting the virus expressing the Cre protein in the chimeric animal model of step (b).
  • the manufacturing method may include (c) crossing the chimeric animal model of step (b) with an astrocyte specific animal protein production animal model.
  • the present invention comprises the steps of: (a) treating a candidate substance in an inflammatory LCN2 animal model; (b) measuring the expression level of LCN2 in the subject; And (c) selecting a candidate substance for reducing the expression level of LCN2 as an inflammatory treatment substance, compared to a non-treatment group of candidate substances.
  • a subject for measuring the expression level of LCN2 may be separated from one or more selected from the group consisting of tissue, cells, whole blood, blood, saliva, sputum, cerebrospinal fluid, and urine.
  • the present invention comprises the steps of: (a) treating a candidate substance in a subject of an inflammatory lipocalin-2 (LCN2) animal model; (b) observing a change in the shape of astrocytes and microglia in the subject; And (c) selecting a candidate substance for reducing morphological changes of astrocytes and microglial cells as an inflammatory treatment substance, compared to a non-treatment group of candidate substances.
  • LN2 inflammatory lipocalin-2
  • a subject for observing changes in the shape of astrocytes and microglia can be separated from one or more selected from the group consisting of tissues and cells.
  • the animal may be a mouse.
  • the inflammation may be local inflammation in the brain.
  • the present inventors have made extensive studies to produce animal models overexpressing the lipocalin-2 (Lipocalin-2; LCN2) gene, and produced transgenic mice that induce overexpression of conditional LCN2 using the Cre-loxP gene expression system. And, it was confirmed that the transgenic mouse induced brain local inflammation. It is expected to be of great help in revealing the true function of LCN2 in the brain using the mouse produced with the present invention, and by overexpressing the cell-specific LCN2 gene in the brain, the cell-level function of LCN2 was previously impossible. It is expected that research will be possible to find out the role of brain inflammation, and it is expected to be able to study changes in inflammatory responses in regions of the brain by inducing local inflammatory responses. In addition, it is expected that the high level of LCN2 research and the presentation of new inflammatory animal models will greatly contribute to the study of the pathogenesis of brain inflammation and the development of treatment methods.
  • LCN2 lipocalin-2
  • 1A is a diagram showing a DNA structure design for selectively overexpressing a lipocalin-2 (LCN2) gene.
  • Figure 1b is a diagram showing a work flow (work flow) for the production of selective overexpression LCN2 transgenic mouse.
  • 2A is a diagram showing a DNA structure vector map for selectively overexpressing the LCN2 gene.
  • Figure 2b is a diagram showing the results of cutting with restriction enzymes for DNA preparation for microinjection to the male pronucleus in the DNA structure vector.
  • Figure 2c is a diagram showing the results of genomic DNA genotype PCR (genomic DNA genotype PCR) to confirm the production of the founder (founder) mouse.
  • Figure 3a is a diagram showing the results of astrocyte culture (primary astrocyte culture) experiment, in order to identify (validation) the transformed mouse produced from the founder mouse.
  • FIG. 3B is a diagram showing the results of brain stereotaxic injection of AAV-GFAP-mCh-Cre in order to confirm (validation) a transgenic mouse produced from a founder mouse.
  • FIG. 4 is a diagram showing the results of a local brain inflammation induction experiment using the identified selective overexpression LCN2 transgenic mice.
  • 5 is a diagram showing a hybrid mouse production design.
  • FIG. 6 is a diagram showing the results of LCN2 measurement in blood serum and weight change of astrocyte-scpcific LCN2 Overexpression (ALO) in astrocyte selective LCN2.
  • ALO astrocyte-scpcific LCN2 Overexpression
  • FIG. 7 is a diagram showing the results of LCN2 expression in the brain of ALO mice.
  • FIG. 8 is a view showing the results of observation of the ventricular expansion of ALO mice.
  • the present inventors have made extensive studies to produce animal models overexpressing the lipocalin-2 (Lipocalin-2; LCN2) gene, and produced transgenic mice that induce overexpression of conditional LCN2 using the Cre-loxP gene expression system. Then, it was confirmed that the transgenic mice induce brain local inflammation, and based on this, the present invention was completed.
  • LCN2 lipocalin-2
  • a DNA structural vector was prepared, cut using a restriction enzyme, and the size was confirmed, and a chimeric mouse was prepared. It was confirmed by screening whether the chimeric mouse was properly manufactured (see Example 1).
  • the selective overexpression LCN2 transgenic mouse in order to confirm whether LCN2 is overexpressed in a Cre protein-dependent mouse, is infected with AAV-Cre-mCh and AAV-GFAP-mCh-Cre after culturing main astrocytes of the chimeric mouse. It was observed whether LCN2 and EGFP are specifically expressed in brain astrocytes using ELISA and immunohistochemistry (see Example 2).
  • transgenic mouse produced in the present invention in order to confirm whether the transgenic mouse produced in the present invention can induce a local inflammatory response in the brain depending on the expression of Cre protein, brain stereotactic surgery on the unilateral hippocampal CA1 site After a local infection of AAV-GFAP-Cre-EGFP with (brain stereotaxic surgery), changes in the shape of astrocytes and microglia were observed using immunohistochemistry (see Example 3).
  • the present invention is a CAG (Cytomegalovirus early enhancer / chicken beta action) promoter; A first loxP (locus of X-over P1) site; Stop codon sites; And a lipocalin-2 (LCN2) gene targeting vector for producing an inflammatory animal model comprising a DNA sequence consisting of a sequence of a second loxP site.
  • CAG Cytomegalovirus early enhancer / chicken beta action
  • LCN2 after the second loxP site, LCN2, p2A (porcine teschovirus-1), enhanced green fluorescent protein (EGFP), woodchuck hepatitis virus transcription control factor (Woodchuck hepatitis) virus post-transcriptional regulatory element (WPRE), and a DNA sequence consisting of the sequence of SV40 polyA signal (pA).
  • p2A porcine teschovirus-1
  • EGFP enhanced green fluorescent protein
  • WPRE woodchuck hepatitis virus transcription control factor
  • pA SV40 polyA signal
  • the vector may have the cleavage map disclosed in FIG. 2A, but is not limited thereto.
  • the "LCN2" is preferably a gene comprising the nucleotide sequence of SEQ ID NO: 1 and most preferably consists of the nucleotide sequence represented by SEQ ID NO: 1, but 80% or more of the sequence of SEQ ID NO: 1, More preferably, it may include a nucleotide sequence having a sequence homology of 90% or more, and more preferably 95% or more.
  • the “CAG promoter” is preferably a gene comprising the nucleotide sequence of SEQ ID NO: 2 and most preferably consists of the nucleotide sequence represented by SEQ ID NO: 2, but 80% or more of the sequence of SEQ ID NO: 2 , More preferably 90% or more, and more preferably 95% or more.
  • the "loxP" is preferably a gene comprising the nucleotide sequence of SEQ ID NO: 3 and most preferably consists of the nucleotide sequence represented by SEQ ID NO: 3, but not less than 80% of the sequence of SEQ ID NO: 3, More preferably, it may include a nucleotide sequence having a sequence homology of 90% or more, and more preferably 95% or more.
  • the "p2A” is preferably a gene comprising the nucleotide sequence of SEQ ID NO: 4 and most preferably consists of the nucleotide sequence represented by SEQ ID NO: 4, but not less than 80% of the sequence of SEQ ID NO: 4, More preferably, it may include a nucleotide sequence having a sequence homology of 90% or more, and more preferably 95% or more.
  • the "EGFP" is preferably a gene comprising the nucleotide sequence of SEQ ID NO: 5 and most preferably consists of the nucleotide sequence represented by SEQ ID NO: 5, but 80% or more of the sequence of SEQ ID NO: 5, More preferably, it may include a nucleotide sequence having a sequence homology of 90% or more, and more preferably 95% or more.
  • the "WPRE” is preferably a gene comprising the nucleotide sequence of SEQ ID NO: 6 and most preferably consists of the nucleotide sequence represented by SEQ ID NO: 6, but not less than 80% of the sequence of SEQ ID NO: 6, More preferably, it may include a nucleotide sequence having a sequence homology of 90% or more, and more preferably 95% or more.
  • vector refers to a vector capable of expressing a peptide or protein encoded by a heterologous nucleic acid inserted in a vector, preferably a vector prepared to express LCN2.
  • vector refers to any medium for introduction and / or transfer of a base into a host cell in vitro, in vivo, or in vivo, and a replication unit capable of binding to other DNA fragments to obtain replication of the bound fragment ( replicon), and "replicating units” are any genetic units (eg, plasmids, phages, cosmids) that function as autologous units of DNA replication in vivo, that is, are replicable by their own control.
  • the recombinant expression vector of the present invention is preferably a transcription initiation factor to which RNA polymerase binds, a promoter, any operator sequence for regulating transcription, a sequence encoding a suitable mRNA ribosomal binding site, and termination of transcription and translation. It may include a sequence that controls, terminators, and the like.
  • the vector used in the present invention may be, for example, an Ai6 vector, but is not limited thereto.
  • EGFP used in the present invention means a basic (constitutive fluorescence) green fluorescent protein extracted from Aequorea victoria, and is known as a weak dimer with weak alkalinity and rapidly maturing.
  • a 2A-EGFP linker was inserted downstream of the LCN2 gene to implement a fluorescent label.
  • reporter proteins known in the art can be used instead of the EGFP, for example, Green fluorescent prtein (GFP), UnaG, dsRed, eqFp611, Dronpa, TagRFPs, KFP, EosFP, Dendra, IrisFP, or smURFP It may be a reporter protein, such as, but is not limited thereto.
  • GFP Green fluorescent prtein
  • UnaG UnaG
  • dsRed eqFp611, Dronpa
  • TagRFPs KFP
  • EosFP EosFP
  • Dendra IrisFP
  • smURFP smURFP
  • WPRE used in the present invention is a DNA sequence in which a third structure is expressed during transcription. It is commonly used in molecular biology to increase the expression of genes delivered by viral vectors. WPRE is a triad that usually contains gamma, alpha and beta elements.
  • post-transcriptional regulatory elements known in the art can be used instead of the WPRE, for example, post-transcriptional regulatory elements such as HPRE, CREs, or TREs, but are not limited thereto.
  • the Cre-loxP system using the Cre recombinase and loxP is a method of inserting a loxP site into a target target genome and expressing Cre recombinase to induce a mutation in the target gene.
  • the two loxP sites are inserted into the target gene at regular intervals, for example, in or around the intron, and recombination occurs between the loxP sites in the presence of the Cre enzyme. , The gene in between is deleted or inversion.
  • the first and second loxP sites can flank the stop codon of LCN2, and as a result, LCN2 is overexpressed in the presence of Cre. In the absence of Cre, the stop codon of the floxed LCN2 is not affected.
  • the present invention provides an animal cell for producing an inflammatory animal model in which the LCN2 gene target vector has been transformed.
  • the animal cells are transformed, transfected, Agrobacterium-mediated transformed, particle gun bombardment, sonication, electroporation And PEG (Polyethylen glycol) -mediated transformation method, but is not limited as long as it is a method capable of injecting the vector of the present invention.
  • PEG Polyethylen glycol
  • the animal cells may be male pronuclei, but are not limited thereto.
  • the present invention provides an inflammatory lipocalin-2 (LCN2) chimeric animal model prepared by injecting animal cells into a surrogate mother.
  • LN2 inflammatory lipocalin-2
  • the LCN2 gene target vector is transduced into animal cells, and then the animal cells are injected into an animal into a gastrola to make a chimeric animal model.
  • the present invention provides an inflammatory overexpression LCN2 animal model prepared by infecting a virus expressing Cre protein in a chimeric animal model or crossing it with an astrocyte specific Cre protein producing animal model.
  • the virus is a carrier protein capable of expressing Cre proteins in a cell-specific manner, for example, AAV-Cre-mCh, AAV-GFAP-mCh-Cre, AAV-GFAP-Cre-EGFP, Ad-CMV-iCre, etc.
  • AAV-Cre-mCh preferably AAV-Cre-mCh, AAV-GFAP-mCh-Cre, or AAV-GFAP-Cre-EGFP, but is not limited thereto.
  • the 'cell-specific' may be a specific site or tissue, for example, brain cell-specific, preferably astrocytes or microglia, but is not limited thereto.
  • the 'viral infection' site may be tissue, cell, systemic, or local, for example, the local part of the brain, preferably one-sided inner habenula, but is not limited thereto.
  • a carrier for expressing the Cre protein in addition to the viral infection, and is not limited to the virus for expressing the Cre protein.
  • AAV-GFAP-mCh-Cre is infected to specifically express Cre protein only in the astrocytes
  • AAV-GFAP-mCh-Cre is locally infected with unilateral medial habenula to express Cre protein only in the astrocytes, but is not limited thereto.
  • the 'Cre protein-producing animal model' may be an animal model capable of expressing Cre protein specifically, but is not limited thereto.
