KR102156089B1 - Inflammation-forming transgenic animal model through selective over-expression of lipocalin-2 and a method for producing the same - Google Patents

Abstract

The present invention relates to a transgenic animal model that induces inflammation through selective overexpression of Lipocalin-2 (LCN2), and the present inventors have developed an animal model that overexpresses the Lipocalin-2 (LCN2) gene. As a result of intensive research to produce, a transgenic mouse inducing conditional overexpression of LCN2 was prepared using the Cre-loxP gene expression system, and it was confirmed that the transgenic mouse induces local brain inflammation. The use of the mouse made with the present invention is expected to be of great help in revealing the true function of LCN2 in the brain, and by overexpressing the LCN2 gene specifically in the brain, the function of the cell level of LCN2, which was previously impossible, and It is believed that research to find out the role of brain inflammation will be possible, and it is expected that it will be possible to study changes in the inflammatory response by region in the brain by inducing a local inflammatory response. In addition, high-level LCN2 research and the presentation of a new animal model for inflammation are expected to greatly contribute to the development of pathogenesis and treatment for brain inflammation.

Description

Inflammation-forming transgenic animal model through selective over-expression of lipocalin-2 and a method for producing the same}

The present invention relates to an inflammatory transgenic animal model through selective overexpression of Lipocalin-2 (LCN2), and more specifically, a targeting vector comprising sequences inducing selective overexpression of LCN2 , Animal cells for producing an inflammatory animal model generated using the target vector, an inflammatory LCN2 animal model obtained by using the same, a method for preparing the same, and a method for screening an inflammatory treatment substance using the same.

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 yet been clearly elucidated, and in particular, research on brain inflammation is insufficient.

Inflammation is a type of recovery mechanism or defensive reaction that attempts to repair and regenerate the damaged area when invasion that causes organic changes in a living body or tissue is applied. When stimulation is applied, inflammatory components such as inflammatory cytokines locally increase. And inflammation is induced. When inflammation occurs, symptoms such as redness, fever, swelling, pain, and dysfunction appear, and symptoms that can be confirmed morphologically include degenerative lesions and circulation. Disorders and excessive tissue proliferation. There are various causes of inflammation such as physical damage, damage by chemical substances, and infection of microorganisms such as bacteria and viruses.

Among them, brain inflammation is also called encephalitis. In other words, the brain is contained in our skull (skull), and this brain is protected from the hard skull of the head, is supplied with nutrients from the blood, and is called the meninges that discharges wastes generated from the brain. It is surrounded by a thin film, where inflammation of the brain itself 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, and is a typical nervous system infection. When it comes to encephalitis, symptoms mainly affect brain tissue. The most representative encephalitis is Japanese encephalitis. In addition, there are many cases of bacterial and viral encephalitis, and herpetic encephalitis is the most common. Most of them are epidemics and have strong infectious powers, and because there is no special treatment, it is a disease that has sequelae such as mental disorders and decreased intelligence. There may be various causes, but it can be commonly caused by respiratory diseases such as colds or ear diseases such as otitis media, and bacterial diseases such as pneumonia and syphilis, or diseases such as tuberculosis and mold may enter the head through blood. In addition, although it is generally viral, it may be caused by allergies or autoimmune abnormalities regardless of bacteria or viruses. Encephalitis generally affects children under the age of 10, but the mortality rate is higher in adults than in children. The incubation period from infection to onset depends on the type of virus or bacteria. Most of them develop between 1 week and 1 month after infection. However, there are also slow progressing viral encephalitis, which can take months to onset.

On the other hand, for the study of LCN2, animal models lacking the corresponding 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 microglia, and the development of a technical tool for this is urgently needed.

Republic of Korea Patent Publication No. 10-0806914

An 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 site; A second loxP site; And it is to provide a lipocalin-2 (Lipocalin-2; LCN2) gene targeting vector (targeting vector) for the production of inflammation-forming animal model comprising a DNA sequence consisting of the sequence of LCN2.

In addition, it is an object of the present invention to provide an animal cell for producing an inflammatory animal model transformed with the LCN2 gene target vector.

In addition, an object of the present invention is to provide an inflammatory LCN2 chimeric animal model prepared by injecting animal cells into a surrogate mother.

In addition, an object of the present invention is to provide an inflammatory LCN2 animal model prepared by infecting a virus expressing Cre protein in a chimeric animal model or crossing it with an animal model producing a Cre protein specific for astrocytes.

In addition, the object of the present invention is (a) preparing an animal cell transformed with the LCN2 gene target vector; And (b) injecting the animal cells of step (a) into a surrogate mother to prepare a chimeric animal model.

