JP2015204839A - Knockout nonhuman animal - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a nonhuman model animal of diabetes, cardiac failure or the like, usable for the screening of a diabetes-treating agent, a cardiac failure-treating agent or the like.SOLUTION: A model animal of diabetes, cardiac failure or the like, or a part thereof comprises a knockout nonhuman animal or a part thereof from which the whole or a part of the functions of at least one gene selected from the group consisting of EBI3 gene, IL27-p28 gene and WSX-1 gene is deleted.

Description

  The present invention relates to the use of a knockout non-human animal as a disease model animal.

Diabetes is one of lifestyle-related diseases, and it is said that there are an estimated 16 million affected people in Japan. Diabetes is caused by insufficient insulin action, that is, insufficient supply of insulin, and decreased sensitivity in the target organ of the insulin, resulting in too much glucose in the blood (hyperglycemia) and excessive sugar in the urine ( Diabetes). In practice, the diagnosis is made by measuring the amount of glucose in the blood (blood glucose level). Diabetes is also known to cause complications such as neuropathy, retinopathy, nephropathy, and other metabolic syndromes that overlap with other lifestyle diseases such as obesity, hyperlipidemia and hypertension. (Metabolic syndrome) is said to be a major cause of cardiovascular diseases such as myocardial infarction and cerebral infarction. Therefore, it is extremely important to elucidate the cause of diabetes and to establish fundamental preventive and therapeutic methods based on it, and it has been desired to produce a model animal for diabetes.

Moreover, heart failure is a general term for syndromes that occur because the heart cannot pump blood that meets the demand of each organ, and is a disease with a very poor prognosis that the 5-year survival rate is about 50%. Currently, diuretics, digitalis, catecholamines, angiotensin converting enzyme inhibitors, β-blockers, etc. are used for the treatment of heart failure, but the treatment that completely suppresses the progression of the disease state or recovers to a completely healthy body There is a need for the discovery of new and useful drugs for heart failure that do not exist other than heart transplantation. Screening using heart failure model animals is important for the discovery of new therapeutic agents for heart failure, and the creation of heart failure model animals has been desired.

By the way, interleukin 27 (IL27) is composed of IL27-p28 and Epstein-
Barr virus (EBV)-a cytokine containing two different subunits of induced gene 3 (EBI3). On the other hand, IL27 receptor (IL27R) is composed of a heterodimer of WSX-1 and glycoprotein 130. I
L27 is expressed on antigen presenting cells such as monocytes, macrophages and dendritic cells. IL27 expressed in antigen presenting cells promotes the proliferation of CD4 ⁺ naive T cells. I
L27, together with IL12, acts on CD4 ⁺ naive T cells and promotes the production of TH1-type cytokine, interferon-γ (IFNγ). Furthermore, IL27 up-regulates T-bet, a transcription factor specific for the TH1-type immune response, thereby producing TH
GATA-3, a transcription factor specific for type 2 immune response, is downregulated. IL
27 suppresses the production of IL2 necessary for the proliferation of immune cells, and suppresses the differentiation of TH17 type cells that play an important role in the pathogenesis of diseases involving inflammation. IL27 also has an action of suppressing the production of various inflammatory cytokines. Therefore, IL27 has an action of suppressing various immune / inflammatory reactions.

Here, in Patent Document 1: Japanese Translation of PCT International Publication No. 2007-523169, IL27R knockout mice have lost the TH1-type immune response. For example, (a) the production amount of IFNγ decreases, (b) Leishmania, (C) a TH1-type T cell-dependent antibody (IgG2a) that becomes hypersensitive to intracellular pathogens such as Listeria and Trypanosoma
It is described that symptoms such as a decrease in the production amount of (subtype) and (d) formation of abnormal granuloma in response to Bacillus are observed. Furthermore, it is also described that administration of an antagonist to IL27R can improve skin inflammatory conditions, arthritis, Crohn's disease, airway hypersensitivity, airway inflammation, atherosclerosis and the like.

Patent Document 2: JP-T-2007-525415 describes that an immune disease or a helper T cell-mediated disease can be treated by using a ligand for IL27R.

Non-Patent Document 1: Immunity 15, 569-78 describes a decrease in Th1-type immune response after infection with Leishmania major in mice in which the action of IL27 has been lost.

Non-patent document 2: Immunity 19, 657-667 describes that overproduction of inflammatory cytokines occurs after trypanosoma cruzi infection in mice in which the action of IL27 is lost. Non-patent document 3: J Immunol 17
7, 5377-85 describes that IL27 acts on lymphocytes to suppress the production of various cytokines.

Special table 2007-523169 gazette Special table 2007-525415 gazette

Yoshida, H .; Hamano, S .; Senaldi, G .; , Covey, T .; , Faggioni, R .; , Mu, S .; Xia, M .; , Wakeham, A .; C. Nishina, H .; Potter, J .; , Saris, C.I. J. et al. Mak, T .; W. (2001) WSX-1 Is Required for the Initiation of Th1 Responses and Resistance to L. major Infection. Immunity 15, 569-78. Hamano, S.H. Himeno, K .; , Miyazaki, Y. et al. , Ishii, K .; Yamanaka, A .; Takeda, A .; , Zhang, M .; , Hisaeda, H .; Mak, T .; W. Yoshimura, A .; , Yoshida, H .; (2003) WSX-1 Is Required for Resistance to Trypanoma cruzi Infection by Regulation of Prophylaxis Cytokine Production. Immunity 19, 657-667. Yoshimura, T .; Takeda, A .; Hamano, S .; , Miyazaki, Y. et al. Kinjo, I .; Ishibashi, T .; Yoshimura, A .; , Yoshida, H .; (2006) Two-Sided Roles of IL-27: Induction of Th1 Differentiation on Naive CD4 + T Cells versus Suppression of Proinflammatory Cytokine Production Including IL-23-Induced IL-17 on Activated CD4 + T Cells Partially Through STAT3-Dependent Mechanism. J Immunol 177, 5377-85.

In the above situation, there has been a demand for model non-human animals such as diabetes and heart failure that can be used for screening for therapeutic agents for diabetes and heart failure.

As a result of intensive studies to solve the above problems, the present inventors have found that IL27 or IL2
The present inventors have found that a knockout non-human animal in which the function of all or a part of the 7R gene has been lost is useful for screening for a therapeutic agent for diabetes, a therapeutic agent for heart failure, and the like.

That is, the present invention provides the following model non-human animals such as diabetes and heart failure, screening methods for therapeutic agents for diabetes, therapeutic agents for heart failure and the like using the non-human animals.
(1) IL27 signal consisting of a knockout non-human animal or a part thereof in which the function of all or part of at least one gene selected from the group consisting of EBI3 gene, IL27-p28 gene and WSX-1 gene is lost An inactivation-related disease model animal or a part thereof.
(2) An IL27 signal inactivation-related disease model animal or a part thereof, comprising a knockdown non-human animal or a part thereof in which the expression level of IL27 and / or IL27R gene is reduced compared to the wild type.
(3) The animal according to (1) or (2) above or a part thereof, wherein the IL27 signal inactivation-related disease is at least one disease selected from the group consisting of diabetes, diabetic complications, and heart failure .
(4) A screening method for a therapeutic agent for diabetes or diabetic complications,
(A) A test substance is brought into contact with the animal according to any one of (1) to (3) above or a part thereof,
(B) comparing the state of diabetes or diabetic complications in the animal or part thereof contacted with the test substance and the state of diabetes or diabetic complications as a control;
(C) Based on the comparison result of (b), evaluate the diabetes improvement effect or diabetic complication improvement effect of the test substance,
(D) selecting the test substance evaluated to have a diabetic improvement effect or a diabetic complication improvement effect.
(5) A screening method for a therapeutic agent for heart failure,
(A) A test substance is brought into contact with the animal according to any one of (1) to (3) above or a part thereof,
(B) comparing the state of heart failure in the animal contacted with the test substance or a part thereof with the state of heart failure in the control;
(C) Based on the comparison result of (b), evaluate the heart failure improving effect of the test substance,
(D) selecting the test substance evaluated to have an effect of improving heart failure.