  • the Cre protein production animal model may be, for example, one or more selected from the group consisting of Aldh1l1-Cre / ERT2, GFAP-Cre / ERT2, Cx3cr1-Cre / ERT2 and Nestin-Cre / ERT2.
  • step (a) producing a target vector transformed animal cells; And (b) preparing a chimeric animal model by injecting the animal cell of step (a) into a surrogate mother.
  • the preparation method may include, but is not limited to, (c) infecting a virus expressing the cre protein in the chimeric animal model of step (b).
  • the manufacturing method may include, but is not limited to, (c) crossing the chimeric animal model of step (b) with an astrocyte specific animal protein production animal model; .
  • LCN2 inflammation overexpression lipocalin-2
  • the subject for measuring the expression level of LCN2 may be tissue, cells, whole blood, blood, saliva, sputum, cerebrospinal fluid, or urine, but is not limited thereto.
  • inflammation overexpression lipocalin-2 (Lipocalin-2; LCN2) processing a candidate substance in a subject of an animal model; (b) observing a change in the shape of astrocytes and microglia in the subject; And (c) selecting a candidate substance for reducing morphological changes of astrocytes and microglial cells as an inflammatory treatment substance, compared to a non-treatment group of candidate substances.
  • the subject for observing changes in the form of astrocytes and microglia may be tissues, cells, whole blood, blood, saliva, sputum, cerebrospinal fluid, or urine, and preferably tissues or cells. It is not limited.
  • test substance used while referring to the screening method of the present invention refers to an unknown material used in screening to test whether or not the present invention has an effect of treating inflammation in the brain.
  • the test substance may include small interference RNA (siRNA), short hairpin RNA (shRNA), microRNA (miRNA), ribozyme, DNAzyme, peptide nucleic acids (PNA), antisense oligonucleotides, antibodies, aptamers, natural extracts, or Chemicals include, but are not limited to.
  • the treatment of the candidate substance in step (a) includes parenteral or oral administration, stereotactic injection, but is not limited thereto, and a person skilled in the art will be able to select an appropriate method for testing the test substance in animals.
  • the step (b) is polymerase chain reaction (PCR), microarray (microarray), northern blotting (northern blotting), western blotting (western blotting), enzyme immunoassay (ELISA), immunoprecipitation (immunoprecipitation) ), Immunochemical staining (immunohistochemistry), or immunofluorescence staining (immunofluorescence) characterized in that the measurement using, but is not limited to.
  • the candidate substance refers to an unknown substance used in screening to measure the expression level of LCN2, preferably one or more selected from the group consisting of compounds, microbial cultures or extracts, natural product extracts, nucleic acids, and peptides Characterized in that, but is not limited to, the nucleic acid is at least one selected from the group consisting of aptamer (aptamer), LNA (locked nucleic acid), PNA (peptide nucleic acid), and morpholino (morpholino) However, it is not limited thereto.
  • the measurement of the expression level of LCN2 in the subject was measured by polymerase chain reaction (PCR), microarray, northern blotting, western blotting, and enzyme immunoassay (ELISA). It is characterized in that it is measured using immunoprecipitation (immunoprecipitation), immunochemical staining (immunohistochemistry), or immunofluorescence staining (immunofluorescence), but is not limited thereto.
  • PCR polymerase chain reaction
  • ELISA enzyme immunoassay
  • the animal model may be manufactured using mammals other than humans, and mammals other than humans may be monkeys, rats, mice, rabbits, dogs, primates, and the like, preferably muridae animals. However, it is not limited thereto.
  • the 'inflammatory formation' is meant to encompass the onset of inflammation, activating inflammation or overexpressing inflammation, and is not limited to any one that causes inflammation.
  • the inflammation may be at least one selected from the group consisting of local inflammation in the brain, allergy, autoimmune disease, prostatitis, glomerulonephritis, colitis, pelvicitis, and rheumatoid arthritis, preferably local inflammation in the brain. , But is not limited to this.
  • the site where the inflammation occurs may be at least one selected from the group consisting of brain, prostate, glomerulonephritis, colon, pelvis, and joints, preferably brain, but is not limited thereto.
  • the brain inflammation includes, for example, meningitis, meningitis, meningitis, brain abscess, encephalitis and inflammation accompanying brain diseases, but is not limited thereto.
  • the term “combination (s)” included in the expression of the marki form means one or more mixtures or combinations selected from the group consisting of the components described in the expression of the marki form, It means to include one or more selected from the group consisting of the above components.
  • a designed LCN2 DNA construct was constructed. As shown in Fig. 2a, for the selective and stable LCN2 gene overexpression in the Ai6 vector, a Cytomegalovirus early enhancer / chicken beta action (CAG) promoter widely used for strong expression is used, and downstream (down-stream) A stop codon, which was floxed, was inserted. When the stop codon is deleted in the presence of the Cre protein using the Cre-loxP system, LCN2-2A-EGFP is expressed, and then LCN2 and the marker protein (maker protein) EGFP are separated and function as a single protein. Is done.
  • CAG Cytomegalovirus early enhancer / chicken beta action
  • AAV-GFAP-mCh-Cre (a virus vector capable of specifically expressing Cre protein only in astrocytes) is subjected to unilateral medial habenula through brain stereotaxic surgery. ) was injected locally.
  • Figure 3b after 4 weeks after the mouse was sacrificed, and confirmed through immunohistochemistry (immunohistochemistry) technology, LCN2 and EGFP expression was specifically observed only at the site of the virus.
  • AAV-GFAP-Cre-EGFP was subjected to unilateral hippocampal CA1 (unilateral hippocampal) through brain stereotaxic surgery.
  • CA1 was injected locally.
  • specific astroglial cells GFAP staining
  • Iba1 stained microglial cells
  • Astrocyte-scpcific LCN2 Overexpression (ALO), a astrocytic selective LCN2 overexpressing transgenic mouse (Astrocyte-scpcific LCN2 Overexpression, ALO) converted by Tamoxifen treatment by crossing the produced LCN2 selective overexpressing transgenic mouse and astrocyte specific Cre producing mouse (Aldh1l1-cre / ERT2) It was produced. Specifically, 20 mg / ml Tamoxifen solution dissolved in corn-oil was intraperitoneally injected into the hybrid mice hybridized with 100 ul for 5 days every day. After 4 weeks of treatment, the brain was excised by sacrifice, and LCN2 expression and ventricular morphology were observed.
  • ALO Astrocyte-scpcific LCN2 Overexpression
  • the present inventors made extensive studies to produce animal models that overexpress the lipocalin-2 (Lipocalin-2; LCN2) gene, and produced transgenic mice that induce overexpression of conditional LCN2 using the Cre-loxP gene expression system And, it was confirmed that the transgenic mouse induced brain local inflammation. It is expected to be of great help in revealing the true function of LCN2 in the brain by using the mouse produced with the present invention. It is expected that research will be possible to find out the role of brain inflammation, and it is expected to be able to study changes in inflammatory responses in regions of the brain by inducing local inflammatory responses. In addition, the high level of LCN2 research and the presentation of new inflammatory animal models are expected to greatly contribute to the study of the pathogenesis of brain inflammation and the development of treatment methods.
  • LCN2 lipocalin-2
  • Cre-loxP Cre-loxP gene expression system

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Abstract

The present invention relates to an inflammation-inducing transgenic animal model through selective overexpression of lipocalin-2 (LCN2). As a result of earnest study to produce an animal model that overexpresses a lipocalin-2 (LCN2) gene, the present inventors have produced a transgenic mouse that induces conditional LCN2 overexpression by using the Cre-loxP gene expression system, and have confirmed that the transgenic mouse induces a local inflammation of the brain. Use of the mouse produced by the present invention is expected to be a great help in discovering the real function of the LCN2 in the brain, and by overexpressing the LCN2 gene specifically to cells in the brain, it would be possible to perform study to find out the function of the LCN2 at a cellular level and the role of the LCN2 with regard to a brain inflammation, which was impossible in the past. Also, by inducing local inflammatory responses, it is expected that it is possible to study changes regarding inflammatory responses in each region in the brain. In addition, high level study of the LCN2 and the presentation of a new inflammatory animal model are expected to greatly contribute to the pathogenesis study and the development of treatment for brain inflammations.

Description

리포칼린-2의 선택적 과발현을 통한 염증형성 형질전환 동물모델 및 그의 제조방법Inflammatory transgenic animal model through selective overexpression of lipocalin-2 and its manufacturing method
본 발명은 리포칼린-2(Lipocalin-2; LCN2)의 선택적 과발현을 통한 염증형성 형질전환 동물모델에 관한 것으로서, 보다 구체적으로는 LCN2의 선택적 과발현을 유도하는 서열들을 포함하는 표적벡터(targeting vector), 상기 표적벡터를 이용하여 생성된 염증형성 동물모델 제작용 동물세포, 이를 이용하여 얻은 염증형성 LCN2 동물모델, 그 제조방법 및 이를 이용한 염증 치료물질의 스크리닝 방법 등에 관한 것이다.The present invention relates to an inflammatory animal model transforming inflammation through selective overexpression of Lipocalin-2 (LCN2), and more specifically, a targeting vector comprising sequences that induce selective overexpression of LCN2. , Animal cells for producing an inflammatory animal model produced using the target vector, an inflammatory LCN2 animal model obtained using the same, a method for manufacturing the same, and a method for screening an inflammatory treatment substance using the same.
본 출원은 2018년 10월 31일에 출원된 한국특허출원 제10-2018-0132582호 및 2019년 10월 21일에 출원된 한국특허출원 제10-2019-0130699호에 기초한 우선권을 주장하며, 해당 출원의 명세서 및 도면에 개시된 모든 내용은 본 출원에 원용된다.This application claims priority based on Korean Patent Application No. 10-2018-0132582 filed on October 31, 2018 and Korean Patent Application No. 10-2019-0130699 filed on October 21, 2019. All contents disclosed in the specification and drawings of the application are incorporated in this application.
리포칼린-2(Lipocalin-2; LCN2)는 oncogene 24p3 또는 neutrophil gelatinase-associated lipocalin(NGAL)이라고도 알려져 있다. LCN2의 염증 상황에서의 역할은 아직 명확하게 규명되지 않고 있으며 특히 뇌 염증에 관한 연구는 미비한 실정이다.Lipocalin-2 (LCN2) is also known as oncogene 24p3 or neutrophil gelatinase-associated lipocalin (NGAL). The role of LCN2 in the inflammatory situation has not been elucidated yet, and research on brain inflammation is incomplete.
염증은 생체 또는 조직에 기질적 변화를 가져오는 침습이 가해질 때 그 손상 부위를 수복 및 재생하려는 복구기전 또는 방어적 반응의 일종으로서, 자극이 가해지면 국소적으로 염증성 사이토카인 등의 염증성 성분이 증가되며 염증이 유발된다. 염증이 발생하면 발적(redness), 발열(fever), 종창(swelling), 동통(pain), 기능장애(dysfunction) 등의 증상이 나타나며, 형태학적으로 진행을 확인할 수 있는 증상으로는 퇴행성 병변, 순환장애 및 조직의 과도한 증식 등이 있다. 염증의 발생 원인으로는 물리학적 손상, 화학적 물질에 의한 손상 및, 세균 및 바이러스 등 미생물의 감염 등 다양한 원인이 있다.Inflammation is a type of repair mechanism or defensive response that attempts to repair and repair the damaged area when an invasion that causes a substrate change in a living body or tissue is applied, and when stimulation is applied, inflammatory components such as inflammatory cytokines increase locally And inflammation. When inflammation occurs, symptoms such as redness, fever, swelling, pain, and dysfunction appear. Degenerative lesions, circulation Disorders and excessive proliferation of tissues. The causes of inflammation include various causes such as physical damage, damage caused by chemical substances, and infection of microorganisms such as bacteria and viruses.