In addition, an object of the present invention is (a) treating a candidate substance to a subject of an inflammatory LCN2 animal model; (b) measuring the expression level of LCN2 in the subject; And (c) selecting a candidate substance whose expression level of LCN2 is reduced as compared to the non-treated group of candidate substances as an inflammatory treatment substance.

In addition, an object of the present invention is (a) treating a candidate substance to a subject of an inflammation-forming lipocalin-2 (LCN2) animal model; (b) observing a change in the morphology of the astrocytes and microglia of the subject; And (c) selecting a candidate substance whose morphology of astrocytes and microglia is reduced compared to the non-treated group as a candidate substance as an inflammatory treatment substance.

However, the technical task to be achieved by the present invention is not limited to the above-mentioned tasks, and other tasks not mentioned can be clearly understood by those of ordinary skill in the technical field to which the present invention belongs from the following description. There will be.

In order to achieve the object of the present invention as described above, the present invention provides 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 site; A second loxP site; And it provides a lipocalin-2 (Lipocalin-2; LCN2) gene targeting vector for producing an inflammation-forming animal model comprising a DNA sequence consisting of the sequence of LCN2 (SEQ ID NO: 1).

In one embodiment of the present invention, after LCN2, p2A (porcine teschovirus-1 2A) (SEQ ID NO: 4), enhanced green fluorescent protein (EGFP) (SEQ ID NO: 5), control elements after transcription of Woodchuck hepatitis virus (Woodchuck hepatitis virus posttranscriptional regulatory element; WPRE) (SEQ ID NO: 6), and a DNA sequence consisting of the SV40 polyA signal (pA) (SEQ ID NO: 7).

In addition, the present invention provides an animal cell for producing an inflammatory animal model transformed with the LCN2 gene target vector.

In addition, the present invention provides an inflammatory LCN2 chimeric animal model prepared by injecting animal cells into a surrogate mother.

In addition, the present invention provides an inflammatory LCN2 animal model prepared by infecting a chimeric animal model with a virus expressing the Cre protein or crossing it with an animal model producing a Cre protein specific for astrocytes.

In addition, the present invention (a) preparing an animal cell transformed with the LCN2 gene target vector; And (b) injecting the animal cells of step (a) into a surrogate mother to prepare a chimeric animal model.

In one embodiment of the present invention, the manufacturing method may include (c) infecting the chimeric animal model of step (b) with a virus expressing Cre protein.

In another embodiment of the present invention, the manufacturing method may include (c) crossing the chimeric animal model of step (b) with an animal model for producing astrocyte-specific Cre protein.

In addition, the present invention comprises the steps of: (a) treating a candidate substance to a subject of an inflammatory LCN2 animal model; (b) measuring the expression level of LCN2 in the subject; And (c) selecting a candidate substance whose expression level of LCN2 is reduced as compared to the non-treatment group of the candidate substance, as an inflammatory treatment substance.

In one embodiment of the present invention, a subject for measuring the expression level of LCN2 may be isolated from at least one selected from the group consisting of tissues, cells, whole blood, blood, saliva, sputum, cerebrospinal fluid, and urine.

In addition, the present invention comprises the steps of: (a) treating a candidate substance to a subject of an inflammatory lipocalin-2 (LCN2) animal model; (b) observing a change in the morphology of the astrocytes and microglia of the subject; And (c) selecting a candidate substance whose morphology of astrocytes and microglia is reduced compared to the non-treated group as a candidate substance as an inflammatory treatment substance.

In one embodiment of the present invention, a subject for observing a change in the morphology of astrocytes and microglia may be isolated from at least one selected from the group consisting of tissues and cells.

In one embodiment of the present invention, the animal may be a mouse.

In another embodiment of the present invention, the inflammation may be local inflammation in the brain.

The present inventors have conducted extensive research to create an animal model that overexpresses the Lipocalin-2 (LCN2) gene. As a result, the present inventors produced a transgenic mouse that induces conditional LCN2 overexpression using the Cre-loxP gene expression system. And, it was confirmed that the transgenic mice induce local brain inflammation. It is expected that it will be of great help in discovering the true function of LCN2 in the brain using the mouse produced by the present invention, and by overexpressing the LCN2 gene specifically in the brain, the function of the cell level of LCN2, which was previously impossible, and It is believed that research to find out the role of brain inflammation will be possible, and it is expected that it will be possible to study changes in the inflammatory response by region in the brain by inducing a local inflammatory response. In addition, high-level LCN2 research and the presentation of a new animal model for inflammation are expected to greatly contribute to the development of pathogenesis and treatment for brain inflammation.