The present invention provides a model animal for diseases such as diabetes and heart failure, or a part thereof, comprising a knockout non-human animal in which the function of all or part of the IL27 and / or IL27R gene is lost or a part thereof. The model animal can be used, for example, for screening for a therapeutic drug for diabetes, a therapeutic drug for heart failure, and the like.

FIG. 1 is a graph showing fasting blood glucose before streptozotocin administration (day 0) and on days 7, 14, 21, and 28 after administration. Higher fasting blood glucose at 7, 14, 21, and 28 days after streptozotocin administration in EBI3 knockout mice (EBI3 ^{− / −} ) and WSX-1 knockout mice (WSX ^{− / −} ) compared to wild type mice (WT) It shows that there is. “*” Indicates that the blood glucose of the EBI3 knockout mouse on day 28 (EBI3 ^{− / −} ) is significantly higher than that of the wild type mouse (WT) on the same day (p <0.05). ** ”indicates that the blood glucose of WSX-1 knockout mice (WSX-1 ^{− / −} ) on day 14 and 28 is higher than that on wild type mice (WT) on days 14 and 28, respectively. Significantly higher (p <0.05). FIG. 2 is a diagram showing hematoxylin-eosin (HE) staining (upper: HE) of pancreatic islets of Langerhans 14 days after administration of streptozotocin and a fluorescent antibody image against proinsulin (lower: prosulin). More leukocyte infiltration occurred in HE staining in EBI3 knockout mice (EBI3 ^{− / −} ) and WSX-1 knockout mice (WSX ^{− / −} ) than in wild type mice (WT), and a fluorescent antibody against proinsulin The picture shows a decrease in proinsulin. FIG. 3 is a graph showing the quantification results of the amount of insulin precursor (proinsulin amount) in pancreatic islets of Langerhans 14 days after administration of streptozotocin. Proinsulin is decreased in EBI3 knockout mice (EBI3 ^{− / −} ) and WSX-1 knockout mice (WSX ^{− / −} ) compared to wild type mice (WT). FIG. 4 is a graph showing fasting blood glucose when streptozotocin (STZ) and recombinant mouse IL27 or a control solvent were administered to wild type mice (WT) and EBI3 knockout mice (EBI3 ^{− / −} ). Fasting blood glucose was measured before the start of administration of streptozotocin (day 0) and 7-14 days after administration. In FIG. 4, “+” indicates a recombinant mouse IL27 administration group, and “−” indicates a control solvent administration group. Fasting blood glucose was reduced by recombinant mouse IL27 administration in both wild-type mice and EBI3 knockout. “*” Indicates that in wild-type mice (WT), blood glucose was higher in the control solvent administration group 14 days after administration of streptozotocin (STZ) than in the recombinant mouse IL27 administration group (p <0.05). , “**” indicates that in the EBI3 knockout mouse (EBI3 ^{− / −} ), the control solvent-administered group 7 days and 14 days after the administration of streptozotocin (STZ) had higher blood glucose than the recombinant mouse IL27-administered group ( p <0.05). FIG. 5 shows stained images of MOMA2 when streptozotocin (STZ) and recombinant mouse IL27 (rIL27) or a control vehicle were administered to wild type mice (WT) and EBI3 knockout mice (EBI3 ^{− / −} ). It is a graph (B) which shows the result of having measured the number of cells of a figure (A) and a MOMA2 positive cell. As shown in FIG. 5, the number of MOMA2-positive macrophages infiltrating the islets of Langerhans was higher in EBI3 knockout mice (EBI3 ^{− / −} ) than in wild type mice (WT). Type mice (WT) and EBI3 knockout mice (EBI3 ^{− / −} ) decreased the number of infiltrating macrophages. FIG. 6 shows stained images of CD4 antigen when streptozotocin (STZ) and recombinant mouse IL27 (rIL27) or a control solvent were administered to wild type mice (WT) and EBI3 knockout mice (EBI3 ^{− / −} ). It is a graph (B) which shows the result of having shown the figure (A) to show, and the cell number of the CD4 antigen positive cell. As shown in FIG. 6, the number of CD4-positive T lymphocytes infiltrating the pancreatic islets of Langerhans was higher in EBI3 knockout mice (EBI3 ^{− / −} ) than in wild type mice (WT), and administration of recombinant mouse IL27 was performed. Reduced the number of infiltrating CD4-positive T lymphocytes in both wild type mice (WT) and EBI3 knockout mice (EBI3 ^{− / −} ). “*” Indicates that the number of CD4-positive T lymphocytes in EBI3 knockout mice (EBI3 − / −) was higher in the streptozotocin (STZ) administration group than in wild type mice (WT) (p <0.05). ). FIG. 7 shows isoproterenol (ISO (+)) or control solvent (ISO (−) in wild type mice (WT), EBI3 knockout mice (EBI3 ^{− / −} ) and WSX-1 knockout mice (WSX ^{− /} −). ) And recombinant mouse IL27 (rIL27 (+)) or control solvent (rIL27 (−)), and the measurement results of the weight (body weight correction value) of the heart excised 7 days later. In EBI3 knockout mice (EBI3 ^{− / −} ) and WSX-1 knockout mice (WSX ^{− / −} ), the increase in heart weight due to isoproterenol administration tended to be larger than in wild type mice (WT). Moreover, the increase in the heart weight by isoproterenol administration was suppressed by administration of recombinant mouse IL27 (rIL27). “*” Indicates that the body weight corrected heart weight is significantly heavier than that of wild type mice (WT) administered with the control vehicle (p <0.05), and “**” indicates administration of isoproterenol. The body weight corrected heart weight is significantly heavier than that of wild type mice (WT) (p <0.05). FIG. 8 shows isoproterenol (ISO (+)) or control solvent (ISO (−) in wild type mice (WT), EBI3 knockout mice (EBI3 ^{− / −} ) and WSX-1 knockout mice (WSX ^{− /} −). ) And recombinant mouse IL27 (rIL27 (+)) or control solvent (rIL27 (−)), and are graphs showing the measurement results of the cardiac myocyte size of the heart excised 7 days later. In EBI3 knockout mice (EBI3 ^{− / −} ) and WSX-1 knockout mice (WSX ^{− / −} ), the increase in cardiomyocyte size by isoproterenol administration tended to be larger than in wild type mice (WT). In addition, the increase in cardiomyocyte size due to administration of isoproterenol was suppressed by administration of recombinant mouse IL27 (rIL27). “*” Indicates significantly larger cardiomyocyte size (p <0.05) compared to wild-type mice (WT) that received control vehicle, and “**” received isoproterenol. Shows significantly larger cardiomyocyte size compared to wild type mice (WT) (p <0.05). FIG. 9 shows a photograph (A) in which recombinant IL27 (rIL27 (+)) or control solvent (rIL27 (−)) was administered to neonatal rat cardiomyocytes, and the cardiomyocytes were observed with a phase contrast microscope, and the cardiomyocytes It is a graph (B) which shows the result of having measured the cell surface area. Recombinant IL27 inhibited an increase in the surface area of cultured cardiomyocytes by administration of isoproterenol (ISO). In FIG. 9B, the unit of the vertical axis is a pixel. “*” Indicates isoproterenol (rIL27) in the absence of recombinant IL27 (rIL27) in the absence of recombinant IL27 (rIL27) as compared to cardiomyocytes administered with a control vehicle instead of isoproterenol (ISO). ISO) indicates that the cell surface area of cardiomyocytes is significantly large, and “**” indicates that the cell surface area of cardiomyocytes administered with recombinant IL27 (rIL27) in the presence of isoproterenol (ISO) It is shown that it is significantly smaller than cardiomyocytes administered with a control solvent instead of recombinant IL27 (rIL27) in the absence of nor (ISO).

Hereinafter, the present invention will be described in detail.
It should be noted that all documents and publications described in this specification are incorporated herein by reference in their entirety regardless of their purposes.

The knockout non-human animal of the present invention may contain IL27 and / or IL27 receptor (IL27R
) It was created so that the function of all or part of the gene is lost.

When streptozotocin is administered to the knockout non-human animal of the present invention to induce insulin production decrease and hyperglycemia, the insulin production decrease and hyperglycemia induced to the knockout non-human animal of the present invention are induced in wild type non-human animals It was found to be severe compared to reduced insulin production and hyperglycemia. In addition, when a knockout non-human animal of the present invention is administered with a β1 adrenergic agent to induce cardiac hypertrophy, the cardiac hypertrophy induced in the knockout non-human animal of the present invention is the cardiac hypertrophy induced in a wild-type non-human animal. It was found to be more severe than From these facts, the animal of the present invention can be used as a model animal representing a pathological condition similar to diabetes, heart failure and the like.