그 중에서도, 뇌 염증은 뇌염이라고도 불린다. 즉, 우리의 머리뼈(두개골) 속에는 뇌가 들어 있고, 이 뇌는 머리의 단단한 두개골로부터 보호받고 있고, 혈액 속에서의 영양분을 공급받으며, 뇌에서 발생하는 노폐물을 배출하는 역할을 하는 뇌막이라고 부르는 얇은 막에 의해 둘러싸여 있게 되는데, 여기서 뇌 자체에 염증이 생기는 경우를 뇌염이라고 부른다. 뇌염은 뇌 실질(intraventricular masses)이 세균이나 바이러스가 원인이 되는 염증 반응에 의하여 파괴되는 질환으로 대표적인 신경계 감염증이다. 뇌염이라고 하면 주로 뇌 조직을 침범한 증상을 보인다. 가장 대표적인 뇌염에는 일본뇌염이 있다. 그 외에도 세균에 의한 뇌염, 바이러스에 의한 경우가 많고 가장 흔한 것이 헤르페스성 뇌염이다. 대개가 유행성으로 감염력이 강하고, 특별한 치료법이 없기 때문에 정신장애와 지능저하 등의 후유증이 남는 병이다. 여러 가지 원인이 있을 수 있지만, 감기 등의 호흡기 질환이나 중이염 등의 귀 질환에 의해서 흔히 발생할 수 있고, 폐렴, 매독 등의 세균성 질환이나 결핵, 곰팡이 등의 질환이 피를 타고 머리로 들어갈 수도 있다. 또, 일반적으로 바이러스성이지만 세균이나 바이러스와는 관계없이 알레르기나 자가면역 이상으로 발병하는 경우도 있다. 뇌염은 일반적으로 10세 이하의 어린이들에게 발병하는 수가 많지만, 성인이 걸리게 되는 경우에 사망률은 어린이에 비해 높아진다. 감염으로부터 발병까지의 잠복 기간은 바이러스나 세균 종류에 따라 달라진다. 대부분은 감염 후 1주일에서 1개월 사이에 발병한다. 그러나 진행이 느린 바이러스성 뇌염도 있어서, 이 경우는 발병할 때까지 몇 달이나 걸리기도 한다.Among them, brain inflammation is also called encephalitis. In other words, our head bone (cranium) contains the brain, which is protected from the hard skull of the head, is supplied with nutrients in the blood, and is called the brain membrane, which serves to discharge waste products from the brain. It is surrounded by a thin film, and the case where the brain itself becomes inflamed is called encephalitis. Encephalitis is a disease in which the brain parenchyma (intraventricular masses) is destroyed by an inflammatory reaction caused by bacteria or viruses. Speaking of encephalitis, it mainly shows symptoms of brain tissue. The most typical encephalitis is Japanese encephalitis. In addition, there are many cases of encephalitis caused by bacteria and viruses, and the most common is herpetic encephalitis. Most of them are epidemic and have strong infectious power, and there are no special treatments, resulting in mental disorders and deterioration of intelligence. Although there may be various causes, it may be commonly caused by respiratory diseases such as a cold or ear diseases such as otitis media, and bacterial diseases such as pneumonia and syphilis, and diseases such as tuberculosis and fungi may enter the head with blood. In addition, although it is generally viral, it may be caused by allergies or autoimmune abnormalities regardless of bacteria or viruses. Encephalitis usually affects children under the age of 10, but the mortality rate is higher in children with adulthood. The period of incubation from infection to onset depends on the type of virus or bacteria. Most develop from 1 week to 1 month after infection. However, there is also a slow progression of viral encephalitis, which can take months to develop.
한편, LCN2의 연구를 위하여 해당 유전자가 결핍된 동물모델은 보고되고 있으나 그 유전자의 과발현되는 동물모델은 없어 제작의 필요성이 요구되었다. 또한, LCN2는 신경세포, 별아교세포, 미세아교세포 등 뇌 내 중요 세포에서의 기능이 서로 다를 것이라 예상되고 있으며 이에 대한 기술적인 툴(tool)의 개발이 절실한 실정이었다.On the other hand, for the study of LCN2, animal models lacking the gene have been reported, but there is no animal model overexpressing the gene, so the necessity of production was required. In addition, LCN2 is expected to have different functions in important cells in the brain, such as neurons, astrocytes, and microglial cells, and the development of a technical tool for this is urgently needed.
본 발명의 목적은 CAG(Cytomegalovirus early enhancer/chicken beta action) 프로모터; 제 1 loxP(locus of X-over P1) 부위; 정지 코돈(stop codon) 부위; 제 2 loxP 부위; 및 LCN2의 순서로 이루어진 DNA 서열을 포함하는 염증형성 동물모델 제작용 리포칼린-2(Lipocalin-2; LCN2) 유전자 표적벡터(targeting vector)를 제공하는 것이다.The object of the present invention is a CAG (Cytomegalovirus early enhancer / chicken beta action) promoter; A first loxP (locus of X-over P1) site; Stop codon sites; A second loxP site; And it provides a lipocalin-2 (Lipocalin-2; LCN2) gene targeting vector for producing an inflammatory animal model comprising a DNA sequence consisting of LCN2.
또한, 본 발명의 목적은 LCN2 유전자 표적벡터가 형질 전환된 염증형성 동물모델 제작용 동물세포를 제공하는 것이다.In addition, an object of the present invention is to provide an animal cell for producing an inflammatory animal model in which the LCN2 gene target vector has been transformed.
또한, 본 발명의 목적은 동물세포를 대리모에 주입하여 제조한 염증형성 LCN2 키메라 동물모델을 제공하는 것이다.In addition, an object of the present invention is to provide an inflammatory LCN2 chimeric animal model prepared by injecting animal cells into surrogate mothers.
또한, 본 발명의 목적은 키메라 동물모델에 Cre 단백질을 발현시키는 바이러스를 감염시키거나 성상교세포 특이적 Cre 단백질 생산 동물모델과 교배시켜 제조한 염증형성 LCN2 동물모델을 제공하는 것이다.In addition, an object of the present invention is to provide an inflammatory LCN2 animal model prepared by infecting a virus expressing the Cre protein in a chimeric animal model or crossing it with an astrocyte specific Cre protein producing animal model.
또한, 본 발명의 목적은 (a) 상기 표적벡터가 형질 전환된 동물세포를 제조하는 단계; 및 (b) 상기 (a)단계의 동물세포를 대리모에 주입하여 키메라 동물모델을 제조하는 단계를 포함하는 염증형성 LCN2 동물모델 제조방법을 제공하는 것이다.In addition, the object of the present invention (a) preparing a target vector transformed animal cells; And (b) preparing a chimeric animal model by injecting the animal cell of step (a) into a surrogate mother.
또한, 본 발명의 목적은 (a) 염증형성 LCN2 동물모델의 피검체에 후보물질을 처리하는 단계; (b) 상기 피검체의 LCN2의 발현정도를 측정하는 단계; 및 (c) 후보물질 비처리군에 비해, LCN2의 발현정도가 감소되는 후보물질을 염증 치료물질로 선정하는 단계를 포함하는 염증 치료물질의 스크리닝 방법을 제공하는 것이다.In addition, the object of the present invention is (a) treating the candidate substance to the subject of the inflammatory LCN2 animal model; (b) measuring the expression level of LCN2 in the subject; And (c) to provide a screening method of an inflammatory treatment material comprising the step of selecting a candidate substance for reducing the expression level of LCN2 as an inflammatory treatment substance, compared to the non-treatment group of candidate substances.
또한, 본 발명의 목적은 (a) 염증형성 리포칼린-2(Lipocalin-2; LCN2) 동물모델의 피검체에 후보물질을 처리하는 단계; (b) 상기 피검체의 성상아교세포 및 미세아교세포의 형태의 변화를 관찰하는 단계; 및 (c) 후보물질 비처리군에 비해, 성상아교세포 및 미세아교세포의 형태의 변화가 감소되는 후보물질을 염증 치료물질로 선정하는 단계를 포함하는 염증 치료물질의 스크리닝 방법을 제공하는 것이다.In addition, the object of the present invention (a) inflammatory lipocalin-2 (Lipocalin-2; LCN2) treating a candidate substance in a subject of an animal model; (b) observing a change in the shape of astrocytes and microglia in the subject; And (c) to provide a screening method of an inflammatory treatment material comprising the step of selecting a candidate substance for reducing the change in the form of astrocytes and microglia compared to the untreated group of candidate substances as an inflammatory treatment substance.
그러나, 본 발명이 이루고자 하는 기술적 과제는 이상에서 언급한 과제에 제한되지 않으며, 언급되지 않은 또 다른 과제들은 아래의 기재로부터 본 발명이 속하는 기술 분야의 통상의 지식을 가진 자에게 명확하게 이해될 수 있을 것이다.However, the technical problem to be achieved by the present invention is not limited to the above-mentioned problems, and other problems not mentioned can be clearly understood by those skilled in the art from the following description. There will be.
상기와 같은 본 발명의 목적을 달성하기 위하여, 본 발명은 CAG(Cytomegalovirus early enhancer/chicken beta action) 프로모터(서열번호 2); 제 1 loxP(locus of X-over P1) 부위(서열번호 3); 정지 코돈(stop codon) 부위; 제 2 loxP 부위; 및 LCN2(서열번호 1)의 순서로 이루어진 DNA 서열을 포함하는 염증형성 동물모델 제작용 리포칼린-2(Lipocalin-2; LCN2) 유전자 표적벡터(targeting vector)를 제공한다.In order to achieve the object of the present invention as described above, the present invention is a CAG (Cytomegalovirus early enhancer / chicken beta action) promoter (SEQ ID NO: 2); A first loxP (locus of X-over P1) site (SEQ ID NO: 3); Stop codon sites; A second loxP site; And LCN2 (SEQ ID NO: 1) provides a lipocalin-2 (Lipocalin-2; LCN2) gene targeting vector for producing an inflammatory animal model comprising a DNA sequence.
본 발명의 일 구현 예로, LCN2 뒤에, p2A(porcine teschovirus-1 2A)(서열번호 4), 향상된 녹색형광단백질(Enhanced green fluorescent protein; EGFP)(서열번호 5), 우드척 간염 바이러스 전사 후 조절요소 (Woodchuck hepatitis virus posttranscriptional regulatory element; WPRE)(서열번호 6), 및 SV40 polyA signal(pA)(서열번호 7)의 순서로 이루어진 DNA 서열을 포함할 수 있다.An embodiment of the present invention, after LCN2, p2A (porcine teschovirus-1 2A) (SEQ ID NO: 4), enhanced green fluorescent protein (Enhanced green fluorescent protein; EGFP) (SEQ ID NO: 5), woodchuck hepatitis virus transcription factor after transcription (Woodchuck hepatitis virus posttranscriptional regulatory element; WPRE) (SEQ ID NO: 6), and may include a DNA sequence consisting of SV40 polyA signal (pA) (SEQ ID NO: 7).
또한, 본 발명은 LCN2 유전자 표적벡터가 형질 전환된 염증형성 동물모델 제작용 동물세포를 제공한다.In addition, the present invention provides an animal cell for producing an inflammatory animal model in which the LCN2 gene target vector has been transformed.
또한, 본 발명은 동물세포를 대리모에 주입하여 제조한 염증형성 LCN2 키메라 동물모델을 제공한다.In addition, the present invention provides an inflammatory LCN2 chimeric animal model prepared by injecting animal cells into surrogate mothers.
또한, 본 발명은 키메라 동물모델에 Cre 단백질을 발현시키는 바이러스를 감염시키거나 성상교세포 특이적 Cre 단백질 생산 동물모델과 교배시켜 제조한 염증형성 LCN2 동물모델을 제공한다.In addition, the present invention provides an inflammatory LCN2 animal model prepared by infecting a virus expressing Cre protein with a chimeric animal model or crossing it with an astrocyte specific Cre protein producing animal model.
또한, 본 발명은 (a) 상기 표적벡터가 형질 전환된 동물세포를 제조하는 단계; 및 (b) 상기 (a)단계의 동물세포를 대리모에 주입하여 키메라 동물모델을 제조하는 단계를 포함하는 염증형성 LCN2 동물모델 제조방법을 제공한다.In addition, the present invention comprises the steps of: (a) preparing an animal cell transformed with the target vector; And (b) preparing a chimeric animal model by injecting the animal cell of step (a) into a surrogate mother.
본 발명의 일 구현예로, 상기 제조방법은 (c) 상기 (b)단계의 키메라 동물모델에 Cre 단백질을 발현시키는 바이러스를 감염시키는 단계;를 포함하는 것일 수 있다.In one embodiment of the present invention, the manufacturing method may include (c) infecting the virus expressing the Cre protein in the chimeric animal model of step (b).
본 발명의 다른 구현예로, 상기 제조방법은 (c) 상기 (b)단계의 키메라 동물모델을 성상교세포 특이적 Cre 단백질 생산 동물모델과 교배하는 단계;를 포함하는 것일 수 있다.In another embodiment of the present invention, the manufacturing method may include (c) crossing the chimeric animal model of step (b) with an astrocyte specific animal protein production animal model.
또한, 본 발명은 (a) 염증형성 LCN2 동물모델의 피검체에 후보물질을 처리하는 단계; (b) 상기 피검체의 LCN2의 발현정도를 측정하는 단계; 및 (c) 후보물질 비처리군에 비해, LCN2의 발현정도가 감소되는 후보물질을 염증 치료물질로 선정하는 단계를 포함하는 염증 치료물질의 스크리닝 방법을 제공한다.In addition, the present invention comprises the steps of: (a) treating a candidate substance in an inflammatory LCN2 animal model; (b) measuring the expression level of LCN2 in the subject; And (c) selecting a candidate substance for reducing the expression level of LCN2 as an inflammatory treatment substance, compared to a non-treatment group of candidate substances.
본 발명의 일 구현 예로, LCN2의 발현정도를 측정하기 위한 피검체는 조직, 세포, 전혈, 혈액, 타액, 객담, 뇌척수액, 및 뇨(urine)로 이루어진 군에서 선택된 하나 이상에서 분리될 수 있다.As an embodiment of the present invention, a subject for measuring the expression level of LCN2 may be separated from one or more selected from the group consisting of tissue, cells, whole blood, blood, saliva, sputum, cerebrospinal fluid, and urine.