1A is a diagram showing a DNA construct design for selectively overexpressing a Lipocalin-2 (LCN2) gene.
Figure 1b is a diagram showing the work flow (work flow) for the production of selective overexpressing LCN2 transgenic mice.
2A is a diagram showing a DNA structure vector map for selectively overexpressing the LCN2 gene.
Figure 2b is a diagram showing the result of cutting with a restriction enzyme to prepare DNA for microinjection for male pronucleus in a 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 made founder mouse.
3A is a diagram showing the results of a primary astrocyte culture experiment in order to validate a transgenic mouse produced from a founder mouse.
3B is a diagram showing the results of brain stereotaxic injection of AAV-GFAP-mCh-Cre in order to validate a transgenic mouse produced from a founder mouse.
Figure 4 is a diagram showing the results of a local brain inflammation induction experiment using the identified selective overexpression LCN2 transgenic mice.
Figure 5 is a diagram showing a hybrid mouse manufacturing design.
Figure 6 is a diagram showing the weight change and measurement results of LCN2 in blood serum of astrocyte-scpcific LCN2 overexpression (ALO) mice.
7 is a diagram showing the results of LCN2 expression in the brain of ALO mice.
8 is a diagram showing the results of observation of ventricular expansion in ALO mice.

The present inventors have conducted extensive research to create an animal model that overexpresses the Lipocalin-2 (LCN2) gene. As a result, the present inventors produced a transgenic mouse that induces conditional LCN2 overexpression using the Cre-loxP gene expression system. And, it was confirmed that the transgenic mouse induces local brain inflammation, and the present invention was completed based on this.

Hereinafter, the present invention will be described in detail.

In an embodiment of the present invention, in order to produce a selective overexpression LCN2 transgenic mouse, a DNA structure vector was prepared, cut using a restriction enzyme, and then the size was confirmed, a chimeric mouse was prepared, and the PCR technique was used as described above. It was confirmed by screening whether the chimeric mouse was properly manufactured (see Example 1).

In another embodiment of the present invention, in order to determine whether the selectively overexpressing LCN2 transgenic mouse overexpresses LCN2 depending on Cre protein, after culturing major astrocytes of chimeric mice, AAV-Cre-mCh and AAV-GFAP-mCh-Cre were infected. It was observed whether LCN2 and EGFP were specifically expressed in brain astrocytes using ELISA and immunohistochemistry techniques (see Example 2).

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 dependent on the Cre protein expression, stereotactic surgery on one side of the hippocampus CA1 (unilateral hippocampal CA1) After local infection with AAV-GFAP-Cre-EGFP by (brain stereotaxic surgery), changes in the morphology of astrocytes and microglia were observed using immunohistochemistry techniques (see Example 3).

Accordingly, the present invention is a CAG (Cytomegalovirus early enhancer/chicken beta action) promoter; A first loxP (locus of X-over P1) site; Stop codon site; And it provides a Lipocalin-2 (Lipocalin-2; LCN2) gene targeting vector (targeting vector) for the production of an inflammation-forming animal model comprising a DNA sequence consisting of the sequence of the second loxP site.

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 transcriptional regulatory element (Woodchuck hepatitis) Virus post-transcriptional regulatory element (WPRE), and SV40 polyA signal (pA) may include, but is not limited to, a DNA sequence consisting of the sequence.

In the present specification, the vector may have a cleavage map disclosed in FIG. 2A, but is not limited thereto.

In the present specification, the "LCN2" is preferably a gene including the nucleotide sequence of SEQ ID NO: 1 and most preferably consists of the nucleotide sequence represented by SEQ ID NO: 1, but is 80% or more of the sequence of SEQ ID NO: 1, More preferably, it may include a base sequence having a sequence homology of 90% or more, more preferably 95% or more.

In the present specification, the "CAG promoter" is preferably a gene including the nucleotide sequence of SEQ ID NO: 2, and most preferably consists of a nucleotide sequence represented by SEQ ID NO: 2, but at least 80% of the sequence of SEQ ID NO: 2 , More preferably, it may include a base sequence having a sequence homology of 90% or more, more preferably 95% or more.

In the present specification, the “loxP” is preferably a gene including the nucleotide sequence of SEQ ID NO: 3 and most preferably consists of a nucleotide sequence represented by SEQ ID NO: 3, but is 80% or more of the sequence of SEQ ID NO: 3, More preferably, it may include a base sequence having a sequence homology of 90% or more, more preferably 95% or more.

In the present specification, the “p2A” is preferably a gene including the nucleotide sequence of SEQ ID NO: 4 and most preferably consists of the nucleotide sequence represented by SEQ ID NO: 4, but is 80% or more of the sequence of SEQ ID NO: 4, More preferably, it may include a base sequence having a sequence homology of 90% or more, more preferably 95% or more.

In the present specification, the "EGFP" is preferably a gene including the nucleotide sequence of SEQ ID NO: 5 and most preferably consists of the nucleotide sequence represented by SEQ ID NO: 5, but is 80% or more of the sequence of SEQ ID NO: 5, More preferably, it may include a base sequence having a sequence homology of 90% or more, more preferably 95% or more.