1. The knockout or knockdown non-human animal of the present invention or a part thereof The knockout non-human animal of the present invention is the one in which all or a part of the IL27 and / or IL27R gene of the animal is destroyed so as not to exhibit its original function. Means an animal that has been or has been recombined. The IL27 and / or IL27R gene may be a hetero knockout (heterozygote) disrupted or mutated so that one allele on the genome does not function, or a homo knockout (homozygote) in which both alleles are disrupted or mutated But you can.
The knockout non-human animals of the present invention also include the offspring of such animals.

“The entire function of the gene has been lost” means that the gene has been completely lost, and “the partial function of the gene has been lost” means that the gene has been lost due to the lack of part of the gene. It means that the function of is in a state of being reduced as compared with the wild type. Therefore, “does not perform its original function” means that the expression of IL27 and / or IL27R itself is not performed, or the protein activity is reduced or lost even if expressed. .

“Knockdown” means to suppress the expression level of a specific gene. Suppression of the gene expression level can be achieved, for example, by reducing the transcription level of the gene or inhibiting translation. The knockdown non-human animal of the present invention refers to an animal in which the expression level of the animal IL27 and / or IL27R gene is reduced as compared to the wild type.

The IL27 gene is a gene encoding IL27, and the IL27R gene is IL2
It is a gene encoding 7R. IL27 is composed of IL27-p28 and Epstein-Ba.
rr virus (EBV)-a cytokine comprising two different subunits of induced gene 3 (EBI3). On the other hand, IL27 receptor (IL27R)
It consists of a heterodimer of SX-1 and glycoprotein 130. IL2
7 is expressed in antigen presenting cells such as monocytes, macrophages and dendritic cells.

In the present invention, the IL27 gene to be knocked out or knocked down is, for example, the EBI3 gene and / or the IL27-p28 gene. In the present invention, the IL27R gene to be knocked out or knocked down is the WSX-1 gene. Therefore, the “IL27 and / or IL27R gene” to be knocked out or knocked down in the present invention refers to the EBI3 gene, IL27-p28 gene and WSX.
Means at least one gene selected from the group consisting of -1 genes. “IL27 and / or IL27R gene” to be knocked out or knocked down in the present invention
Is preferably the EBI3 gene, IL27-p28 gene, or WSX-1 gene.

In the present invention, the EBI3 gene, IL27, which is the target of knockout or knockdown
The -p28 gene and WSX-1 gene are known, and the base sequence information and amino acid sequence information can be known from the accession number such as GenBank. Below, EBI
The accession numbers indicating the base sequence information and amino acid sequence information of the 3 genes, IL27-p28 gene and WSX-1 gene are exemplified.

-DNA sequence of mouse EBI3 gene: NM_015766 (SEQ ID NO: 1),
-Amino acid sequence of mouse EBI3: NP_056581 (SEQ ID NO: 2),
-DNA sequence of rat EBI3 gene: NM_001109421 (SEQ ID NO: 3),
-Amino acid sequence of rat EBI3: NP_001102891 (SEQ ID NO: 4),

-DNA sequence of mouse IL27-p28 gene: NM_145636 (SEQ ID NO: 5)
,
-Amino acid sequence of mouse IL27-p28: NP_663611 (SEQ ID NO: 6),

-DNA sequence of mouse WSX-1 gene: NM_0166671 (SEQ ID NO: 7),
-Amino acid sequence of mouse WSX-1: NP_0578880 (SEQ ID NO: 8).

In the present invention, the non-human animal to be knocked out or knocked down may be any animal other than a human having the IL27 gene and IL27R gene, but a non-human mammal is preferred. Examples of non-human mammals include cattle, minipigs, pigs, sheep, goats, rabbits, dogs, cats, guinea pigs, hamsters, mice, rats, monkeys, and the like. Among non-human mammals, rodents, particularly mice or rats, which have relatively short ontogeny and biological cycle and are easy to reproduce, are particularly preferable from the viewpoint of producing pathological animal model systems.

In the present invention, a part of an animal means a tissue, a cell, a crushed or extract thereof, and a body fluid derived from the animal, and is not particularly limited. As tissue derived from non-human animals,
For example, heart, lung, kidney, gallbladder, liver, pancreas, spleen, intestine, testis (testis), ovary, uterus, placenta, muscle, blood vessel, brain, bone marrow, thyroid, thymus, breast, lymph node, blood, etc. It is done.

In the present invention, the animal-derived cell means a cell contained in the tissue, organ or body fluid, and includes a cultured cell obtained by isolation and culture. Examples of the cells include various cells such as macrophages, vascular endothelial cells, vascular smooth muscle cells, cardiomyocytes, bone marrow cells, and lymphocytes. The cultured cells include both primary cultured cells and established cell lines thereof. Tissues, organs, cells, debris or extracts and body fluids in the developmental stage (embryonic stage) are also included in some non-human animals.

Furthermore, body fluids such as animal-derived blood, lymph and urine are also included as part of the animal in the present invention.

The knockout non-human animal of the present invention causes a decrease in insulin production and hyperglycemia, such as when streptozotocin is administered. The resulting decrease in insulin production and hyperglycemia is caused by a decrease in insulin production and hyperglycemia induced in wild-type non-human animals. Is severe compared to Therefore,
The knockout or knockdown non-human animal of the present invention can be used as a diabetic or diabetic complication model animal or a part thereof.

Examples of diabetic complications include diabetic neuropathy, retinopathy, nephropathy, vascular disorders including arteriosclerosis, diabetic coma, and the like.

Further, cardiac hypertrophy occurs when β1 adrenergic agonist is administered to the knockout non-human animal of the present invention, and the resulting cardiac hypertrophy is severe compared to cardiac hypertrophy induced in wild-type non-human animals. . Therefore, the knockout or knockdown non-human animal of the present invention can be used as a heart failure model animal or a part thereof.

Examples of heart failure include cardiac hypertrophy, decreased cardiac function, arrhythmia, heart failure-related death, and the like.

2. Production of knockout non-human animals or knock-down non-human animals 2.1. Production of Knockout Non-Human Animal The knockout non-human animal of the present invention can be produced and obtained by a standard method, for example.
As a standard method, for example, a method by producing an ES cell in which IL27 and / or IL27R gene is inactivated can be mentioned.

The method is
(A) producing an ES cell in which all or a part of the functions of the IL27 gene and / or IL27R gene are deleted by gene targeting;
(B) producing a chimeric non-human animal using ES cells;
(C) crossing a chimeric non-human animal with a wild-type mouse to produce a hetero-knockout non-human animal;
(D) crossing hetero-knockout non-human animals to produce homo-knock-out non-human animals.
Hereafter, each said process is demonstrated in detail.

Step (a): In gene targeting gene targeting, first, a target gene (IL27 gene and / or IL27R)
Gene) loses its function, or is disrupted or recombined so that its function is reduced
A fragment is prepared. The target gene targeting vector destroys the target gene, thereby losing all or a part of the gene to completely lose the function or to reduce the function. “Loss all of the gene” means that the gene is completely lost, and “Lose part of the gene” means that the function of the gene is It means that it is in a state of being lowered in comparison. Therefore, “all or part of a gene is lost” means that the expression of the protein encoded by the target gene is not performed, or the activity of the protein is lost even when expressed. It means that it is lower than the mold.

The targeting vector is preferably constructed so as to facilitate screening of recombinants after homologous recombination has occurred. For example, a selection marker such as a drug resistance gene or a toxin gene can be linked to the vector for positive-negative selection. Positive-negative selection methods are well known in the art. In other words, positive selection utilizes the fact that cells that have not incorporated the selectable marker gene die when they are cultured in a culture medium containing a drug because they do not contain a resistance gene, and the negative selection method uses random integration. In the cells that occurred in the above, the fact that the cells die is used to express the negative selection gene. As a result, only cells that have undergone homologous recombination survive and are selected.