또한, 본 발명은 (a) 염증형성 리포칼린-2(Lipocalin-2; LCN2) 동물모델의 피검체에 후보물질을 처리하는 단계; (b) 상기 피검체의 성상아교세포 및 미세아교세포의 형태의 변화를 관찰하는 단계; 및 (c) 후보물질 비처리군에 비해, 성상아교세포 및 미세아교세포의 형태의 변화가 감소되는 후보물질을 염증 치료물질로 선정하는 단계를 포함하는 염증 치료물질의 스크리닝 방법을 제공한다.In addition, the present invention comprises the steps of: (a) treating a candidate substance in a subject of an inflammatory lipocalin-2 (LCN2) animal model; (b) observing a change in the shape of astrocytes and microglia in the subject; And (c) selecting a candidate substance for reducing morphological changes of astrocytes and microglial cells as an inflammatory treatment substance, compared to a non-treatment group of candidate substances.
본 발명의 일 구현 예로, 성상아교세포 및 미세아교세포의 형태의 변화를 관찰하기 위한 피검체는 조직 및 세포로 이루어진 군에서 선택된 하나 이상에서 분리될 수 있다.As an exemplary embodiment of the present invention, a subject for observing changes in the shape of astrocytes and microglia can be separated from one or more selected from the group consisting of tissues and cells.
본 발명의 일 구현 예로, 상기 동물은 마우스일 수 있다.In one embodiment of the present invention, the animal may be a mouse.
본 발명의 다른 구현 예로, 상기 염증은 뇌 내 국부염증일 수 있다.In another embodiment of the present invention, the inflammation may be local inflammation in the brain.
본 발명자들은 리포칼린-2(Lipocalin-2; LCN2) 유전자를 과발현 시키는 동물모델을 제작하기 위해 예의 연구한 결과, Cre-loxP 유전자 발현 시스템을 이용하여 조건부 LCN2의 과발현을 유도하는 형질전환 마우스를 제작하고, 상기 형질전환 마우스가 뇌 국부 염증을 유도하는 것을 확인하였다. 본 발명으로 제작된 마우스를 이용하여 진정한 뇌 내 LCN2의 기능을 밝혀내는 데에 큰 도움이 될 것으로 기대되고, 뇌 내 세포 특이적으로 LCN2 유전자를 과발현 시킴으로써 기존에는 불가능 하였던 LCN2의 세포수준의 기능과 뇌 염증에 대한 역할을 알아내는 연구를 가능할 것으로 판단되며, 국부적인 염증반응을 유도함으로써 뇌 내 지역별 염증반응에 대한 변화를 연구할 수 있을 것이라 기대된다. 또한, 높은 수준의 LCN2 연구와 새로운 염증 동물모델의 제시로 뇌 염증에 대한 발병기전 연구 및 치료법 개발에 크게 기여할 것으로 기대된다.The present inventors have made extensive studies to produce animal models overexpressing the lipocalin-2 (Lipocalin-2; LCN2) gene, and produced transgenic mice that induce overexpression of conditional LCN2 using the Cre-loxP gene expression system. And, it was confirmed that the transgenic mouse induced brain local inflammation. It is expected to be of great help in revealing the true function of LCN2 in the brain using the mouse produced with the present invention, and by overexpressing the cell-specific LCN2 gene in the brain, the cell-level function of LCN2 was previously impossible. It is expected that research will be possible to find out the role of brain inflammation, and it is expected to be able to study changes in inflammatory responses in regions of the brain by inducing local inflammatory responses. In addition, it is expected that the high level of LCN2 research and the presentation of new inflammatory animal models will greatly contribute to the study of the pathogenesis of brain inflammation and the development of treatment methods.
도 1a는 리포칼린-2(Lipocalin-2; LCN2) 유전자를 선택적으로 과발현 시키기 위한 DNA 구조(construct) 디자인을 나타낸 도이다.1A is a diagram showing a DNA structure design for selectively overexpressing a lipocalin-2 (LCN2) gene.
도 1b는 선택적 과발현 LCN2 형질전환 마우스 제작을 위한 작업 흐름(work flow)을 나타낸 도이다.Figure 1b is a diagram showing a work flow (work flow) for the production of selective overexpression LCN2 transgenic mouse.
도 2a는 LCN2 유전자를 선택적으로 과발현 시키기 위한 DNA 구조 벡터 맵(vector map)을 나타낸 도이다.2A is a diagram showing a DNA structure vector map for selectively overexpressing the LCN2 gene.
도 2b는 DNA 구조 벡터에서 웅성전핵(Male pronucleus)에 대한 미세주입(microinjection)용 DNA 준비를 위하여 제한효소로 절단한 결과를 나타낸 도이다.Figure 2b is a diagram showing the results of cutting with restriction enzymes for DNA preparation for microinjection to the male pronucleus in the DNA structure vector.
도 2c는 만들어진 파운더(founder) 마우스의 제작 확인을 위하여, 게놈 DNA 유전자형 PCR(genomic DNA genotype PCR) 결과를 나타낸 도이다.Figure 2c is a diagram showing the results of genomic DNA genotype PCR (genomic DNA genotype PCR) to confirm the production of the founder (founder) mouse.
도 3a는 파운더 마우스로부터 생산된 형질전환 마우스를 확인(validation)하기 위하여, 성상세포 배양(primary astrocyte culture) 실험 결과를 나타낸 도이다.Figure 3a is a diagram showing the results of astrocyte culture (primary astrocyte culture) experiment, in order to identify (validation) the transformed mouse produced from the founder mouse.
도 3b는 파운더 마우스로부터 생산된 형질전환 마우스를 확인(validation)하기 위하여, AAV-GFAP-mCh-Cre를 뇌 정위 주입(brain stereotaxic injection)한 결과를 나타낸 도이다.FIG. 3B is a diagram showing the results of brain stereotaxic injection of AAV-GFAP-mCh-Cre in order to confirm (validation) a transgenic mouse produced from a founder mouse.
도 4는 확인된 선택적 과발현 LCN2 형질전환 마우스를 이용하여, 국부적 뇌 염증 유도 실험한 결과를 나타낸 도이다.4 is a diagram showing the results of a local brain inflammation induction experiment using the identified selective overexpression LCN2 transgenic mice.
도 5는 하이브리드 생쥐 제작 디자인을 나타낸 도이다.5 is a diagram showing a hybrid mouse production design.
도 6은 성상교세포 선택적 LCN2 과발현 생쥐(Astrocyte-scpcific LCN2 Overexpression, ALO) 의 체중변화 및 혈액 serum안 LCN2 측정 결과를 나타낸 도이다.6 is a diagram showing the results of LCN2 measurement in blood serum and weight change of astrocyte-scpcific LCN2 Overexpression (ALO) in astrocyte selective LCN2.
도 7은 ALO 생쥐의 뇌 내 LCN2 발현 결과를 나타낸 도이다.7 is a diagram showing the results of LCN2 expression in the brain of ALO mice.
도 8은 ALO 생쥐의 뇌실 확장 관찰 결과를 나타낸 도이다.8 is a view showing the results of observation of the ventricular expansion of ALO mice.
본 발명자들은 리포칼린-2(Lipocalin-2; LCN2) 유전자를 과발현 시키는 동물모델을 제작하기 위해 예의 연구한 결과, Cre-loxP 유전자 발현 시스템을 이용하여 조건부 LCN2의 과발현을 유도하는 형질전환 마우스를 제작하고, 상기 형질전환 마우스가 뇌 국부 염증을 유도하는 것을 확인하고, 이에 기초하여 본 발명을 완성하였다.The present inventors have made extensive studies to produce animal models overexpressing the lipocalin-2 (Lipocalin-2; LCN2) gene, and produced transgenic mice that induce overexpression of conditional LCN2 using the Cre-loxP gene expression system. Then, it was confirmed that the transgenic mice induce brain local inflammation, and based on this, the present invention was completed.
이하, 본 발명을 자세히 설명한다.Hereinafter, the present invention will be described in detail.
본 발명의 일 실시 예에서는, 선택적 과발현 LCN2 형질전환 마우스를 제작하기 위하여, DNA 구조 벡터를 제작하고, 제한효소를 이용하여 자른 후 그 크기를 확인하였으며, 키메라 마우스를 제작하고, PCR기술을 이용해 상기 키메라 마우스가 제대로 제작되었는지 여부를 스크리닝으로 확인하였다(실시예 1 참조).In one embodiment of the present invention, in order to produce a selective overexpression LCN2 transgenic mouse, a DNA structural vector was prepared, cut using a restriction enzyme, and the size was confirmed, and a chimeric mouse was prepared. It was confirmed by screening whether the chimeric mouse was properly manufactured (see Example 1).
본 발명의 다른 실시 예에서는 선택적 과발현 LCN2 형질전환 마우스가 Cre 단백질 의존적으로 LCN2가 과발현되는지 확인하기 위하여, 키메라 마우스의 주요 성상세포 배양 후 AAV-Cre-mCh 및 AAV-GFAP-mCh-Cre를 감염시켜 ELISA 및 면역조직화학 기술을 이용하여 뇌 성상아교세포 특이적으로 LCN2와 EGFP가 발현되는지 관찰하였다(실시예 2 참조).In another embodiment of the present invention, in order to confirm whether LCN2 is overexpressed in a Cre protein-dependent mouse, the selective overexpression LCN2 transgenic mouse is infected with AAV-Cre-mCh and AAV-GFAP-mCh-Cre after culturing main astrocytes of the chimeric mouse. It was observed whether LCN2 and EGFP are specifically expressed in brain astrocytes using ELISA and immunohistochemistry (see Example 2).
본 발명의 또 다른 실시 예에서는 본 발명에서 제작한 형질전환 마우스가 Cre 단백질 발현에 의존적으로 뇌 내 국부 염증 반응을 유도할 수 있는지 확인하기 위해, 일측 해마 CA1 (unilateral hippocampal CA1) 부위에 뇌 정위 수술(brain stereotaxic surgery)로 AAV-GFAP-Cre-EGFP를 국부 감염한 뒤, 면역조직화학 기술을 이용하여 성상아교세포와 미세아교세포의 형태의 변화를 관찰하였다(실시예 3 참조).In another embodiment of the present invention, in order to confirm whether the transgenic mouse produced in the present invention can induce a local inflammatory response in the brain depending on the expression of Cre protein, brain stereotactic surgery on the unilateral hippocampal CA1 site After a local infection of AAV-GFAP-Cre-EGFP with (brain stereotaxic surgery), changes in the shape of astrocytes and microglia were observed using immunohistochemistry (see Example 3).
따라서, 본 발명은 CAG(Cytomegalovirus early enhancer/chicken beta action) 프로모터; 제 1 loxP(locus of X-over P1) 부위; 정지 코돈(stop codon) 부위; 및 제 2 loxP 부위의 순서로 이루어진 DNA 서열을 포함하는 염증형성 동물모델 제작용 리포칼린-2(Lipocalin-2; LCN2) 유전자 표적벡터(targeting vector)를 제공한다.Therefore, the present invention is a CAG (Cytomegalovirus early enhancer / chicken beta action) promoter; A first loxP (locus of X-over P1) site; Stop codon sites; And a lipocalin-2 (LCN2) gene targeting vector for producing an inflammatory animal model comprising a DNA sequence consisting of a sequence of a second loxP site.
본 발명의 구체적 실시 예에 따르면, 상기 제 2 loxP 부위 뒤에, LCN2, p2A(porcine teschovirus-1), 향상된 녹색형광단백질(Enhanced green fluorescent protein; EGFP), 우드척 간염 바이러스 전사 후 조절요소(Woodchuck hepatitis virus post-transcriptional regulatory element; WPRE), 및 SV40 polyA signal(pA)의 순서로 이루어진 DNA 서열을 포함할 수 있으나 이에 제한되지 않는다.According to a specific embodiment of the present invention, after the second loxP site, LCN2, p2A (porcine teschovirus-1), enhanced green fluorescent protein (EGFP), woodchuck hepatitis virus transcription control factor (Woodchuck hepatitis) virus post-transcriptional regulatory element (WPRE), and a DNA sequence consisting of the sequence of SV40 polyA signal (pA).
본 명세서에서 있어서, 상기 벡터는 도 2a에 개시된 개열지도를 가질 수 있으나, 이에 제한되는 것은 아니다.In the present specification, the vector may have the cleavage map disclosed in FIG. 2A, but is not limited thereto.
본 명세서에 있어서, 상기 “LCN2”는 바람직하게는 서열번호 1의 염기서열을 포함하는 유전자이고 가장 바람직하게는 서열번호 1로 표시되는 염기서열로 이루어지나, 서열번호 1의 서열과 80 % 이상, 더욱 바람직하게는 90 % 이상, 더욱 바람직하게는 95 % 이상의 서열 상동성을 가지는 염기서열을 포함할 수 있다. In this specification, the "LCN2" is preferably a gene comprising the nucleotide sequence of SEQ ID NO: 1 and most preferably consists of the nucleotide sequence represented by SEQ ID NO: 1, but 80% or more of the sequence of SEQ ID NO: 1, More preferably, it may include a nucleotide sequence having a sequence homology of 90% or more, and more preferably 95% or more.