In the present specification, the "WPRE" is preferably a gene including the nucleotide sequence of SEQ ID NO: 6 and most preferably consists of the nucleotide sequence represented by SEQ ID NO: 6, but is 80% or more of the sequence of SEQ ID NO: 6, More preferably, it may include a base sequence having a sequence homology of 90% or more, more preferably 95% or more.

In the present specification, the term "expression vector" refers to a vector capable of expressing a peptide or protein encoded by a heterogeneous nucleic acid inserted into the vector, and preferably a vector prepared to express LCN2 Means. The "vector" refers to an arbitrary medium for the introduction and/or transfer of a base into a host cell in vitro, in vivo or in vivo, and a replication unit capable of bringing about replication of the bound fragment by binding other DNA fragments ( replicon), and the "replication unit" refers to any genetic unit (e.g., plasmid, phage, cosmid, etc.) that functions as a self-unit of DNA replication in vivo, that is, replicable by its own regulation. Chromosomes, viruses, etc.). The recombinant expression vector of the present invention is preferably a promoter, which is a transcription initiation factor to which RNA polymerase binds, an arbitrary operator sequence for regulating transcription, a sequence encoding a suitable mRNA ribosome binding site, and termination of transcription and translation. It may include a sequence that controls, terminator, and the like. The vector used in the present invention may be, for example, an Ai6 vector, but is not limited thereto.

The term "EGFP" used in the present invention refers to a basic (constituent fluorescence) green fluorescent protein extracted from Aequorea victoria, and is known as a weak dimer that has weak alkaline acid sensitivity and matures rapidly. In order to mark the cells overexpressed with LCN2, a 2A-EGFP linker was inserted into the LCN2 gene downstream to implement fluorescent labeling.

In the present invention, instead of the EGFP, various reporter proteins known in the art may be used, for example, GFP (Green fluorescent prtein), UnaG, dsRed, eqFp611, Dronpa, TagRFPs, KFP, EosFP, Dendra, IrisFP, or smURFP. It may be a reporter protein such as, but is not limited thereto.

The term "WPRE" used in the present invention is a DNA sequence in which a third structure is expressed upon transcription. It is commonly used in molecular biology to increase the expression of genes carried by viral vectors. WPRE is a three-way regulator, usually containing gamma, alpha and beta elements.

In the present invention, instead of the WPRE, various post-translational regulatory elements known in the art may be used, and may be, for example, post-translational regulatory elements such as HPRE, CREs, or TREs, but are not limited thereto.

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 genome and expressing a Cre recombinase to induce a mutation in the target gene. When the target gene is floxed, two loxP sites are inserted inside the target gene at regular intervals, for example, at or around the intron site, and recombination occurs between the loxP sites in the presence of the Cre enzyme. , The genes in between are 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. If Cre does not exist, the stop codon of the floxed LCN2 is not affected.

In addition, the present invention provides an animal cell for producing an inflammatory animal model transformed with the LCN2 gene target vector.

The animal cells are transformed (transformation), transfection (transfection), Agrobacterium (Agrobacterium)-mediated transformation method, particle gun bombardment (particle gun bombardment), sonication (sonication), electroporation (electroporation) And polyethylen glycol (PEG)-mediated transformation method, etc., but there is no limitation as long as it is a method capable of injecting the vector of the present invention.

The animal cell may be a male pronucleus, but is not limited thereto.

In addition, the present invention provides a chimeric animal model of inflammation-forming lipocalin-2 (LCN2) prepared by injecting animal cells into a surrogate mother.

In the present invention, instead of injecting the animal cells into a surrogate mother, the LCN2 gene target vector is transduced into an animal cell, and the animal cells are then injected into a gastrula to create a chimeric animal model, and the model The inflammatory LCN2 animal model can be prepared by crossing the animal model with Cre protein, but is not limited thereto.

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 animal model producing a Cre protein specific for astrocytes.

The virus is a carrier protein capable of expressing a Cre protein cell-specifically, for example, AAV-Cre-mCh, AAV-GFAP-mCh-Cre, AAV-GFAP-Cre-EGFP, Ad-CMV-iCre, etc. May be used, 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, a local area of the brain, and preferably, a unilateral inner habenula, but is not limited thereto.

In addition, in addition to the viral infection, it is possible to use a delivery system for expressing the Cre protein, and is not limited to a virus for expressing the Cre protein.

According to a specific embodiment of the present invention, after primary astrocyte culture of the chimeric animal model, the Cre protein can be specifically expressed only in the astrocytes by infecting AAV-GFAP-mCh-Cre, Preferably, the Cre protein can be expressed only in the astrocytes by local infection with AAV-GFAP-mCh-Cre to unilateral medial habenula, but is not limited thereto.