For example, a neomycin resistance gene (ne
o) a hygromycin B phosphotransferase gene, an ampicillin resistance gene,
A tetracycline resistance gene or the like can be used, and herpes virus thymidine kinase gene (HSV-tk), diphtheria toxin A gene, or the like can be used for negative selection.

Homologous recombination is performed using the targeting vector prepared by the above method. In the currently established gene targeting methods, it is desirable to use ES cells when producing knockout non-human animals. As ES cells, TT2 cells, AB-1 cells,
J1 cells, R1 cells and the like can be appropriately selected and used.

In order to cause homologous recombination, a targeting vector in which the function of the target gene is lost is introduced into the cell. As a method for introducing a targeting vector into a cell, an electroporation method, a calcium phosphate method, a DEAE-dextran method, a liposome method, or the like can be used. The electroporation method is preferably used in consideration of efficiency, ease of work, and the like. Then, the target genomic DNA sequence in the cell is replaced by homologous recombination of the targeting vector.

In order to increase the homologous recombination efficiency, for example, the Cre-loxP system, which is a recombination system derived from bacteriophage P1, can be used. Cre is a recombination enzyme that recognizes a 34-base sequence called loxP and allows recombination to occur at this site. Therefore, the gene (target gene) to be targeted is assigned to the loxP sequence and lo
By inserting the Cre recombinase gene downstream of the specific promoter between the xP sequences, Cre is produced in a site-specific and time-specific manner, and the gene sandwiched between loxPs is cut out (ie, the function of the target gene is lost) Can).

Thereafter, whether or not the target gene is targeted is confirmed by PCR, Southern blotting, or the like.

In PCR, the primer is, for example, genomic DNA outside the targeting vector.
It can be designed on the region and on the drug resistance gene in the targeting vector sequence.

Southern blot analysis can be performed, for example, as follows. One or two types of probes that recognize genomic regions not included in the targeting vector are designed. By hybridizing a DNA fragment obtained by treating a clone containing the target gene genome with an appropriate restriction enzyme and the probe, the presence or absence of the target gene can be confirmed.

Step (b): Production of Chimeric Non-Human Animal Recombinant ES cells obtained as a result of homologous recombination are transplanted into the 8-cell stage or blastocyst embryo.
This ES cell transplanted embryo is transplanted into the womb of a pseudopregnant foster parent to give birth, thereby producing a chimeric animal.

Known methods such as a microinjection method and an agglutination method can be used as a method for transplanting ES cells into the embryo.

In the case of a mouse, first, a female mouse subjected to superovulation treatment with a hormonal agent is mated with a male mouse. Thereafter, early embryos are collected from the oviduct or uterus on day 2.5 after fertilization when using an 8-cell stage embryo, and on day 3.5 after fertilization when using a blastocyst, respectively. ES cells that have undergone homologous recombination are injected into the collected embryos to produce chimeric embryos.

On the other hand, a pseudopregnant female mouse for use as a foster parent can be obtained by mating a female mouse having a normal cycle with a male mouse castrated by vagina ligation or the like. A chimeric mouse can be produced by transplanting the chimeric embryo produced by the above-mentioned method in utero to the produced pseudopregnant mouse and then giving birth.

For non-human animals other than mice, chimeric non-human animals can be prepared in the same manner as described above.

Step (c): Production of hetero knockout non-human animal From the chimeric non-human animal obtained as described above, a male chimeric non-human animal derived from an ES cell-transplanted embryo is selected. In the case of a mouse, after the male mouse derived from the selected ES cell-transplanted embryo has matured, this mouse is mated with a female mouse of a pure mouse strain. And to the born mouse,
When the coat color of a mouse derived from an ES cell (a mouse having a genome incorporated into the ES cell) appears, it can be confirmed that the ES cell has been introduced into the germ line of the chimeric mouse. Then, the target gene-deficient hetero knockout non-human animal in which the recombinant ES cells transplanted into the embryo are introduced into the germ line is bred.

Step (d): Production of homo-knockout non-human animal The gene-deficient homo-knock-out non-human animal can be obtained by crossing the target gene hetero-knock-out non-human animals obtained as described above.

Confirmation that a knockout non-human animal has been obtained in step (c) or (d) can be carried out by extracting chromosomal DNA from the tissue and then using Southern blotting or PCR. Furthermore, RNA can be extracted from the tissue, and the gene expression pattern can be analyzed by Northern blot analysis. Western blotting may be performed using an antibody against the protein encoded by the target gene.

In addition, the phenotypes of hetero knockout non-human animals and homo knock-out non-human animals can be analyzed for the established animal lines. The phenotype is analyzed by macroscopic observation, internal observation by anatomy, tissue section of each organ, observation by X-ray imaging, observation of behavior and memory, blood test and serum biochemical test. The analysis period may be any period from the embryonic period to the adult period, and is not particularly limited.

In addition, the production method of EBI3 gene knockout mouse is described in, for example, Igawa, T .; ,
Nakashima, H .; , Sadanaga, A .; , Masutani,
K. , Miyake, K .; Shimizu, S .; Takeda, A .; ,
Hamano, S.H. , Yoshida, H .; (2009) Defiien
cy in EBV-induced gene 3 (EBI3) in MRL / l
pr mice results in pathological alterati
on of autoimmune globeronephritis and
sialadenitis. Mod Rheumatol 19, 33-41. Are also described. A method for producing a WSX-1 gene knockout mouse is described in, for example, Yoshida, H. et al. Hamano, S .; Senaldi, G .; , C
ovey, T.M. , Faggioni, R .; , Mu, S .; Xia, M .; ,
Wakeham, A.D. C. Nishina, H .; Potter, J .; ,
Saris, C.I. J. et al. Mak, T .; W. (2001) WSX-1 Is
Required for the Initiation of Th1 Respo
nses and Resistance to L. major Infectio
n. Immunity 15, 569-78. Are also described.

2.2. Production of Knockdown Non-Human Animal The knockdown non-human animal of the present invention can be produced by suppressing the expression of IL27 gene and / or IL27R gene of the non-human animal according to a known method.

In order to suppress the expression of IL27 gene and / or IL27R gene, for example, RNA interference (RN
Ai) can be used, but is not particularly limited thereto. For example, IL2
SiRNA (small interface) against 7 genes and / or IL27R gene
ring RNA) can be designed and synthesized, and this can be incorporated into a retrovirus vector or adenovirus vector and introduced into a non-human animal to cause RNAi.

RNAi is a phenomenon in which dsRNA (double-strand RNA) specifically and selectively binds to a target gene and efficiently cleaves the target gene by cleaving the target gene. For example, when dsRNA is introduced into a non-human animal, expression of a gene having a sequence homologous to that RNA is suppressed (knocked down).

(1) siRNA
siRNA can be designed as follows.
(A) It is not particularly limited as long as it is a gene encoding IL27 or IL27R, and any region can be used as a candidate. (B) A sequence beginning with AA from a selected region. 18-30 bases in length, or 18-30 bases when AA is added to the 5 'side, preferably 19-23
Select the base sequence. The GC content of the sequence may be selected to be, for example, 30 to 70%, and is preferably around 50%. Further, it is preferable to select a GC distribution that has no bias.
(C) An overhang of 2 residues of dT or U may be added to the 3 ′ side of the selected sequence.
(D) A BLAST search is preferably performed to confirm that there is no protein having a sequence similar to the selected sequence.

Furthermore, by introducing several types of siRNA simultaneously into non-human animals, gene expression may be more effectively suppressed.

Another method for synthesizing siRNA is the Dicer method. The outline of the Dicer method is as follows. First, a gene sequence of 500 bp to 1 kbp is incorporated into a vector or the like from the start codon of the gene sequence encoding IL27 or IL27R, and the sense RNA and antisense RNA of the gene sequence are transcribed and then annealed to dsRNA. Is made. Next, this ds-RNA is processed by a Dicer Enzyme belonging to the RNase III family into a 21-23 base siRNA having a 2 base protruding end on the 3 ′ end side.