본 명세서에 있어서, 상기 “CAG 프로모터”는 바람직하게는 서열번호 2의 염기서열을 포함하는 유전자이고 가장 바람직하게는 서열번호 2로 표시되는 염기서열로 이루어지나, 서열번호 2의 서열과 80 % 이상, 더욱 바람직하게는 90 % 이상, 더욱 바람직하게는 95 % 이상의 서열 상동성을 가지는 염기서열을 포함할 수 있다. In the present specification, the “CAG promoter” is preferably a gene comprising the nucleotide sequence of SEQ ID NO: 2 and most preferably consists of the nucleotide sequence represented by SEQ ID NO: 2, but 80% or more of the sequence of SEQ ID NO: 2 , More preferably 90% or more, and more preferably 95% or more.
본 명세서에 있어서, 상기 “loxP”는 바람직하게는 서열번호 3의 염기서열을 포함하는 유전자이고 가장 바람직하게는 서열번호 3으로 표시되는 염기서열로 이루어지나, 서열번호 3의 서열과 80 % 이상, 더욱 바람직하게는 90 % 이상, 더욱 바람직하게는 95 % 이상의 서열 상동성을 가지는 염기서열을 포함할 수 있다. In the present specification, the "loxP" is preferably a gene comprising the nucleotide sequence of SEQ ID NO: 3 and most preferably consists of the nucleotide sequence represented by SEQ ID NO: 3, but not less than 80% of the sequence of SEQ ID NO: 3, More preferably, it may include a nucleotide sequence having a sequence homology of 90% or more, and more preferably 95% or more.
본 명세서에 있어서, 상기 “p2A”는 바람직하게는 서열번호 4의 염기서열을 포함하는 유전자이고 가장 바람직하게는 서열번호 4로 표시되는 염기서열로 이루어지나, 서열번호 4의 서열과 80 % 이상, 더욱 바람직하게는 90 % 이상, 더욱 바람직하게는 95 % 이상의 서열 상동성을 가지는 염기서열을 포함할 수 있다. In the present specification, the "p2A" is preferably a gene comprising the nucleotide sequence of SEQ ID NO: 4 and most preferably consists of the nucleotide sequence represented by SEQ ID NO: 4, but not less than 80% of the sequence of SEQ ID NO: 4, More preferably, it may include a nucleotide sequence having a sequence homology of 90% or more, and more preferably 95% or more.
본 명세서에 있어서, 상기 “EGFP”는 바람직하게는 서열번호 5의 염기서열을 포함하는 유전자이고 가장 바람직하게는 서열번호 5로 표시되는 염기서열로 이루어지나, 서열번호 5의 서열과 80 % 이상, 더욱 바람직하게는 90 % 이상, 더욱 바람직하게는 95 % 이상의 서열 상동성을 가지는 염기서열을 포함할 수 있다. In the present specification, the "EGFP" is preferably a gene comprising the nucleotide sequence of SEQ ID NO: 5 and most preferably consists of the nucleotide sequence represented by SEQ ID NO: 5, but 80% or more of the sequence of SEQ ID NO: 5, More preferably, it may include a nucleotide sequence having a sequence homology of 90% or more, and more preferably 95% or more.
본 명세서에 있어서, 상기 “WPRE”는 바람직하게는 서열번호 6의 염기서열을 포함하는 유전자이고 가장 바람직하게는 서열번호 6으로 표시되는 염기서열로 이루어지나, 서열번호 6의 서열과 80 % 이상, 더욱 바람직하게는 90 % 이상, 더욱 바람직하게는 95 % 이상의 서열 상동성을 가지는 염기서열을 포함할 수 있다. In the present specification, the "WPRE" is preferably a gene comprising the nucleotide sequence of SEQ ID NO: 6 and most preferably consists of the nucleotide sequence represented by SEQ ID NO: 6, but not less than 80% of the sequence of SEQ ID NO: 6, More preferably, it may include a nucleotide sequence having a sequence homology of 90% or more, and more preferably 95% or more.
본 명세서에 있어서, “벡터(expression vector)”란 벡터 내에 삽입된 이종의 핵산에 의해 코딩되는 펩타이드 또는 단백질을 발현할 수 있는 벡터를 지칭하는 것으로, 바람직하게는 LCN2를 발현할 수 있도록 제조된 벡터를 의미한다. 상기 "벡터"는 시험관 내, 생체 왜 또는 생체 내에서 숙주 세포로 염기의 도입 및/또는 전이를 위한 임의의 매개물을 말하며, 다른 DNA 단편이 결합하여 결합된 단편의 복제를 가져올 수 있는 복제단위(replicon)일 수 있으며, "복제 단위"란 생체 내에서 DNA 복제의 자가 유닛으로서 기능하는, 즉, 스스로의 조절에 의해 복제가능한, 임의의 유전적 단위(예를 들면, 플라스미드, 파지, 코스미드, 염색체, 바이러스 등)를 말한다. 본 발명의 재조합 발현 벡터는 바람직하게는 RNA 중합효소가 결합하는 전사 개시 인자인 프로모터(promoter), 전사를 조절하기 위한 임의의 오퍼레이터 서열, 적합한 mRNA 리보좀 결합 부위를 코딩하는 서열과 전사 및 해독의 종결을 조절하는 서열, 터미네이터 등을 포함할 수 있다. 본 발명에서 사용되는 벡터는 예를 들어 Ai6 벡터일 수 있으나, 이에 제한되는 것은 아니다.In the present specification, "vector (expression vector)" refers to a vector capable of expressing a peptide or protein encoded by a heterologous nucleic acid inserted in a vector, preferably a vector prepared to express LCN2. Means The "vector" refers to any medium for introduction and / or transfer of a base into a host cell in vitro, in vivo, or in vivo, and a replication unit capable of binding to other DNA fragments to obtain replication of the bound fragment ( replicon), and "replicating units" are any genetic units (eg, plasmids, phages, cosmids) that function as autologous units of DNA replication in vivo, that is, are replicable by their own control. Chromosome, virus, etc.). The recombinant expression vector of the present invention is preferably a transcription initiation factor to which RNA polymerase binds, a promoter, any operator sequence for regulating transcription, a sequence encoding a suitable mRNA ribosomal binding site, and termination of transcription and translation. It may include a sequence that controls, terminators, and the like. The vector used in the present invention may be, for example, an Ai6 vector, but is not limited thereto.
본 발명에 사용되는 용어 “EGFP”란, Aequorea victoria에서 추출한 기본 (구성 형광) 녹색 형광 단백질을 의미하며, 알칼리성산 민감도가 약하고 빠르게 성숙하는 약한 이량체로 알려져 있다. LCN2가 과발현된 세포의 표식을 위하여 2A-EGFP linker를 LCN2 유전자 하류(down-stream)에 삽입하여 형광 표식을 구현하였다.The term “EGFP” used in the present invention means a basic (constitutive fluorescence) green fluorescent protein extracted from Aequorea victoria, and is known as a weak dimer with weak alkalinity and rapidly maturing. For labeling of cells overexpressed with LCN2, a 2A-EGFP linker was inserted downstream of the LCN2 gene to implement a fluorescent label.
본 발명에 있어서, 상기 EGFP 대신 당업계에 공지된 다양한 리포터 단백질을 사용할 수 있으며, 예컨대, GFP(Green fluorescent prtein), UnaG, dsRed, eqFp611, Dronpa, TagRFPs, KFP, EosFP, Dendra, IrisFP, 또는 smURFP 등의 리포터 단백질일 수 있으나, 이에 제한되지 않는다.In the present invention, various reporter proteins known in the art can be used instead of the EGFP, for example, Green fluorescent prtein (GFP), UnaG, dsRed, eqFp611, Dronpa, TagRFPs, KFP, EosFP, Dendra, IrisFP, or smURFP It may be a reporter protein, such as, but is not limited thereto.
본 발명에 사용되는 용어 “WPRE”란, 전사시 제 3의 구조가 발현되는 DNA 서열이다. 바이러스 벡터에 의해 전달되는 유전자의 발현을 증가시키기 위해 분자 생물학에서 일반적으로 사용된다. WPRE는 보통 감마, 알파 및 베타 요소가 포함된 삼자 조절요소이다.The term “WPRE” used in the present invention is a DNA sequence in which a third structure is expressed during transcription. It is commonly used in molecular biology to increase the expression of genes delivered by viral vectors. WPRE is a triad that usually contains gamma, alpha and beta elements.
본 발명에 있어서, 상기 WPRE 대신 당업계에 공지된 다양한 전사 후 조절요소를 사용할 수 있으며, 예컨대, HPRE, CREs, 또는 TREs 등의 전사 후 조절요소일 수 있으나, 이에 제한되지 않는다.In the present invention, various post-transcriptional regulatory elements known in the art can be used instead of the WPRE, for example, post-transcriptional regulatory elements such as HPRE, CREs, or TREs, but are not limited thereto.
본 발명에 있어서, 상기 Cre 재조합효소와 loxP를 이용한 Cre-loxP 시스템은 loxP 부위를 목적하는 표적 유전체에 삽입하고, Cre 재조합효소를 발현시켜 표적 유전자에 돌연변이를 유발하는 방법이다. 표적하는 유전자가 플록스드(floxed)된 경우, 두 개의 loxP 사이트는 일정 간격을 두고 목적하는 유전자의 내부, 예를 들면 인트론 부위 또는 그 주위에 삽입되고, Cre 효소의 존재시 loxP 사이트간에 재조합이 일어나, 그 사이의 유전자가 결실(deletion) 또는 역위(inversion)된다. 본 발명에서 제 1 및 제 2 loxP 부위는 LCN2의 정지코돈을 플랭크(flank) 할 수 있고, 그 결과 Cre의 존재시 LCN2가 과발현된다. Cre가 존재하지 않는 경우, 플록스(floxed)된 LCN2의 정지코돈은 영향을 받지 않는다.In the present invention, the Cre-loxP system using the Cre recombinase and loxP is a method of inserting a loxP site into a target target genome and expressing Cre recombinase to induce a mutation in the target gene. When the target gene is floxed, the two loxP sites are inserted into the target gene at regular intervals, for example, in or around the intron, and recombination occurs between the loxP sites in the presence of the Cre enzyme. , The gene in between is deleted or inversion. In the present invention, the first and second loxP sites can flank the stop codon of LCN2, and as a result, LCN2 is overexpressed in the presence of Cre. In the absence of Cre, the stop codon of the floxed LCN2 is not affected.
또한, 본 발명은 LCN2 유전자 표적벡터가 형질 전환된 염증형성 동물모델 제작용 동물세포를 제공한다.In addition, the present invention provides an animal cell for producing an inflammatory animal model in which the LCN2 gene target vector has been transformed.
상기 동물세포는 형질전환(transformation), 형질감염(transfection), 아그로박테리움(Agrobacterium)-매개 형질전환 방법, 입자 총 충격법(particle gun bombardment), 초음파 처리법(sonication), 전기충격법(electroporation) 및 PEG(Polyethylen glycol)-매개 형질전환 방법 등의 방법으로 제조될 수 있으나, 본 발명의 벡터를 주입할 수 있는 방법이라면 제한이 없다.The animal cells are transformed, transfected, Agrobacterium-mediated transformed, particle gun bombardment, sonication, electroporation And PEG (Polyethylen glycol) -mediated transformation method, but is not limited as long as it is a method capable of injecting the vector of the present invention.
상기 동물세포는 웅성전핵일 수 있으나 이에 제한되지 않는다.The animal cells may be male pronuclei, but are not limited thereto.
또한, 본 발명은 동물세포를 대리모에 주입하여 제조한 염증형성 리포칼린-2(Lipocalin-2; LCN2) 키메라 동물모델을 제공한다.In addition, the present invention provides an inflammatory lipocalin-2 (LCN2) chimeric animal model prepared by injecting animal cells into a surrogate mother.
본 발명에 있어서, 상기 동물세포를 대리모에 주입하는 것 대신, LCN2 유전자 표적벡터를 동물세포로 형질도입한 후, 그 동물세포를 동물에 낭배(gastrula)에 주입하여 키메라 동물모델을 만들고, 그 모델을 Cre 단백질이 있는 동물모델과 교배시켜 염증형성 LCN2 동물모델을 제조할 수 있으나, 이에 제한되지 않는다. In the present invention, instead of injecting the animal cells into a surrogate mother, the LCN2 gene target vector is transduced into animal cells, and then the animal cells are injected into an animal into a gastrola to make a chimeric animal model. Can be crossed with an animal model with Cre protein to produce an inflammatory LCN2 animal model, but is not limited thereto.