The'Cre protein producing animal model' may be an animal model capable of expressing a Cre protein specifically, but is not limited thereto. The Cre protein-producing 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.

In addition, as another aspect of the present invention, (a) preparing an animal cell transformed with the LCN2 gene target vector; And (b) injecting the animal cells of step (a) into a surrogate mother to prepare a chimeric animal model.

In one embodiment of the present invention, the manufacturing method may include, but is not limited to, (c) infecting a virus expressing Cre protein in the chimeric animal model of step (b).

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 animal model producing Cre protein specific for astrocytes. .

In addition, as another aspect of the present invention, (a) treating a candidate substance to a subject of an inflammatory overexpression Lipocalin-2 (LCN2) animal model; (b) measuring the expression level of LCN2 in the subject; And (c) selecting a candidate substance whose expression level of LCN2 is reduced as compared to the non-treatment group as a candidate substance as an inflammatory treatment substance.

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.

In addition, as another aspect of the present invention, (a) treating a candidate substance to a subject of an inflammatory overexpression Lipocalin-2 (LCN2) animal model; (b) observing a change in the morphology of the astrocytes and microglia of the subject; And (c) selecting a candidate substance whose morphology of astrocytes and microglia is reduced as compared to the non-treated group as a candidate substance as an inflammatory substance.

The subject for observing the change in the morphology of astrocytic cells and microglia may be tissues, cells, whole blood, blood, saliva, sputum, cerebrospinal fluid, urine, etc., preferably tissues or cells. Not limited.

The term "candidate" used while referring to the screening method of the present invention refers to an unknown substance used in screening to test whether or not it exhibits the effect of treating inflammation in the brain of the present invention. The test substances are siRNA (small interference RNA), shRNA (short hairpin RNA), miRNA (microRNA), ribozyme, DNAzyme, PNA (peptide nucleic acids), antisense oligonucleotides, antibodies, aptamers, natural extracts, or Including chemicals, but not limited thereto.

In addition, the treatment of the candidate substance in step (a) includes parenteral or oral administration, and stereotactic injection, but is not limited thereto, and those skilled in the art will be able to select an appropriate method for testing the test substance on an animal.

In addition, the (b) step is a polymerase chain reaction (PCR), microarray, northern blotting, western blotting, enzyme immunoassay (ELISA), immunoprecipitation method (immunoprecipitation). ), immunochemical staining (immunohistochemistry), or immunofluorescence staining (immunofluorescence) is characterized by the measurement using, but is not limited thereto.

In addition, the candidate substance refers to an unknown substance used in screening to measure the expression level of LCN2, and preferably, one or more selected from the group consisting of a compound, a microorganism culture solution or extract, a natural product extract, a nucleic acid, and a peptide. The nucleic acid is not limited thereto, and the nucleic acid is at least one selected from the group consisting of an aptamer, a locked nucleic acid (LNA), a peptide nucleic acid (PNA), and a morpholino. However, it is not limited thereto.

In addition, the measurement of the level of expression of LCN2 in the subject is polymerase chain reaction (PCR), microarray, northern blotting, western blotting, enzyme immunoassay (ELISA), It is characterized by using immunoprecipitation, immunohistochemistry, or immunofluorescence, but is not limited thereto.

The animal model may be manufactured using mammals other than humans, and the mammals other than humans may be monkeys, rats, mice, rabbits, dogs, primates, and the like, and preferably may be murine animals. However, it is not limited thereto.

The'inflammation formation' is meant to encompass the onset of inflammation, activation of inflammation, or overexpression of inflammation, and is not limited to any one that causes inflammation.

In addition, the inflammation may be one or more selected from the group consisting of local inflammation in the brain, allergies, autoimmune diseases, prostatitis, glomerulonephritis, colitis, pelvic inflammation, and rheumatoid arthritis, and preferably local inflammation in the brain. , Is not limited thereto.

In addition, the area where the inflammation occurs may be at least one selected from the group consisting of brain, prostate, glomerulonephritis, large intestine, 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.

Throughout the specification of the present application, when a certain part "includes" a certain constituent element, it means that other constituent elements may be further included rather than excluding other constituent elements unless otherwise specified. The terms "about", "substantially", etc. of the degree used throughout the present specification are used at or close to the numerical value when manufacturing and material tolerances specific to the stated meaning are presented, and understanding of the present invention To aid in, accurate or absolute figures are used to prevent unfair use of the stated disclosure by unscrupulous infringers. As used throughout the specification of the present application, the term "step (to)" or "step of" does not mean "step for".