(2) shRNA
The present invention can also use shRNA to bring about the RNAi effect.
shRNA is called short hairpin RNA and is an RNA molecule having a stem-loop structure so that a part of a single strand forms a complementary strand with another region. RNA molecules having this stem-loop structure are dicers in vivo.
Etc., and siRNA is produced.

shRNA can be designed so that a part thereof forms a stem-loop structure.
For example, assuming that the sequence of a certain region is “sequence A” and the complementary strand to sequence A is “sequence B”, these sequences are present in a single RNA strand in the order of sequence A, spacer, and sequence B. Thus, the length is designed to be 42 to 120 bases as a whole. The sequence A is a sequence of a partial region of a gene encoding IL27 or IL27R to be a target, and the target region is not particularly limited, and any region can be a candidate. And array A
Is 18 to 50 bases, preferably 21 to 30 bases.

In addition, for suppression of IL27 gene and / or IL27R gene expression, microRNA (
miRNA) can also be used. miRNA is a small RNA, and can inhibit the expression of a specific gene by inhibiting translation of mRNA having a complementary sequence to protein into a protein.

miRNA can be designed to recognize mRNA of a specific gene. The length of the base sequence of miRNA designed as such is, for example, 18 to 25 bases (eg, about 22 bases).

3. The present invention relates to a knockout or knockdown non-human animal of the present invention that has developed diabetes or diabetic complications or a part thereof (hereinafter referred to as the knockout non-human animal of the present invention, etc.). And a candidate drug substance (test substance) for treating diabetes or diabetic complications can be screened for a therapeutic drug for diabetes or a therapeutic drug for diabetic complications. For example, a drug candidate substance is brought into contact with a knockout non-human animal or the like of the present invention that has developed diabetes or diabetic complications, and the target disease is correlated with the target disease in the non-human animal or the like of the present invention that is contacted with the candidate substance. Measure indicators (eg, degree of inflammation of pancreatic islets of Langerhans, insulin production, blood glucose level, etc.), compare with controls, and reduce or eliminate symptoms of diabetes or diabetic complications based on the comparison results It is possible to screen candidate substances by confirming whether or not.

Examples of the method for bringing the knockout or knockdown non-human animal of the present invention into contact with a candidate substance include oral administration, intravenous injection, application, subcutaneous administration, intradermal administration, and intraperitoneal administration. It can be appropriately selected according to the nature of the substance. The dosage of the candidate substance can be appropriately selected according to the administration method, the nature of the candidate substance, and the like.

Examples of the method of contacting a part of the knockout or knockdown non-human animal of the present invention with a candidate substance include, for example, the same reaction system or culture system for a part of the knockout or knockdown non-human animal of the present invention and the candidate substance. Can be used. More specifically, a candidate substance is added to a culture container of a part of the knockout or knockdown non-human animal of the present invention, and a part of the knockout or knockdown nonhuman animal of the present invention and the candidate substance are mixed. And culturing a part of the knockout or knockdown non-human animal of the present invention in the presence of a candidate substance. The amount of candidate substance to be contacted can be appropriately selected according to the administration method, the nature of the candidate substance, and the like.

Diabetes can be induced in, for example, the knockout non-human animal of the present invention and wild-type non-human animals by administering streptozotocin, for example.

Controls for comparison include, for example, a wild-type non-human animal or a part thereof that is not contacted with a test substance, a wild-type non-human animal or a part thereof that is contacted with a test substance, and has an effect of improving diabetes or diabetic complications Wild-type non-human animals or parts thereof that have been contacted with known substances (such as known anti-diabetic drugs), knock-out or knock-down non-human animals or parts of the present invention that are not contacted with test substances, known It means any of the knockout or knockdown non-human animals of the present invention contacted with a therapeutic agent for diabetes or the like, or a part thereof, and at least one control can be appropriately selected according to the purpose.

Here, the “wild-type non-human animal or a part thereof” means a non-human animal or a part thereof in which both the IL27 gene and the IL27R gene are normally expressed and function.

For example, if the control is a knockout or knockdown non-human animal of the present invention that is not contacted with a test substance, the degree of inflammation of pancreatic islets of the knockout or knockdown nonhuman animal of the present invention that is contacted with a candidate substance, insulin Production amount or blood glucose level (hereinafter,
The degree of inflammation of the pancreatic islets of Langerhans. ) And the measured degree of inflammation of the pancreatic islets of the pancreas is improved over the degree of inflammation of the pancreatic islets of the pancreas in the control, the candidate substance is used as a therapeutic agent for diabetes or diabetic complications. You can choose. “Improvement” in this case refers to a reduction in the degree of inflammation of the pancreatic islets of the pancreatic islets of the knockout or knockdown non-human animals of the present invention (eg, 10% or more,
20% or more, decrease by 30% or more), increase in insulin production (for example, 5% or more,
10% or more, 20% or more increase), or blood glucose level decreases (for example, 5% or more, 10%
That is, it is reduced by 20% or more).

Alternatively, when the control is a wild-type non-human animal that is not contacted with a test substance, the degree of inflammation, insulin production, or blood glucose level of the pancreatic islets of the knockout or knock-down non-human animal of the present invention that is contacted with the candidate substance (Hereinafter referred to as the degree of inflammation of the pancreatic islets of Langerhans) was measured, and the degree of inflammation of the pancreatic islets of Langerhans was measured to the same level as or higher than the degree of inflammation of the pancreatic islets of Langerhans in the control. Can also be selected as a therapeutic agent for diabetes or diabetic complications. “Improvement” in this case means that the degree of inflammation of pancreatic islets of the pancreatic islets of the knockout or knockdown non-human animal of the present invention is the same or reduced (for example, 5% or more, 10% More than 15% or more than 20%), the amount of insulin production is comparable or increased (for example, more than 5%, more than 10%, more than 15% or more than 20%), or blood sugar The value becomes comparable or decreases (for example, 5% or more, 10% or more, 15%
Or more than 20%).

Candidate substances include, for example, peptides, proteins, non-peptidic compounds, synthetic compounds,
Fermentation products, cell extracts, cell culture supernatants, plant extracts, mammalian tissue extracts (eg, mice, rats, pigs, cows, sheep, monkeys, humans, etc.), plasma, etc. May be a novel compound or a known compound. These candidate substances may form salts, and salts of candidate substances include physiologically acceptable acids (for example,
A salt with an organic acid or inorganic acid) or a base (such as a metal acid) is used, and a physiologically acceptable acid addition salt is particularly preferable. Examples of such salts include salts with inorganic acids (eg, hydrochloric acid, phosphoric acid, hydrobromic acid, sulfuric acid, etc.), or organic acids (eg, acetic acid, formic acid, propionic acid, fumaric acid, maleic acid, And salts with succinic acid, tartaric acid, citric acid, malic acid, succinic acid, benzoic acid, methanesulfonic acid, benzenesulfonic acid, and the like.

For example, if a candidate substance is administered and results confirming that the symptoms of diabetes or diabetic complications have been alleviated or eliminated, the candidate substance used is selected as a therapeutic agent for diabetes or diabetic complications. Is possible.

4). The present invention relates to a method for screening for heart failure in a knockout or knockdown non-human animal of the present invention or a part thereof (hereinafter referred to as the knockout non-human animal of the present invention) that has developed heart failure. A drug for treating heart failure can be screened by contacting a drug candidate substance (test substance). For example, a drug candidate substance is brought into contact with a knockout non-human animal or the like of the present invention that has developed heart failure, and an index having a correlation with a target disease in the knockout non-human animal or the like of the present invention that is contacted with the candidate substance (for example, , Degree of cardiac hypertrophy, cardiac function, mortality, etc.)
The candidate substance can be screened by determining whether or not the symptoms of heart failure are reduced or eliminated based on the comparison result.

Examples of the method of bringing the knockout or knockdown non-human animal of the present invention into contact with a candidate substance include the same methods as those described in the above section “Screening method for therapeutic agent for diabetes or diabetic complications”.

Examples of the method of contacting a part of the knockout or knockdown non-human animal of the present invention with a candidate substance include the same methods as described in the above-mentioned “Method for screening for therapeutic agent for diabetes or diabetic complications”. it can.

Among heart failure, cardiac hypertrophy can be induced in, for example, the knockout non-human animal of the present invention and wild type non-human animal by administering a β1 adrenergic receptor agonist (eg, isoproterenol).