또한, 본 발명은 키메라 동물모델에 Cre 단백질을 발현시키는 바이러스를 감염시키거나 성상교세포 특이적 Cre 단백질 생산 동물모델과 교배시켜 제조한 염증형성 과발현 LCN2 동물모델을 제공한다.In addition, the present invention provides an inflammatory overexpression LCN2 animal model prepared by infecting a virus expressing Cre protein in a chimeric animal model or crossing it with an astrocyte specific Cre protein producing animal model.
상기 바이러스는 세포특이적으로 Cre 단백질을 발현시킬 수 있는 전달체 단백질로서, 예를 들어, AAV-Cre-mCh, AAV-GFAP-mCh-Cre, AAV-GFAP-Cre-EGFP, Ad-CMV-iCre 등이 사용될 수 있고, 바람직하게는 AAV-Cre-mCh, AAV-GFAP-mCh-Cre, 또는 AAV-GFAP-Cre-EGFP 이나 이에 제한되지 않는다.The virus is a carrier protein capable of expressing Cre proteins in a cell-specific manner, for example, AAV-Cre-mCh, AAV-GFAP-mCh-Cre, AAV-GFAP-Cre-EGFP, Ad-CMV-iCre, etc. This can be used, preferably AAV-Cre-mCh, AAV-GFAP-mCh-Cre, or AAV-GFAP-Cre-EGFP, but is not limited thereto.
상기 '세포특이적'이란, 특이적인 부위 또는 조직일 수 있고, 예를 들어, 뇌세포 특이적일 수 있으며, 바람직하게는 성상세포 또는 미세아교세포일 수 있으나, 이에 제한되지 않는다.The 'cell-specific' may be a specific site or tissue, for example, brain cell-specific, preferably astrocytes or microglia, but is not limited thereto.
상기 '바이러스 감염' 부위는 조직, 세포, 전신, 또는 국부일 수 있고, 예를 들어, 뇌의 국부일 수 있고, 바람직하게는 일방 내측 고삐핵(habenula)일 수 있으나 이에 제한되지 않는다.The 'viral infection' site may be tissue, cell, systemic, or local, for example, the local part of the brain, preferably one-sided inner habenula, but is not limited thereto.
또한, 상기 바이러스 감염 외에도 Cre 단백질을 발현시키기 위한 전달체를 사용하는 것이라면 가능하며, 상기 Cre 단백질을 발현시키기 위한 바이러스에 한정되지 않는다. In addition, it is possible to use a carrier for expressing the Cre protein in addition to the viral infection, and is not limited to the virus for expressing the Cre protein.
본 발명의 구체적 실시예 따라, 상기 키메라 동물모델을 주요 성상세포 배양(primary astrocyte culture)한 후, AAV-GFAP-mCh-Cre를 감염시켜 특이적으로 상기 성상세포에만 Cre 단백질을 발현시킬 수 있고, 바람직하게는 일방 내측 하베눌라(unilateral medial habenula)에 AAV-GFAP-mCh-Cre를 국부감염시켜 상기 성상세포에만 Cre 단백질을 발현시킬 수 있으나, 이에 제한되지 않는다.According to a specific embodiment of the present invention, after the primary astrocyte culture of the chimeric animal model, AAV-GFAP-mCh-Cre is infected to specifically express Cre protein only in the astrocytes, Preferably, AAV-GFAP-mCh-Cre is locally infected with unilateral medial habenula to express Cre protein only in the astrocytes, but is not limited thereto.
상기 ‘Cre 단백질 생산 동물모델’은 세포특이적으로 Cre 단백질을 발현시킬 수 있는 동물 모델일 수 있으나, 이에 제한되지 않는다. 상기 Cre 단백질 생산 동물모델은 예를 들어 Aldh1l1-Cre/ERT2, GFAP-Cre/ERT2, Cx3cr1-Cre/ERT2 및 Nestin-Cre/ERT2로 이루어진 군으로부터 선택된 하나 이상일 수 있다.The 'Cre protein-producing animal model' may be an animal model capable of expressing Cre protein specifically, but is not limited thereto. The Cre protein production animal model may be, for example, one or more selected from the group consisting of Aldh1l1-Cre / ERT2, GFAP-Cre / ERT2, Cx3cr1-Cre / ERT2 and Nestin-Cre / ERT2.
또한, 본 발명의 다른 양태로서, (a) 상기 표적벡터가 형질 전환된 동물세포를 제조하는 단계; 및 (b) 상기 (a)단계의 동물세포를 대리모에 주입하여 키메라 동물모델을 제조하는 단계를 포함하는 염증형성 LCN2 동물모델 제조방법을 제공한다.In addition, as another aspect of the present invention, (a) producing a target vector transformed animal cells; And (b) preparing a chimeric animal model by injecting the animal cell of step (a) into a surrogate mother.
본 발명의 일 구현예로, 상기 제조방법은 (c) 상기 (b)단계의 키메라 동물모델에 cre 단백질을 발현시키는 바이러스를 감염시키는 단계;를 포함하는 것일 수 있으나, 이에 제한되지 않는다.In one embodiment of the present invention, the preparation method may include, but is not limited to, (c) infecting a virus expressing the cre protein in the chimeric animal model of step (b).
본 발명의 다른 구현예로, 상기 제조방법은 (c) 상기 (b)단계의 키메라 동물모델을 성상교세포 특이적 Cre 단백질 생산 동물모델과 교배하는 단계;를 포함하는 것일 수 있으나, 이에 제한되지 않는다.In another embodiment of the present invention, the manufacturing method may include, but is not limited to, (c) crossing the chimeric animal model of step (b) with an astrocyte specific animal protein production animal model; .
또한, 본 발명의 다른 양태로서, (a) 염증형성 과발현 리포칼린-2(Lipocalin-2; LCN2) 동물모델의 피검체에 후보물질을 처리하는 단계; (b) 상기 피검체의 LCN2의 발현정도를 측정하는 단계; 및 (c) 후보물질 비처리군에 비해, LCN2의 발현정도가 감소되는 후보물질을 염증 치료물질로 선정하는 단계를 포함하는, 염증 치료물질의 스크리닝 방법을 제공한다.In addition, as another aspect of the present invention, (a) inflammation overexpression lipocalin-2 (Lipocalin-2; LCN2) treating a candidate substance in a subject of an animal model; (b) measuring the expression level of LCN2 in the subject; And (c) selecting a candidate substance for reducing the expression level of LCN2 as an inflammatory treatment substance, compared to a non-treatment group for the candidate substance.
LCN2의 발현정도를 측정하기 위한 상기 피검체는 조직, 세포, 전혈, 혈액, 타액, 객담, 뇌척수액, 또는 뇨 등일 수 있으나, 이에 제한되지 않는다.The subject for measuring the expression level of LCN2 may be tissue, cells, whole blood, blood, saliva, sputum, cerebrospinal fluid, or urine, but is not limited thereto.
또한, 본 발명의 또 다른 양태로서, (a) 염증형성 과발현 리포칼린-2(Lipocalin-2; LCN2) 동물모델의 피검체에 후보물질을 처리하는 단계; (b) 상기 피검체의 성상아교세포 및 미세아교세포의 형태의 변화를 관찰하는 단계; 및 (c) 후보물질 비처리군에 비해, 성상아교세포 및 미세아교세포의 형태의 변화가 감소되는 후보물질을 염증 치료물질로 선정하는 단계를 포함하는, 염증 치료물질의 스크리닝 방법을 제공한다.In addition, as another aspect of the present invention, (a) inflammation overexpression lipocalin-2 (Lipocalin-2; LCN2) processing a candidate substance in a subject of an animal model; (b) observing a change in the shape of astrocytes and microglia in the subject; And (c) selecting a candidate substance for reducing morphological changes of astrocytes and microglial cells as an inflammatory treatment substance, compared to a non-treatment group of candidate substances.
성상아교세포 및 미세아교세포의 형태의 변화를 관찰하기 위한 상기 피검체는 조직, 세포, 전혈, 혈액, 타액, 객담, 뇌척수액, 또는 뇨 등일 수 있고, 바람직하게는 조직 또는 세포일 수 있으나, 이에 제한되지 않는다.The subject for observing changes in the form of astrocytes and microglia may be tissues, cells, whole blood, blood, saliva, sputum, cerebrospinal fluid, or urine, and preferably tissues or cells. It is not limited.
본 발명 스크리닝 방법을 언급하면서 사용되는 용어 “후보물질”은 본 발명의 뇌 내 염증 치료 효과를 나타내는지 여부를 검사하기 위하여 스크리닝에서 이용되는 미지의 물질을 의미한다. 상기 시험물질은 siRNA(small interference RNA), shRNA(short hairpin RNA), miRNA(microRNA), 리보자임(ribozyme), DNAzyme, PNA(peptide nucleic acids), 안티센스 올리고뉴클레오타이드, 항체, 앱타머, 천연추출물 또는 화학물질을 포함하나, 이에 한정되는 것은 아니다. The term “candidate” used while referring to the screening method of the present invention refers to an unknown material used in screening to test whether or not the present invention has an effect of treating inflammation in the brain. The test substance may include small interference RNA (siRNA), short hairpin RNA (shRNA), microRNA (miRNA), ribozyme, DNAzyme, peptide nucleic acids (PNA), antisense oligonucleotides, antibodies, aptamers, natural extracts, or Chemicals include, but are not limited to.
또한, 상기 (a) 단계에서 후보물질의 처리는 비경구 또는 경구투여, 정위주사를 포함하나, 이로 제한하는 것은 아니며, 당업자라면 시험물질을 동물에게 테스트하기 위해 적절한 방법을 선택할 수 있을 것이다.In addition, the treatment of the candidate substance in step (a) includes parenteral or oral administration, stereotactic injection, but is not limited thereto, and a person skilled in the art will be able to select an appropriate method for testing the test substance in animals.
또한, 상기 (b) 단계는 중합효소연쇄반응(PCR), 마이크로어레이(microarray), 노던 블롯팅(northern blotting), 웨스턴 블롯팅(western blotting), 효소면역분석법(ELISA), 면역침전법(immunoprecipitation), 면역화학염색법(immunohistochemistry), 또는 면역형광염색법(immunofluorescence)을 이용하여 측정하는 것을 특징으로 하나 이에 제한되지 않는다.In addition, the step (b) is polymerase chain reaction (PCR), microarray (microarray), northern blotting (northern blotting), western blotting (western blotting), enzyme immunoassay (ELISA), immunoprecipitation (immunoprecipitation) ), Immunochemical staining (immunohistochemistry), or immunofluorescence staining (immunofluorescence) characterized in that the measurement using, but is not limited to.
또한, 상기 후보물질은 LCN2의 발현정도를 측정하기 위하여 스크리닝에서 사용되는 미지의 물질을 의미하며, 바람직하게는 화합물, 미생물 배양액 또는 추출물, 천연물 추출물, 핵산, 및 펩타이드로 이루어진 군으로부터 선택되는 하나 이상인 것을 특징으로 하나 이에 제한되지 않으며, 상기 핵산은 앱타머(aptamer), LNA(locked nucleic acid), PNA(peptide nucleic acid), 및 모폴리노(morpholino)로 이루어진 군으로부터 선택되는 하나 이상인 것을 특징으로 하나 이에 제한되지 않는다.In addition, the candidate substance refers to an unknown substance used in screening to measure the expression level of LCN2, preferably one or more selected from the group consisting of compounds, microbial cultures or extracts, natural product extracts, nucleic acids, and peptides Characterized in that, but is not limited to, the nucleic acid is at least one selected from the group consisting of aptamer (aptamer), LNA (locked nucleic acid), PNA (peptide nucleic acid), and morpholino (morpholino) However, it is not limited thereto.
또한, 상기 피검체의 LCN2의 발현정도의 측정은 중합효소연쇄반응(PCR), 마이크로어레이(microarray), 노던 블롯팅(northern blotting), 웨스턴 블롯팅(western blotting), 효소면역분석법(ELISA), 면역침전법(immunoprecipitation), 면역화학염색법(immunohistochemistry), 또는 면역형광염색법(immunofluorescence)을 이용하여 측정하는 것을 특징으로 하나 이에 제한되지 않는다.In addition, the measurement of the expression level of LCN2 in the subject was measured by polymerase chain reaction (PCR), microarray, northern blotting, western blotting, and enzyme immunoassay (ELISA). It is characterized in that it is measured using immunoprecipitation (immunoprecipitation), immunochemical staining (immunohistochemistry), or immunofluorescence staining (immunofluorescence), but is not limited thereto.
상기 동물모델은 인간을 제외한 포유동물을 이용하여 제조될 수 있으며, 상기 인간을 제외한 포유동물은 원숭이, 랫트, 생쥐, 토끼, 개, 영장류 등일 수 있으며, 바람직하게는 쥣과(Muridae) 동물일 수 있으나 이에 제한되지는 않는다.The animal model may be manufactured using mammals other than humans, and mammals other than humans may be monkeys, rats, mice, rabbits, dogs, primates, and the like, preferably muridae animals. However, it is not limited thereto.