Throughout the present specification, the term “combination(s) thereof” included in the expression of the Makushi format refers to one or more mixtures or combinations selected from the group consisting of components described in the expression of the Makushi format, It means to include at least one selected from the group consisting of the above components.

Throughout this specification, the description of “A and/or B” means “A or B, or A and B”.

When a certain embodiment can be implemented differently, certain 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 the described order.

Hereinafter, a preferred embodiment is presented to aid the understanding of the present invention. However, the following examples are provided for easier understanding of the present invention, and the contents of the present invention are not limited by the following examples.

[Example]

Example 1. Preparation of selective LCN2 overexpressing transgenic mice

In order to construct a selective LCN2 overexpressing transgenic mouse, a designed LCN2 DNA construct was constructed. As shown in Figure 2a, for selective and stable LCN2 gene overexpression in Ai6 vector, a CAG (Cytomegalovirus early enhancer/chicken beta action) promoter widely used for strong expression was used, and downstream thereof (down-stream) The floxed stop codon (STOP codon) was inserted in the. 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 EGFP, the marker protein, are separated and function as a single protein. Is done.

As shown in Fig. 2b, after cutting the prepared DNA structure using AvrII and SacI restriction enzymes, the cut size was confirmed using gel electroporation. The prepared DNA structure was microinjected into the male pronucleus of a mouse and transplanted into a surrogate mother. Genotype-PCR (genotype-PCR) through a combination of primers that can identify the inserted gene by extracting genomic DNA from the tail when the born mouse is 3 weeks old, as shown in FIG. ) The chimeric mice made correctly using the technique were screened. After maintaining the identified mouse line and selecting only the lines in which germ-line transfer was confirmed, verification was performed to determine whether the designed selective LCN2 overexpression appeared.

Example 2. Verification of selective LCN2 overexpressing transgenic mice

In order to verify whether the chimeric mouse line was expressed as in the design, primary astrocyte culture was performed using P1 pups in the line. After inducing overexpression of LCN2 and EGFP through Cre protein by infecting the cultured astrocytes with AAV-Cre-mCh, the amount of LCN2 in the cell culture media is determined by ELISA technology. It was measured using. As shown in FIG. 3A, a remarkably high LCN2 was detected in astrocytes cultured in chimeric pups.

Next, AAV-GFAP-mCh-Cre (a virus vector that can specifically express Cre protein only in astrocytes) was used in adult chimeric mice by unilateral medial habenula through brain stereotaxic surgery. ) Was injected locally. As a result, as shown in FIG. 3B, after the mice were sacrificed 4 weeks after surgery, as a result of confirming through immunohistochemistry technology, LCN2 and EGFP expression could be observed specifically only at the site containing the virus.

Through the above two experiments, it was confirmed that in the LCN2 selectively transgenic mice designed in the present invention, Cre-dependent LCN2 overexpression appeared as intended.

Example 3. Confirmation of local brain inflammation induction of selective LCN2 overexpressing transgenic mice

In order to confirm whether LCN2 overexpression can induce local brain inflammation in the fabricated selective LCN2 overexpression transgenic mice, AAV-GFAP-Cre-EGFP was used in unilateral hippocampal CA1 (unilateral hippocampal surgery) through brain stereotaxic surgery. CA1) site was injected locally. After the mice were sacrificed 4 weeks after the operation, as a result of confirming through immunohistochemistry technology, as shown in FIG. 4, specific astrocytes (GFAP staining) up to the part where the virus entered as well as in the contralateral part. Morphology) and microglia (Iba1 stained) were observed.

This experimental result is similar to the morphology change of astroglia and microglia in the brain under known inflammatory conditions, and it is possible to induce a local inflammatory response in the brain that is dependent on Cre protein expression in the transgenic mice produced in the present invention. I could confirm that there is.

Example 4. Construction and confirmation of astrocyte-selective LCN2 overexpressing transgenic mice

Astrocyte-scpcific LCN2 overexpression transgenic mice (Astrocyte-scpcific LCN2 Overexpression, ALO) converted by Tamoxifen treatment by crossing the produced LCN2-selective overexpression transgenic mice and astrocyte-specific Cre producing mice (Aldh1l1-Cre/ERT2) Was produced. Specifically, the hybrid mice were intraperitoneally injected with 20mg/ml of Tamoxifen solution dissolved in corn-oil for 5 days every day. After 4 weeks of treatment, the brain was sacrificed and the LCN2 expression and the change in the morphology of the ventricle were observed.

As shown in FIG. 6, it was confirmed that LCN2 overexpression appeared in the blood of ALO mice.

In addition, as shown in FIG. 7, it was confirmed that LCN2 and EGFP were overexpressed in the brain of ALO mice.

In addition, as shown in Figure 8, it was confirmed that the symptoms of enlarged ventricle and hydrocephalus of ALO mice appear.