Controls for comparison include wild-type non-human animals that are not contacted with the test substance, wild-type non-human animals that are contacted with the test substance, and substances that are known to have an effect of improving heart failure (such as known heart failure therapeutic agents). A contacted wild-type non-human animal, a knock-out or knock-down non-human animal of the present invention that is not contacted with a test substance or a part thereof, a knock-out or knock-down non-human animal of the present invention that is contacted with a known heart failure therapeutic agent, or the like Any one of them is meant, and at least one control can be appropriately selected depending on the purpose.

For example, when the control is a knockout or knockdown non-human animal of the present invention that is not contacted with a test substance, the degree of cardiac hypertrophy of the knockout or knockdown non-human animal of the present invention that is contacted with a candidate substance is measured, When the measured degree of cardiac hypertrophy improves over the degree of cardiac hypertrophy in the control, the candidate substance can be selected as a therapeutic agent for heart failure. The degree of cardiac hypertrophy can be quantified, for example, by measuring heart weight (body weight correction value), measuring myocardial cell size, or the like. “Improvement” in this case refers to a decrease in the degree of cardiac hypertrophy in the knockout or knockdown non-human animal of the present invention, for example, a decrease in heart weight (weight correction value) compared to the control (for example, 2 % Or more, 5% or more, 10%
Or 20% or more) or cardiomyocyte size is reduced (for example, 2% or more, 5%
Or more, 10% or more, or 20% or more).

Alternatively, when the control is a wild-type non-human animal that is not contacted with the test substance, the degree of cardiac hypertrophy of the knockout or knock-down non-human animal of the present invention contacted with the candidate substance is measured, and the measured cardiac hypertrophy The candidate substance can be selected as a therapeutic agent for heart failure when the degree of is improved to the same degree or better than the degree of cardiac hypertrophy in the control. “Improvement” in this case means that the degree of cardiac hypertrophy in the knockout or knockdown non-human animal of the present invention is the same or reduced compared to the control, for example, heart weight (weight correction value). Are comparable or reduced (eg, 2% or more, 5% or more, 10% or more reduction), or cardiomyocyte size is comparable or reduced (eg, 2% or more, 5% or more, 10%
It means that it is reduced).

Candidate substances may include the same substances as those described in the above section “Screening method for therapeutic agent for diabetes or diabetic complications”.

For example, when a candidate substance is administered, if a result that confirms that the symptoms of heart failure have been alleviated or eliminated is obtained, the used candidate substance can be selected as a therapeutic agent for heart failure.

The best mode for carrying out the invention and specific examples show preferred embodiments of the present invention and are shown for illustration or description, and the present invention is not limited thereto. Not what you want. It will be apparent to those skilled in the art that various modifications can be made based on the description of the specification within the spirit and scope of the invention disclosed herein.
In the following examples, the test for significant difference was performed by analysis of variance (ANOVA).

Example 1: Preparation of IL27-p28 gene knockout mouse (1) Preparation of IL27-p28 gene knockout ES cell Yoshida, H. et al. Hamano, S .; Senaldi, G .; , C
ovey, T.M. , Faggioni, R .; , Mu, S .; Xia, M .; ,
Wakeham, A.D. C. Nishina, H .; Potter, J .; ,
Saris, C.I. J. et al. Mak, T .; W. (2001) WSX-1 Is
Required for the Initiation of Th1 Respo
nses and Resistance to L. major Infectio
n. Immunity 15, 569-78. According to the described method, IL27-p2
Eight gene knockout ES cells were prepared.

That is, a targeting vector having a structure capable of replacing the first to fourth exons of the mouse IL27-p28 gene with a neomycin resistance gene connected to the pgk promoter sequence is prepared, and this targeting vector is electroporated in ES cells. It introduced into a certain E14 cell. For selection of transgenic cells, cell clones that have acquired resistance by culturing in the presence of neomycin are obtained, and then homologous recombination occurs correctly by PCR, so that the first to fourth exons of the IL27-p28 gene are neomycin resistant genes. The cell clone replaced with was selected, and homologous recombination was confirmed by Southern blotting.

(2) Preparation of IL27-p28 gene-deficient chimeric mouse An IL27-p28 gene-deficient chimeric mouse was prepared as follows. That is, Example 1
The ES cell obtained by (1) in which one-sided aryl (one-sided gene) of the IL27-p28 gene was disrupted was injected into a goblet cyst derived from a C57BL6 mouse, and transplanted into a pseudopregnant mouse. A chimeric mouse consisting of mouse-derived cells was obtained. Male individuals presumed that ES cells having mutations in the IL27-p28 gene have a large contribution were selected by hair color and Southern blotting.

(3) Preparation of IL27-p28 gene hetero knockout mouse An IL27-p28 gene hetero knockout mouse was prepared as follows. That is,
Offspring were obtained by mating the chimeric mice obtained in Example 1 (2) with C57BL6 female mice, and ES cell-derived offspring having mutations in the IL27-p28 gene were selected based on coat color. Further, genomic DNA was extracted from the body tissues of these offsprings, and mice having mutations on one side alleles of the IL27-p28 gene were selected by PCR and Southern blotting to obtain hetero knockout mice.

(4) Preparation of IL27-p28 gene homo knockout mouse An IL27-p28 gene homo knockout mouse was prepared as follows. That is, the hetero knockout mouse obtained in Example 1 (3) was crossed with a wild type C57BL / 6 mouse, and a hetero knockout mouse was selected from its offspring, and this process was repeated 6 times.
IL27-p28 gene hetero knockout mice with a BL / 6 gene background were generated. Offspring are obtained by mating these males and females, genomic DNA is extracted from the body tissues of these offsprings, and mice having mutations in both alleles of the IL27-p28 gene are selected by PCR and Southern blotting, and homo knockout mice Got.

In the following Examples, EBI-3 gene homo knockout mice were prepared in the laboratory of the present inventors (Igawa, T., Nakashima,
H. , Sadanaga, A .; , Masutani, K .; , Miyake,
K. Shimizu, S .; Takeda, A .; Hamano, S .; ,
Yoshida, H .; (2009) Efficiency in EBV-in
Duced gene 3 (EBI3) in MRL / lpr mice resu
lts in pathological alteration of autoim
mune glomerulonephritis and sialadenitis
. Mod Rheumatol 19, 33-41. ).
WSX-1 gene homo-knockout mice used were those distributed by Amgen (Yoshida, H., Hamano, S., Senaldi, G.,).
Covey, T .; , Faggioni, R .; , Mu, S .; , Xia, M
. , Wakeham, A .; C. Nishina, H .; , Potter, J
. , Saris, C .; J. et al. Mak, T .; W. (2001) WSX-1 I
s Required for the Initiation of Th1 Res
Ponses and Resistance to L. major Infect
ion. Immunity 15, 569-78. ).
C57BL6 (Jackson Laboratory) was used as the wild type mouse.

Example 2: Quantification / ratio of the degree of inflammation of pancreatic islets of Langerhans, amount of insulin production and blood glucose level
Compare wild type mice, after the administration of streptozotocin (Sigma) to each knockout mice EBI-3 gene knockout mice, and WSX-1 gene knockout mice,
The degree of inflammation, insulin production, and blood glucose level in the pancreatic islets of Langerhans were compared and examined as follows.

(1) Fasting blood glucose level Streptozotocin (5 μg per body weight) was administered intraperitoneally once a day for 5 days, before starting administration (Day 0) and after 7, 14, and 28 days after starting administration. Blood glucose was measured. Measurement of fasting blood glucose was performed as follows. That is, venous blood was collected after fasting for 12 hours, and the fasting blood glucose level was measured with a blood glucose meter (trade name: Glucose Pilot; manufactured by Technicon International).

The result is shown in FIG.
FIG. 1 is a graph showing fasting blood glucose before streptozotocin administration (day 0) and on days 7, 14, 21, and 28 after administration. As shown in FIG. 1, EBI3 knockout mice (EBI3 ^{− / −} ) and WSX-1 knockout mice (W
SX ^{− / −} ), fasting blood glucose tends to be high on days 7, 14, 21, and 28 after administration of streptozotocin.

(2) Hematoxylin-eosin (HE) -stained image of pancreatic islets of Langerhans A HE-stained image of pancreatic islets of Langerhans on the 14th day after the start of administration of streptozotocin was obtained as follows. That is, after blood removal under anesthesia, it was refluxed and fixed with formalin, and the pancreas was removed. The excised pancreas was fixed in formalin and dehydrated, and then embedded in paraffin. Thereafter, the slices were sliced and stained with HE, and a microscopic image was obtained with a CCD camera (trade name: AxioCam; Zeiss
Obtained).