상기 '염증형성'은 염증의 발병, 염증 활성화 또는 염증 과발현 등을 포괄하는 의미이며, 염증 유발되는 것이라면 어느 하나에 제한되지 않는다.The 'inflammatory formation' is meant to encompass the onset of inflammation, activating inflammation or overexpressing inflammation, and is not limited to any one that causes inflammation.
또한, 상기 염증은 뇌 내 국부염증, 알레르기, 자가면역질환, 전립선염, 사구체콩팥염, 대장염, 골반염, 및 류마티스 관절염으로 이루어진 군에서 선택된 하나 이상일 수 있고, 바람직하게는 뇌 내 국부염증일 수 있으나, 이에 제한되지 않는다.In addition, the inflammation may be at least one selected from the group consisting of local inflammation in the brain, allergy, autoimmune disease, prostatitis, glomerulonephritis, colitis, pelvicitis, and rheumatoid arthritis, preferably local inflammation in the brain. , But is not limited to this.
또한, 상기 염증이 발생하는 부위는 뇌, 전립선, 사구체콩팥염, 대장, 골반, 및 관절로 이루어진 군에서 선택된 하나 이상일 수 있고, 바람직하게는 뇌일 수 있으나 이에 제한되지 않는다.In addition, the site where the inflammation occurs may be at least one selected from the group consisting of brain, prostate, glomerulonephritis, colon, pelvis, and joints, preferably brain, but is not limited thereto.
상기 뇌 염증은 예를 들어 수막염, 뇌막염, 뇌수막염, 뇌 농양, 뇌염 및 뇌 질환으로 인하여 수반되는 염증 등이 있으나, 이에 제한되지 않는다.The brain inflammation includes, for example, meningitis, meningitis, meningitis, brain abscess, encephalitis and inflammation accompanying brain diseases, but is not limited thereto.
본원 명세서 전체에서, 어떤 부분이 어떤 구성 요소를 “포함” 한다고 할 때, 이는 특별히 반대되는 기재가 없는 한 다른 구성 요소를 제외하는 것이 아니라 다른 구성 요소를 더 포함할 수 있는 것을 의미한다. 본원 명세서 전체에서 사용되는 정도의 용어 “약”, “실질적으로” 등은 언급된 의미에 고유한 제조 및 물질 허용오차가 제시될 때 그 수치에서 또는 그 수치에 근접한 의미로 사용되고, 본 발명의 이해를 돕기 위해 정확하거나 절대적인 수치가 언급된 개시 내용을 비양심적인 침해자가 부당하게 이용하는 것을 방지하기 위해 사용된다. 본원 명세서 전체에서 사용되는 정도의 용어 “~(하는) 단계” 또는 “~의 단계”는 “~ 를 위한 단계”를 의미하지 않는다.Throughout the present specification, when a part “includes” a certain component, it means that the component may further include other components, not to exclude other components, unless specifically stated to the contrary. The terms “about”, “substantially”, etc., used to the extent of the present specification are used in the sense of or close to the numerical value when manufacturing and substance tolerances unique to the stated meaning are presented, and understanding of the present invention In order to help, accurate or absolute figures are used to prevent unconscionable abusers from unduly using the disclosures mentioned. The term “~ (steps)” or “steps of” as used in the entire specification does not mean “steps for ~”.
본원 명세서 전체에서, 마쿠시 형식의 표현에 포함된 “이들의 조합(들)”의 용어는 마쿠시 형식의 표현에 기재된 구성 요소들로 이루어진 군에서 선택되는 하나 이상의 혼합 또는 조합을 의미하는 것으로서, 상기 구성 요소들로 이루어진 군에서 선택되는 하나 이상을 포함하는 것을 의미한다.Throughout the present specification, the term “combination (s)” included in the expression of the marki form means one or more mixtures or combinations selected from the group consisting of the components described in the expression of the marki form, It means to include one or more selected from the group consisting of the above components.
본원 명세서 전체에서, “A 및/또는 B”의 기재는 “A 또는 B, 또는 A 및 B”를 의미한다.Throughout this specification, the description of “A and / or B” means “A or B, or A and B”.
어떤 실시예가 달리 구현 가능한 경우에 특정한 단계는 설명되는 순서와 다르게 수행될 수도 있다. 예를 들어, 연속하여 설명되는 두 단계는 실질적으로 동시에 수행될 수도 있고, 설명되는 순서와 반대의 순서로 수행될 수도 있다.When an embodiment is implemented differently, specific steps may be performed differently from the described order. For example, two steps described in succession may be performed substantially simultaneously, or may be performed in an order opposite to that described.
이하, 본 발명의 이해를 돕기 위하여 바람직한 실시예를 제시한다. 그러나 하기의 실시예는 본 발명을 보다 쉽게 이해하기 위하여 제공되는 것일 뿐, 하기 실시예에 의해 본 발명의 내용이 한정되는 것은 아니다.Hereinafter, preferred embodiments are provided to help understanding of the present invention. However, the following examples are only provided to more easily understand the present invention, and the contents of the present invention are not limited by the following examples.
[실시예][Example]
실시예 1. 선택적 LCN2 과발현 형질전환 마우스 제작Example 1. Preparation of selective LCN2 overexpressing transgenic mice
선택적 LCN2 과발현 형질전환 마우스를 제작하기 위하여, 디자인된 LCN2 DNA 구조(construct)를 제작하였다. 도 2a에 나타난 바와 같이, Ai6 벡터(vector)에 선택적이고 안정적인 LCN2 유전자 과발현을 위하여, 강한 발현으로 널리 쓰이는 CAG(Cytomegalovirus early enhancer/chicken beta action) 프로모터를 사용하고, 그 하류에(down-stream)에 플록스(floxed)된 정지 코돈(STOP codon)을 삽입하였다. Cre-loxP 시스템을 이용하여 Cre 단백질의 존재 하에서 정지 코돈이 결실(deletion)되면, LCN2-2A-EGFP가 발현되고, 이후에 LCN2와 마커 단백질(maker protein)인 EGFP는 분리가 되어 단일 단백질로 기능하게 된다.To construct a selective LCN2 overexpressing transgenic mouse, a designed LCN2 DNA construct was constructed. As shown in Fig. 2a, for the selective and stable LCN2 gene overexpression in the Ai6 vector, a Cytomegalovirus early enhancer / chicken beta action (CAG) promoter widely used for strong expression is used, and downstream (down-stream) A stop codon, which was floxed, was inserted. When the stop codon is deleted in the presence of the Cre protein using the Cre-loxP system, LCN2-2A-EGFP is expressed, and then LCN2 and the marker protein (maker protein) EGFP are separated and function as a single protein. Is done.
도 2b에 나타난 바와 같이, 제작된 DNA 구조를 AvrⅡ와 SacⅠ 제한효소를 이용하여 자른 후에, 겔 전기천공법(gel electroporation)을 이용하여 잘려진 크기를 확인하였다. 준비된 DNA 구조를 마우스의 웅성전핵에 미세주입(microinjection) 하고 대리모에 이식하였다. 태어난 마우스가 3주령이 되었을 때 꼬리에서 게놈 DNA(genomic DNA)를 추출하여 도 2c에 나타난 바와 같이, 삽입(insertion)된 유전자를 확인할 수 있는 프라이머(primer) 조합을 통한 유전자형-PCR(genotype-PCR) 기술을 이용하여 올바르게 만들어진 키메라 마우스를 스크리닝(screening) 하였다. 확인된 마우스 계통(line)를 유지하며 생식-계열 전이(germ-line transfer)가 확인되는 계통(line)들만 선별한 뒤, 디자인된 선택적 LCN2 과발현이 나타나는지에 대한 검증을 실시하였다.As shown in Figure 2b, after cutting the produced DNA structure using AvrII and SacI restriction enzymes, the size of the cut was confirmed by gel electroporation. The prepared DNA structure was microinjected into the male pronucleus of the mouse and transplanted into a surrogate mother. Genome type-PCR (genotype-PCR) through a primer (primer) combination to identify the inserted gene, as shown in Figure 2c by extracting genomic DNA (genomic DNA) from the tail when the mouse is 3 weeks of age, born ) Screening of correctly made chimeric mice using technology. After maintaining the identified mouse line and selecting only those lines for which germ-line transfer was confirmed, verification was performed to see whether the designed selective LCN2 overexpression appeared.
실시예 2. 선택적 LCN2 과발현 형질전환 마우스 검증Example 2. Validation of selective LCN2 overexpressing transgenic mice
키메라 마우스 계통이 디자인과 같이 발현을 보이는 지에 대한 검증을 위하여, 해당 계통에서 P1 새끼(pups)들을 이용하여 주요 성상세포 배양(primary astrocyte culture)를 수행하였다. 상기 배양(culture)한 성상세포를 AAV-Cre-mCh를 감염(infection)시켜 Cre 단백질을 통한 LCN2와 EGFP의 과발현을 유도시킨 후, 세포 배양 배지(cell culture media)에서의 LCN2의 양을 ELISA 기술을 이용하여 측정하였다. 도 3a에 나타난 바와 같이, 키메라 새끼에서 배양(culture)한 성상세포에서 월등히 높은 LCN2를 검출하였다.In order to verify whether the chimeric mouse strain shows expression as in the design, primary astrocyte culture was performed using P1 pups in the strain. After incubating the cultured astrocytes with AAV-Cre-mCh to induce overexpression of LCN2 and EGFP through the Cre protein, the amount of LCN2 in cell culture media is measured by ELISA. It was measured using. As shown in FIG. 3A, significantly higher LCN2 was detected in astrocytes cultured in chimeric pups.
다음으로 성체 키메라 마우스에 AAV-GFAP-mCh-Cre(성상아교세포에서만 특이적으로 Cre 단백질을 발현 시킬 수 있는 virus vector)를 뇌 정위 수술(brain stereotaxic surgery)를 통하여 일방 내측 하베눌라(unilateral medial habenula)에 국부 주입을 하였다. 그 결과, 도 3b에 나타난 바와 같이, 수술 4주후에 마우스를 희생시킨 후, 면역조직화학(immunohistochemistry)기술을 통해 확인한 결과, 바이러스가 들어간 부위에서만 특이적으로 LCN2와 EGFP 발현을 관찰할 수 있었다.Next, in adult chimeric mice, AAV-GFAP-mCh-Cre (a virus vector capable of specifically expressing Cre protein only in astrocytes) is subjected to unilateral medial habenula through brain stereotaxic surgery. ) Was injected locally. As a result, as shown in Figure 3b, after 4 weeks after the mouse was sacrificed, and confirmed through immunohistochemistry (immunohistochemistry) technology, LCN2 and EGFP expression was specifically observed only at the site of the virus.
위의 두 실험을 통하여 본 발명에서 디자인한 LCN2 선택적 형질전환 마우스에서, 의도한대로 Cre에 의존적인 LCN2 과발현이 나타나는 것을 확인할 수 있었다.Through the above two experiments, it was confirmed that, in the LCN2 selective transgenic mouse designed in the present invention, Cre-dependent LCN2 overexpression appeared as intended.
실시예 3. 선택적 LCN2 과발현 형질전환 마우스의 국부 뇌 염증 유도 확인Example 3. Confirmation of local brain inflammation induction in selective LCN2 overexpressing transgenic mice
제작이 완성된 선택적 LCN2 과발현 형질전환 마우스에서 LCN2 과발현이 국부적 뇌 염증을 유도할 수 있는지 확인하기 위하여, AAV-GFAP-Cre-EGFP를 뇌 정위 수술(brain stereotaxic surgery)를 통하여 일측 해마 CA1(unilateral hippocampal CA1) 부위에 국부적으로 주입하였다. 수술 4주후에 마우스를 희생한 뒤, 면역조직화학(immunohistochemistry) 기술을 통해 확인한 결과, 도 4에 나타난 바와 같이, 바이러스가 들어간 부위와 더불어 반대측(contralateral) 부위에서까지 특이적인 성상아교세포(GFAP 염색된)와 미세아교세포(Iba1 염색된)의 형태의 변화를 관찰할 수 있었다. In order to confirm whether LCN2 overexpression can induce local brain inflammation in the production-selective selective LCN2 overexpression transgenic mouse, AAV-GFAP-Cre-EGFP was subjected to unilateral hippocampal CA1 (unilateral hippocampal) through brain stereotaxic surgery. CA1) was injected locally. After sacrificing the mouse 4 weeks after surgery, as a result of confirming through immunohistochemistry, as shown in FIG. 4, specific astroglial cells (GFAP staining) from the site containing the virus to the contralateral site (contralateral) ) And microglial cells (Iba1 stained) were observed.
이 실험결과는 이미 알려진 염증 상황에서의 뇌 내 성상아교세포와 미세아교세포의 형태변화와 유사한 결과로서, 본 발명에서 제작한 형질전환 마우스에서 Cre 단백질 발현 의존적인 뇌 내 국부 염증 반응을 유도할 수 있다는 것을 확인할 수 있었다.The results of this experiment are similar to the morphological changes of astrocytes and microglial cells in the brain in a known inflammatory situation. I could confirm that there is.