This experimental result is similar to the morphological change in the brain under known inflammatory conditions. Even when the transgenic mice produced in the present invention are crossed with a mouse-specific Cre protein expression, Cre protein expression-dependent localization in the brain It was confirmed that it can induce an inflammatory response.

The foregoing description of the present invention is for illustrative purposes only, and those of ordinary skill in the art to which the present invention pertains will be able to understand that it is possible to easily transform it into other specific forms without changing the technical spirit or essential features of the present invention. will be. Therefore, it should be understood that the embodiments described above are illustrative in all respects and not limiting.

<110> Korea University Industry and Academy Cooperation Foundation KOREA INSTITUTE OF SCIENCE AND TECHNOLOGY <120> Inflammation-forming transgenic animal model through selective over-expression of lipocalin-2 and a method for producing the same <130> MP19-248 <150> KR 10-2018-0132582 <151> 2018-10-31 <160> 7 <170> KoPatentIn 3.0 <210> 1 <211> 600 <212> DNA <213> Artificial Sequence <220> <223> mLCN2 <400> 1 atggccctga gtgtcatgtg tctgggcctt gccctgcttg gggtcctgca gagccaggcc 60 caggactcaa ctcagaactt gatccctgcc ccatctctgc tcactgtccc cctgcagcca 120 gacttccgga gcgatcagtt ccggggcagg tggtacgttg tgggcctggc aggcaatgcg 180 gtccagaaaa aaacagaagg cagctttacg atgtacagca ccatctatga gctacaagag 240 aacaatagct acaatgtcac ctccatcctg gtcagggacc aggaccaggg ctgtcgctac 300 tggatcagaa catttgttcc aagctccagg gctggccagt tcactctggg aaatatgcac 360 aggtatcctc aggtacagag ctacaatgtg caagtggcca ccacggacta caaccagttc 420 gccatggtat ttttccgaaa gacttctgaa aacaagcaat acttcaaaat taccctgtat 480 ggaagaacca aggagctgtc ccctgaactg aaggaacgtt tcacccgctt tgccaagtct 540 ctgggcctca aggacgacaa catcatcttc tctgtcccca ccgaccaatg cattgacaac 600 600 <210> 2 <211> 583 <212> DNA <213> Artificial Sequence <220> <223> CAG <400> 2 cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc cccgcccatt 60 gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc attgacgtca 120 atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt atcatatgcc 180 aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt atgcccagta 240 catgacctta tgggactttc ctacttggca gtacatctac gtattagtca tcgctattac 300 catggtcgag gtgagcccca cgttctgctt cactctcccc atctcccccc cctccccacc 360 cccaattttg tatttattta ttttttaatt attttgtgca gcgatggggg cggggggggg 420 gggggggcgc gcgccaggcg gggcggggcg gggcgagggg cggggcgggg cgaggcggag 480 aggtgcggcg gcagccaatc agagcggcgc gctccgaaag tttcctttta tggcgaggcg 540 gcggcggcgg cggccctata aaaagcgaag cgcgcggcgg gcg 583 <210> 3 <211> 34 <212> DNA <213> Artificial Sequence <220> <223> LoxP <400> 3 ataacttcgt ataatgtatg ctatacgaag ttat 34 <210> 4 <211> 57 <212> DNA <213> Artificial Sequence <220> <223> p2A <400> 4 gctactaact tcagcctgct gaagcaggct ggagacgtgg aggagaaccc tggacct 57 <210> 5 <211> 720 <212> DNA <213> Artificial Sequence <220> <223> EGFP <400> 5 atggtgagca agggcgagga gctgttcacc ggggtggtgc ccatcctggt cgagctggac 60 ggcgacgtaa acggccacaa gttcagcgtg tccggcgagg gcgagggcga tgccacctac 120 ggcaagctga ccctgaagtt catctgcacc accggcaagc tgcccgtgcc ctggcccacc 180 ctcgtgacca ccctgaccta cggcgtgcag tgcttcagcc gctaccccga ccacatgaag 240 cagcacgact tcttcaagtc cgccatgccc gaaggctacg tccaggagcg caccatcttc 300 ttcaaggacg acggcaacta caagacccgc gccgaggtga agttcgaggg cgacaccctg 360 gtgaaccgca tcgagctgag gggcatcgac ttcaaggagg acggcaacat cctggggcac 420 aagctggagt acaactacaa cagccacaac gtctatatca tggccgacaa gcagaagaac 480 ggcatcaagg tgaacttcaa gatccgccac aacatcgagg acggcagcgt gcagctcgcc 540 gaccactacc agcagaacac ccccatcggc gacggccccg tgctgctgcc cgacaaccac 600 tacctgagca cccagtccgc cctgagcaaa gaccccaacg agaagcgcga tcacatggtc 660 ctgctggagt tcgtgaccgc cgccgggatc actctcggca tggacgagct gtacaagtga 720 720 <210> 6 <211> 600 <212> DNA <213> Artificial Sequence <220> <223> WPRE <400> 6 atggccctga gtgtcatgtg tctgggcctt gccctgcttg gggtcctgca gagccaggcc 60 caggactcaa ctcagaactt gatccctgcc ccatctctgc tcactgtccc cctgcagcca 120 gacttccgga gcgatcagtt ccggggcagg tggtacgttg tgggcctggc aggcaatgcg 180 gtccagaaaa aaacagaagg cagctttacg atgtacagca ccatctatga gctacaagag 240 aacaatagct acaatgtcac ctccatcctg gtcagggacc aggaccaggg ctgtcgctac 300 tggatcagaa catttgttcc aagctccagg gctggccagt tcactctggg aaatatgcac 360 aggtatcctc aggtacagag ctacaatgtg caagtggcca ccacggacta caaccagttc 420 gccatggtat ttttccgaaa gacttctgaa aacaagcaat acttcaaaat taccctgtat 480 ggaagaacca aggagctgtc ccctgaactg aaggaacgtt tcacccgctt tgccaagtct 540 ctgggcctca aggacgacaa catcatcttc tctgtcccca ccgaccaatg cattgacaac 600 600 <210> 7 <211> 135 <212> DNA <213> Artificial Sequence <220> <223> pA <400> 7 aacttgttta ttgcagctta taatggttac aaataaagca atagcatcac aaatttcaca 60 aataaagcat ttttttcact gcattctagt tgtggtttgt ccaaactcat caatgtatct 120 tatcatgtct ggatc 135