  The result is shown in the upper part (HE) of FIG.

FIG. 2 shows EBI3 knockout mice (EBI3 ⁻ //) compared to wild type mice (WT).
^- ) And WSX-1 knockout mouse (WSX ^-/- ) pancreatic islets of Langerhans are shown to accumulate more cells with violet stained nuclei. This means that EBI3 knockout mice (EBI3 ^{− / −} ) and WSX− compared to wild type mice (WT).
It shows that more leukocyte infiltration occurs in one knockout mouse (WSX ^{− / −} ).

(3) Fluorescent antibody image against pancreatic islets of Langerhans (proinsulin) A fluorescent antibody image against proinsulin of pancreatic islets on the 14th day after the start of administration of streptozotocin was obtained as follows. That is, after blood removal under anesthesia, it was refluxed and fixed with formalin, and the pancreas was removed. The excised pancreas is fixed in formalin and then compound (
Product Name Tissue-Tek OCT compound; Sakura Finet
embedded in a chemical company) and snap frozen with liquid nitrogen. This was sliced and stained with an anti-proinsulin antibody (Takara Bio) and a FITC-labeled secondary antibody (Jackson). Fluorescence microscope image is cooled by CCD camera (trade name: AxioCam; Zeiss
).

  The result is shown in the lower part of FIG.

FIG. 2 shows that the amount of fluorescently stained protein in the pancreatic islets of EBI3 knockout mice (EBI3 ^{− / −} ) and WSX-1 knockout mice (WSX ^{− / −} ) is smaller than that in wild type mice (WT). The situation is shown. This indicates that proinsulin is decreased in EBI3 knockout mice (EBI3 ^{− / −} ) and WSX-1 knockout mice (WSX ^{− / −} ) compared to wild type mice (WT).

Next, the amount of insulin precursor (proinsulin amount) in pancreatic islets of Langerhans was quantified by the fluorescent antibody method as follows. That is, from the fluorescent antibody images obtained above, the fluorescence intensity per unit area was measured with NIH Image software distributed free of charge by NIH, USA.

  The result is shown in FIG.

The vertical axis in FIG. 3 shows the relative fluorescence intensity with the fluorescence intensity of wild-type mouse (WT) as 1. As shown in FIG. 3, EBI3 knockout mice (E
BI3 ^{− / −} ) and WSX-1 knockout mice (WSX ^{− / −} ) have a significant decrease in proinsulin (*: p <0.05).

(4) Effect of Recombinant IL27 Administration Recombinant mouse IL27 was obtained from Pflanz, S .; , J. et al. C. Timan
s, J.M. Cheung, R.A. Rosales, H.M. Kanzler, J.M.
Gilbert, L.M. Hibert, T .; Churakova, M .; T
ravis, E.M. Vaisberg, W.M. M.M. Blumenschein,
J. et al. D. Mattson, J.M. L. Wagner, W.W. To, S.M. Z
urawski, T .; K. McClanahan, D.M. M.M. Gorman,
J. et al. F. Bazan, R.A. de Waal Malefyt, D.D. Ren
nick, and R.M. A. Kastelein. 2002. IL-27,
a heterodimeric cytokine composed of EBI
3 and p28 protein, industries promotion
of naive CD4 (+) T cells. Immunity 16:77
9-790. It was produced according to the method described in 1. Specifically, FLAG in CHO cells
Tagged mouse IL27 expression vector was expressed, and recombinant mouse IL27 secreted into the culture supernatant was purified by column method using beads with anti-FLAG tag antibody (manufactured by Sigma).
In addition to those prepared above, a commercially available product from Abnova was used as the recombinant mouse IL27.

Streptozotocin (5 μg per body weight) was administered intraperitoneally once a day for 5 days. Purified recombinant mouse IL27 or control solvent was administered intraperitoneally once a day for 7 days from the start of streptozotocin administration. Titrate buffer (manufactured by Wako Pure Chemical Industries, Ltd.) was used as a control solvent. The single dose of recombinant mouse IL27 is 500 ng / 0.
2 ml and the single dose of control solvent is 0.2 ml. Then, fasting blood glucose was measured before the start of streptozotocin administration (day 0) and 7, 14 days after the start of administration.

In addition, the degree of inflammation of pancreatic islets of Langerhans and the amount of insulin produced by administration of recombinant mouse IL27 were compared as follows. That is, the degree of inflammation of the pancreatic islets of Langerhans was prepared by preparing a tissue section from the excised pancreas, staining the macrophage markers MOMA2 and CD4 antigen by immunohistochemical staining using Nichirei's histofine, and microscopic observation This was determined by counting the number of MOMA2 or CD4 positive cells. Here, a large number of infiltrating MOMA2-positive macrophages and infiltrating CD4-positive T lymphocytes indicates that the degree of inflammation of pancreatic islets of Langerhans is more severe. The amount of insulin produced was determined by measuring the amount of proinsulin in the same manner as in Example 2 (3).

  The results are shown in FIG. 4, FIG. 5 and FIG.

FIG. 4 is a graph showing fasting blood glucose when streptozotocin and recombinant mouse IL27 or a control solvent were administered to wild type mice (WT) and EBI3 knockout mice (EBI3 ^{− / −} ). As shown in FIG. 4, wild type mice and EBs
In both I3 knockout, fasting blood glucose was reduced by administration of recombinant mouse IL27.

FIG. 5 is a diagram (A) and MOMA2 showing stained images of MOMA2 when streptozotocin and recombinant mouse IL27 or a control solvent were administered to wild-type mice (WT) and EBI3 knockout mice (EBI3 ^{− / −} ). It is a graph (B) which shows the result of having measured the number of cells of a positive cell. As shown in FIG. 5, MO infiltrated the islets of Langerhans
The number of MA2-positive macrophages is higher in EBI3 knockout mice (EBI3 ^{− / −} ) than in wild type mice (WT), and by administration of recombinant mouse IL27, wild type mice (WT) and EBI3 knockout mice (EBI3 ^{− / −} ). In both cases, the number of infiltrating macrophages decreased. This indicates the inhibitory action of recombinant mouse IL27 on macrophage infiltration.

FIG. 6A shows a stained image of CD4 antigen when streptozotocin and recombinant mouse IL27 or a control solvent are administered to wild type mice (WT) and EBI3 knockout mice (EBI3 ^{− / −} ). It is a graph (B) which shows the result of having measured the number of cells of a CD4 antigen positive cell. The number of CD4-positive T lymphocytes that infiltrated the pancreatic islets of Langerhans was greater in EBI3 knockout mice (EBI3 ^{− / −} ) than in wild type mice (WT), and administration of recombinant mouse IL27 resulted in wild type mice (WT) and EBI3. Both knockout mice (EBI3 ^{− / −} ) show a decrease in the number of infiltrating CD4-positive T lymphocytes. This indicates that the recombinant mouse IL27 has an inhibitory effect on the infiltration of the number of CD4-positive T lymphocytes.

As shown in the above examples, in EBI-3 gene knockout mice and WSX-1 gene knockout mice, the degree of inflammation of pancreatic islets of Langerhans, decreased insulin production, and hyperglycemia were severe compared to wild-type mice. Met. Wild type mouse, EB
The degree of pancreatic islet inflammation, decreased insulin production, and hyperglycemia in I-3 gene knockout mice were alleviated by administering recombinant IL27. These examples also show that candidate substances for treating diabetes or diabetic complications can be screened from test substances using the state of diabetes or diabetic complications in the knockout non-human animal of the present invention as an index. .