실시예 4. 성상교세포 선택적 LCN2 과발현 형질전환 마우스의 제작 및 확인Example 4. Construction and confirmation of astrocyte selective LCN2 overexpressing transgenic mice
제작된 LCN2 선택적 과발현 형질전환 마우스와 성상교세포 특이적 Cre 생산 마우스 (Aldh1l1-cre/ERT2)를 교배하여 Tamoxifen 처치로 인하여 전환되는 성상교세포 선택적 LCN2 과발현 형질전환 마우스(Astrocyte-scpcific LCN2 Overexpression, ALO)를 제작하였다. 구체적으로 교배된 하이브리드 생쥐에 corn-oil 에 녹인 20mg/ml 의 Tamoxifen 용액을 매일 5일동안 100ul 씩 복강주사하였다. 처치 4주 후에 sacrifice 하여 뇌를 적출 후 LCN2 발현과 뇌실의 형태 변화를 관찰하였다. Astrocyte-scpcific LCN2 Overexpression (ALO), a astrocytic selective LCN2 overexpressing transgenic mouse (Astrocyte-scpcific LCN2 Overexpression, ALO) converted by Tamoxifen treatment by crossing the produced LCN2 selective overexpressing transgenic mouse and astrocyte specific Cre producing mouse (Aldh1l1-cre / ERT2) It was produced. Specifically, 20 mg / ml Tamoxifen solution dissolved in corn-oil was intraperitoneally injected into the hybrid mice hybridized with 100 ul for 5 days every day. After 4 weeks of treatment, the brain was excised by sacrifice, and LCN2 expression and ventricular morphology were observed.
도 6에 나타난 바와 같이, ALO 생쥐의 혈액에서 LCN2 과발현이 나타남을 확인하였다.As shown in FIG. 6, it was confirmed that LCN2 overexpression appeared in the blood of ALO mice.
또한, 도 7에 나타난 바와 같이 ALO 생쥐의 뇌 내에서 LCN2 및 EGFP가 과발현됨을 확인하였다.In addition, it was confirmed that LCN2 and EGFP are overexpressed in the brain of ALO mice as shown in FIG. 7.
또한, 도 8에 나타난 바와 같이, ALO 생쥐의 뇌실 확대증상, 뇌수종 증상 등이 나타남을 확인하였다.In addition, as shown in FIG. 8, it was confirmed that the ventricular enlargement symptoms and hydrocephalus symptoms of ALO mice appeared.
이 실험결과는 이미 알려진 염증 상황에서의 뇌 내 형태변화와 유사한 결과로서, 본 발명에서 제작한 형질전환 마우스를 성상교세포 특이적 Cre 단백질 발현 마우스와 교배한 경우에도, Cre 단백질 발현 의존적인 뇌 내 국부 염증 반응을 유도할 수 있다는 것을 확인할 수 있었다.The results of this experiment are similar to the morphological changes in the brain in an already known inflammatory situation, and even when the transgenic mouse produced in the present invention is crossed with astrocyte specific Cre protein-expressing mice, localization in the Cre protein expression-dependent brain It was confirmed that it can induce an inflammatory reaction.
전술한 본 발명의 설명은 예시를 위한 것이며, 본 발명이 속하는 기술분야의 통상의 지식을 가진 자는 본 발명의 기술적 사상이나 필수적인 특징을 변경하지 않고서 다름 구체적인 형태로 쉽게 변형이 가능하다는 것을 이해할 수 있을 것이다. 그러므로 이상에서 기술한 실시예들은 모든 면에서 예시적인 것이며 한정적인 아닌 것으로 이해해야 한다.The above description of the present invention is for illustration only, and those of ordinary skill in the art to which the present invention pertains can understand that it can be easily modified to other specific forms without changing the technical spirit or essential features of the present invention. will be. Therefore, it should be understood that the above-described embodiments are illustrative in all respects and not restrictive.
본 발명자들은 리포칼린-2(Lipocalin-2; LCN2) 유전자를 과발현 시키는 동물모델을 제작하기 위해 예의 연구한 결과, Cre-loxP 유전자 발현 시스템을 이용하여 조건부 LCN2의 과발현을 유도하는 형질전환 마우스를 제작하고, 상기 형질전환 마우스가 뇌 국부 염증을 유도하는 것을 확인하였다. 본 발명으로 제작된 마우스를 이용하여 진정한 뇌 내 LCN2의 기능을 밝혀내는 데에 큰 도움이 될 것으로 기대되고, 뇌 내 세포 특이적으로 LCN2 유전자를 과발현 시킴으로써 기존에는 불가능 하였던 LCN2의 세포수준의 기능과 뇌 염증에 대한 역할을 알아내는 연구를 가능할 것으로 판단되며, 국부적인 염증반응을 유도함으로써 뇌 내 지역별 염증반응에 대한 변화를 연구할 수 있을 것이라 기대된다. 또한, 높은 수준의 LCN2 연구와 새로운 염증 동물모델의 제시로 뇌 염증에 대한 발병기전 연구 및 치료법 개발에 크게 기여할 것으로 기대되는 바, 산업상 이용가능성이 있다.The present inventors made extensive studies to produce animal models that overexpress the lipocalin-2 (Lipocalin-2; LCN2) gene, and produced transgenic mice that induce overexpression of conditional LCN2 using the Cre-loxP gene expression system And, it was confirmed that the transgenic mouse induced brain local inflammation. It is expected to be of great help in revealing the true function of LCN2 in the brain by using the mouse produced with the present invention. It is expected that research will be possible to find out the role of brain inflammation, and it is expected to be able to study changes in inflammatory responses in regions of the brain by inducing local inflammatory responses. In addition, the high level of LCN2 research and the presentation of new inflammatory animal models are expected to greatly contribute to the study of the pathogenesis of brain inflammation and the development of treatment methods.

Claims (15)

  1. CAG(Cytomegalovirus early enhancer/chicken beta action) 프로모터; 제 1 loxP(locus of X-over P1) 부위; 정지 코돈(stop codon) 부위; 제 2 loxP 부위; 및 LCN2의 순서로 이루어진 DNA 서열을 포함하는 염증형성 동물모델 제작용 리포칼린-2(Lipocalin-2; LCN2) 유전자 표적벡터(targeting vector).CAG (Cytomegalovirus early enhancer / chicken beta action) promoter; A first loxP (locus of X-over P1) site; Stop codon sites; A second loxP site; And Lipocalin-2 (LCN2) gene targeting vector for producing an inflammatory animal model comprising a DNA sequence consisting of LCN2.
  2. 제1항에 있어서,According to claim 1,
    상기 LCN2 뒤에, p2A(porcine teschovirus-1 2A), 향상된 녹색형광단백질(Enhanced green fluorescent protein; EGFP), 우드척 간염 바이러스 전사 후 조절요소 (Woodchuck hepatitis virus posttranscriptional regulatory element; WPRE), 및 SV40 polyA signal(pA)의 순서로 이루어진 DNA 서열을 포함하는 것을 특징으로 하는, 염증형성 동물모델 제작용 LCN2 유전자 표적벡터.After the LCN2, p2A (porcine teschovirus-1 2A), enhanced green fluorescent protein (EGFP), Woodchuck hepatitis virus posttranscriptional regulatory element (WPRE), and SV40 polyA signal ( It characterized in that it comprises a DNA sequence consisting of the sequence of pA, LCN2 gene target vector for the production of inflammatory animal models.
  3. 제1항의 LCN2 유전자 표적벡터가 형질 전환된 염증형성 동물모델 제작용 동물세포.An animal cell for producing an inflammatory animal model in which the LCN2 gene target vector of claim 1 is transformed.
  4. 제3항의 동물세포를 대리모에 주입하여 제조한 염증형성 리포칼린-2(Lipocalin-2; LCN2) 키메라 동물모델.An inflammatory lipocalin-2 (LCN2) chimeric animal model prepared by injecting the animal cell of claim 3 into a surrogate mother.
  5. 제4항의 키메라 동물모델에 Cre 단백질을 발현시키는 바이러스를 감염시키거나 성상교세포 특이적 Cre 단백질 생산 동물모델과 교배시켜 제조한 염증형성 리포칼린-2(Lipocalin-2; LCN2) 동물모델.An inflammatory lipocalin-2 (LCN2) animal model prepared by infecting the chimeric animal model of claim 4 with a virus expressing Cre protein or crossing it with an astrocyte specific Cre protein producing animal model.
  6. 제5항에 있어서,The method of claim 5,
    상기 염증은 뇌 내 국부염증인 것을 특징으로 하는, 염증형성 LCN2 동물모델.The inflammation is characterized in that the local inflammation in the brain, inflammatory LCN2 animal model.
  7. 하기 단계를 포함하는 염증형성 리포칼린-2(Lipocalin-2; LCN2) 동물모델 제조방법:Inflammatory lipocalin-2 (LCN2) animal model manufacturing method comprising the following steps:
    (a) 제1항 또는 제2항의 표적벡터가 형질 전환된 동물세포를 제조하는 단계; 및(a) preparing an animal cell transformed with the target vector of claim 1 or 2; And
    (b) 상기 (a)단계의 동물세포를 대리모에 주입하여 키메라 동물모델을 제조하는 단계.(b) preparing the chimeric animal model by injecting the animal cell of step (a) into a surrogate mother.
  8. 제7항에 있어서,The method of claim 7,
    상기 제조방법은 (c) 상기 (b)단계의 키메라 동물모델에 Cre 단백질을 발현시키는 바이러스를 감염시켜 LCN2 동물모델을 제조하는 단계를 포함하는 것을 특징으로 하는, 제조방법.The manufacturing method is characterized in that it comprises the step of (c) infecting the virus expressing the Cre protein in the chimeric animal model of step (b) to prepare an LCN2 animal model.
  9. 제7항에 있어서,The method of claim 7,
    상기 제조방법은 (c) 상기 (b)단계의 키메라 동물모델을 성상교세포 특이적 Cre 단백질 생산 동물모델과 교배하는 단계를 포함하는 것을 특징으로 하는, 제조방법.The manufacturing method is characterized in that it comprises the step of (c) crossing the chimeric animal model of step (b) with astrocyte specific Cre protein production animal model.
  10. 제7항에 있어서,The method of claim 7,
    상기 동물은 마우스인 것을 특징으로 하는, 제조방법.The animal is characterized in that the mouse, the manufacturing method.
  11. 제7항에 있어서,The method of claim 7,
    상기 염증은 뇌 내 국부염증인 것을 특징으로 하는, 제조방법.The inflammation is characterized in that the local inflammation in the brain, the manufacturing method.
  12. 하기의 단계를 포함하는, 염증 치료물질의 스크리닝 방법:A method of screening for an inflammatory therapeutic agent comprising the following steps:
    (a) 염증형성 리포칼린-2(Lipocalin-2; LCN2) 동물모델의 피검체에 후보물질을 처리하는 단계;(a) treating a candidate substance in a subject of an inflammatory lipocalin-2 (LCN2) animal model;
    (b) 상기 피검체의 LCN2의 발현정도를 측정하는 단계; 및(b) measuring the expression level of LCN2 in the subject; And
    (c) 후보물질 비처리군에 비해, LCN2의 발현정도가 감소되는 후보물질을 염증 치료물질로 선정하는 단계.(c) The step of selecting a candidate substance for reducing the expression level of LCN2 as an inflammatory treatment substance, compared to a non-treatment group of candidate substances.
  13. 하기의 단계를 포함하는, 염증 치료물질의 스크리닝 방법:A method of screening for an inflammatory therapeutic agent comprising the following steps:
    (a) 염증형성 리포칼린-2(Lipocalin-2; LCN2) 동물모델의 피검체에 후보물질을 처리하는 단계;(a) treating a candidate substance in a subject of an inflammatory lipocalin-2 (LCN2) animal model;
    (b) 상기 피검체의 성상아교세포 및 미세아교세포의 형태의 변화를 관찰하는 단계; 및(b) observing a change in the shape of astrocytes and microglia in the subject; And
    (c) 후보물질 비처리군에 비해, 성상아교세포 및 미세아교세포의 형태의 변화가 감소되는 후보물질을 염증 치료물질로 선정하는 단계.(c) The step of selecting a candidate substance for reducing morphological changes of astrocytes and microglial cells as an inflammatory treatment substance, compared to a non-treatment group of candidate substances.
  14. 제12항에 있어서,The method of claim 12,
    상기 피검체는 조직, 세포, 전혈, 혈액, 타액, 객담, 뇌척수액 및 뇨로 이루어진 군에서 선택된 하나 이상인 것을 특징으로 하는 스크리닝 방법.The screening method characterized in that the subject is at least one selected from the group consisting of tissue, cells, whole blood, blood, saliva, sputum, cerebrospinal fluid, and urine.
  15. 제13항에 있어서,The method of claim 13,
    상기 피검체는 조직 및 세포로 이루어진 군에서 선택된 하나 이상인 것을 특징으로 하는 스크리닝 방법.The screening method, characterized in that the subject is at least one selected from the group consisting of tissue and cells.
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