Claims (15)

CAG (Cytomegalovirus early enhancer/chicken beta action) promoter; A first loxP (locus of X-over P1) site; Stop codon site; A second loxP site; And Lipocalin-2 (Lipocalin-2; LCN2) gene targeting vector for the production of hydrocephalus forming animal model comprising a DNA sequence consisting of the sequence of LCN2.
The method of claim 1,
After the LCN2, p2A (porcine teschovirus-1 2A), enhanced green fluorescent protein (EGFP), Woodchuck hepatitis virus posttranscriptional regulatory element (WPRE), and SV40 polyA signal ( The LCN2 gene target vector for producing a hydrocephalus-forming animal model, characterized in that it comprises a DNA sequence consisting of the sequence of pA).
An animal cell for producing a hydrocephalus-forming animal model transformed with the LCN2 gene target vector of claim 1.
Hydrocephalus-forming Lipocalin-2 (LCN2) chimeric animal model prepared by injecting the animal cells of claim 3 into a surrogate mother.
A hydrocephalus-forming lipocalin-2 (LCN2) animal model prepared by infecting a virus expressing Cre protein in the chimeric animal model of claim 4 or crossing it with an animal model producing a Cre protein specific for astrocytes.
delete Hydrocephalus formation Lipocalin-2 (LCN2) animal model manufacturing method comprising the following steps:
(a) preparing an animal cell transformed with the LCN2 gene target vector of claim 1 or 2; And
(b) Injecting the animal cells of step (a) into a surrogate mother to prepare a chimeric animal model.
The method of claim 7,
The manufacturing method comprises the step of (c) infecting the chimeric animal model of step (b) with a virus expressing the Cre protein to prepare an LCN2 animal model.
The method of claim 7,
The manufacturing method comprises the step of (c) crossing the chimeric animal model of the step (b) with an animal model producing a Cre protein specific for astrocytes.
The method of claim 7,
The method of manufacturing, characterized in that the animal is a mouse.
delete A method for screening a substance for treating hydrocephalus, comprising the following steps:
(a) treating the candidate substance to the subject of the animal model of claim 4 or 5;
(b) measuring the expression level of LCN2 in the subject; And
(c) Selecting a candidate substance whose expression level of LCN2 is decreased as compared to the non-treatment group of the candidate substance as a hydrocephalus treatment substance.
A method for screening a substance for treating hydrocephalus, comprising the following steps:
(a) treating the candidate substance to the subject of the animal model of claim 4 or 5;
(b) observing a change in the morphology of the astrocytes and microglia of the subject; And
(c) Selecting a candidate substance whose morphology of astroglia and microglia is reduced as a treatment substance for hydrocephalus compared to the non-treated group of candidate substances.
The method of claim 12,
The subject is a screening method, characterized in that at least one selected from the group consisting of tissue, cells, whole blood, blood, saliva, sputum, cerebrospinal fluid, and urine.
The method of claim 13,
The screening method, characterized in that the subject is at least one selected from the group consisting of tissues and cells.

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