Example 3: Quantification / comparison of degree of cardiac hypertrophy Isoproterenol (manufactured by Sigma) which is a β1 adrenergic agent in each knockout mouse of wild type mouse, EBI-3 gene knockout mouse, and WSX-1 gene knockout mouse or After the control solvent was administered by an osmotic pump, the degree of cardiac hypertrophy was quantitatively compared as follows. Specifically, osmotic pump (trade name: Alzet osm
otic pump (manufactured by Alzet) was administered with isoproterenol or a control solvent for 7 days, and “Tail cuff method” (Models of experimental h
hypertension in mice. Johns C, Gavrs I,
Handy DE, Salomao A, Gavras H. et al. Hypertens
ion. 1996 28: 1064-1069.) And blood pressure and pulse were measured. The dose of isoproterenol at this time is 30 mg / kg / day. The control solvent is physiological saline, and the dose of the control solvent is the same volume as the isoproterenol solution. Seven days after the start of administration of isoproterenol or control solvent, cardiac ultrasound (Ec
hocardiographic evaluation of ventricula
r function in mice. Rotman JN, Ni G, Br
down M.M. Echocardiography. 2007 24: 83-89.)
LV mass was calculated after measuring cardiac parameters. After heart removal, the heart weight (corrected by body weight) was measured, and the cardiomyocyte size (cardiomyocyte cell surface area) in the heart tissue was measured. Here, measurement of heart weight (corrected by body weight) and measurement of cardiomyocyte size were performed as follows. Regarding the heart weight, the weight of the extracted heart was measured using an electronic balance. Then, the measured heart weight (mg) was corrected by the body weight (g) of the mouse measured at the time of survival immediately before the heart removal, that is, the value obtained by dividing the heart weight by the body weight The corrected heart weight (mg / g) was used. Note that the vertical axis in FIG. 7 shows the body weight corrected heart weight (mg / g) obtained in this way as a numerical value. Regarding the myocardial cell size, a section of a tissue specimen prepared from the isolated heart was stained with FITC-lectin manufactured by DAKO, and then the stained tissue specimen section was observed with a microscope, and this was imaged with a CCD camera. NIH Image
The value obtained by analysis by software (free distribution by US NIH), that is, by measuring the area of a single cell recognized by lectin was taken as the myocardial size (nm ² ). Note that FIG.
The cell surface area of the cardiomyocytes is shown in unit pixels instead of the unit nm ² , and this shows the area obtained by computer analysis under the same conditions as above in unit pixels.

Recombinant mouse IL27 (rIL27) was prepared and administered as follows.
In the same manner as in Example 2 (4), a FLAG-tagged mouse IL27 expression vector was expressed in CHO cells, and recombinant mouse IL27 secreted into the culture supernatant was treated with anti-FL.
Purification was performed by a column method using beads with an AG tag antibody (manufactured by Sigma).
Purified recombinant mouse IL27 (rIL27 (+)) or its control solvent (rI
L27 (−) was intraperitoneally administered 1, 3, and 5 days after the start of administration of isoproterenol or its control solvent. Titrate buffer (manufactured by Wako Pure Chemical Industries, Ltd.) was used as a control solvent for recombinant mouse IL27. A single dose of recombinant mouse IL27 is 500 ng /
0.2 ml and the single dose of the control solvent is 0.2 ml.

  The results are shown in FIG. 7, FIG. 8, and FIG.

FIG. 7 shows isoproterenol (ISO (+)) in wild type mice (WT), EBI3 knockout mice (EBI3 ^{− / −} ) and WSX-1 knockout mice (WSX ^{− / −} ).
Alternatively, the measurement results of the weight (weight correction value) of the heart excised 7 days after administration of the control vehicle (ISO (−)) and the recombinant mouse IL27 (rIL27 (+)) or the control vehicle (rIL27 (−)) It is a graph to show. In EBI3 knockout mice (EBI3 ^{− / −} ) and WSX-1 knockout mice (WSX ^{− / −} ), the increase in heart weight due to administration of isoproterenol tended to be larger than in wild type mice. The heart weight increased by isoproterenol (ISO) administration tended to be suppressed in wild-type mice (WT) and EBI3 knockout mice (EBI3 ^{− / −} ) by the administration of recombinant mouse IL27.

FIG. 8 shows isoproterenol (ISO (+)) in wild type mice (WT), EBI3 knockout mice (EBI3 ^{− / −} ) and WSX-1 knockout mice (WSX ^{− / −} ).
Alternatively, 7 days after administration of the control solvent (ISO (−)) and the recombinant mouse IL27 (rIL27 (+)) or the control solvent (rIL27 (−)), the heart was removed, and the cardiomyocyte size of the removed heart was measured. It is a graph which shows a result. As shown in FIG. 8, wild type mice (WT), EBI3 knockout mice (EBI3 ^{− / −} ) and WSX-1 knockout mice (WSX-1 ^{− / −} ) were all treated with isoproterenol (ISO). Cardiomyocyte size increased compared to vehicle administration. The size of cardiomyocytes after ISO administration was statistically significantly larger in EBI3 knockout mice (EBI3 ^{− / −} ) than in wild type mice (WT), and WSX-1 knockout mice (WSX-1 ^{− / −} ). There was a big trend. Also,
As shown in FIG. 8, the cardiomyocyte size increased by isoproterenol (ISO) administration was observed in wild type mice (WT) and EBI3 by administration of recombinant mouse IL27.
There was a tendency to be suppressed in knockout mice (EBI3 ^{− / −} ).

FIG. 9 shows recombinant IL27 (rIL27 (+)) in rat neonatal cardiomyocytes.
Alternatively, a control solvent (rIL27 (−)) was administered and the cardiomyocytes were observed with a phase-contrast microscope (FIG. 9A), and a graph showing the results of measuring the cell surface area of the cardiomyocytes (FIG. 9B). As shown in FIG. 9, recombinant IL27 suppressed the increase in the surface area of cultured cardiomyocytes by administration of isoproterenol (ISO).

As shown in the above Examples, the degree of cardiac hypertrophy was severer in EBI-3 gene knockout mice and WSX-1 gene knockout mice than in wild-type mice. In addition, the degree of cardiac hypertrophy in wild type mice and EBI-3 gene knockout mice was reduced by administering recombinant IL27. These examples also show that candidate substances for therapeutic agents for heart failure can be screened from test substances using the state of heart failure in the knockout non-human animal of the present invention as an index.

[SEQ ID NO: 1] DNA sequence of mouse EBI3 gene (Accession No. NM)
_015766).
[SEQ ID NO: 2] Amino acid sequence of mouse EBI3 (Accession No. NP — 0
56581).
[SEQ ID NO: 3] DNA sequence of the rat EBI3 gene (Accession No. NM)
_001109421).
[SEQ ID NO: 4] Amino acid sequence of rat EBI3 (Accession No. NP — 0
01102891).
[SEQ ID NO: 5] DNA sequence of mouse IL27-p28 gene (Accession N
o. NM_145636).
[SEQ ID NO: 6] Amino acid sequence of mouse IL27-p28 (Accession No.
NP_663611).
[SEQ ID NO: 7] DNA sequence of mouse WSX-1 gene (Accession No. N)
M_016667).
[SEQ ID NO: 8] Amino acid sequence of mouse WSX-1 (Accession No. NP_
057880).

Claims (5)

IL27 signal inactivation comprising a knockout non-human animal in which the function of all or part of at least one gene selected from the group consisting of EBI3 gene, IL27-p28 gene and WSX-1 gene is lost or a part thereof Related disease model animals or parts thereof. An IL27 signal inactivation-related disease model animal or a part thereof, comprising a knockdown non-human animal or a part thereof in which the expression level of IL27 and / or IL27R gene is reduced compared to the wild type. The animal or part thereof according to claim 1 or 2, wherein the IL27 signal inactivation-related disease is at least one disease selected from the group consisting of diabetes, diabetic complications, and heart failure. A screening method for a therapeutic agent for diabetes or diabetic complications,
(A) a test substance is brought into contact with the animal according to any one of claims 1 to 3 or a part thereof;
(B) comparing the state of diabetes or diabetic complications in the animal or part thereof contacted with the test substance and the state of diabetes or diabetic complications as a control;
(C) Based on the comparison result of (b), evaluate the diabetes improvement effect or diabetic complication improvement effect of the test substance,
(D) selecting the test substance evaluated to have a diabetic improvement effect or a diabetic complication improvement effect. A screening method for a therapeutic agent for heart failure,
(A) a test substance is brought into contact with the animal according to any one of claims 1 to 3 or a part thereof;
(B) comparing the state of heart failure in the animal contacted with the test substance or a part thereof with the state of heart failure in the control;
(C) Based on the comparison result of (b), evaluate the heart failure improving effect of the test substance,
(D) selecting the test substance evaluated to have an effect of improving heart failure.