JP5692677B2 - Non-human knockout animal, its use and production method - Google Patents

Description

  The present invention relates to a non-human knockout animal, its use, and its production method. More particularly, the invention relates to the use of non-human knockout animals in the development of means for the treatment and / or prevention of diseases and / or symptoms associated with angiogenesis and / or angiogenesis.

  Mammalian blood vessels, including humans, are formed during the embryonic period. Angiogenesis in mammals begins with the process of vasculogenesis, which refers to the development and differentiation of undifferentiated endothelial progenitor cells, tube formation, and the formation of primitive vascular plexus. After this angiogenesis, blood vessels consisting of the reconstruction of primitive blood vessels such as stabilization of blood vessel structure, fusion of blood vessels, and sprouting-type neovascularization by mobilization of wall cells (pericytes, vascular smooth muscle cells) to the blood vessel wall A vascular network is stretched throughout the body through an angiogenesis process.

In this angiogenesis process, the phosphatidylinositol 3-kinase (PI3K) family plays an extremely important role. Currently, in mammals, there are three known classes of PI3K (I, II, III), and eight isoforms, and these PI3K products, PI (3) P, PI (3, 4) Exhibits a variety of physiological actions via P ₂ and PI (3,4,5) P ₃ . In recent years, the relationship between PI3K and diseases such as cancer and diabetes has attracted attention, and in particular, vigorous research has been conducted on the physiological function of class I PI3K enzymes. For example, in angioplasty, Class I type PI3K, especially p110α and p110β is due to vascular endothelial growth factor (VEGF) stimulation, PI (4,5) PI to _{P 2} as substrates (3,4,5) _{P 3} It is produced on the cell membrane and is responsible for the control of proliferation, survival, and differentiation of endothelial cells via the downstream phosphorylase protein kinase B (Akt) (see, for example, Non-Patent Document 1).

  On the other hand, with regard to class II type PI3K, which has been completely unknown until now, several research teams including the present inventors group have only analyzed. Class II PI3K produces phosphatidylinositol-3-phosphate (PI (3) P) mainly on endosomes, which are intracellular organelles, using phosphatidylinositol (PI) as a substrate in cells. It is assumed to be related to membrane transport. However, there are still many unclear points regarding the physiological role of C2α, which is a type II class PI3K, and the phenotype of the individual when the PI3K-C2α gene encoding C2α is deleted is not clear.

See Narinokawa-Shinozaki et al., Inositol Phospholipids Intracellular Imaging of Signaling Lipids, Protein Nucleic Acid Enzymes, Vol. 49, no. 10 (2004)

  An object of the present invention is to provide a non-human knockout animal deficient in the PI3K-C2α gene and a method for producing the same. Moreover, it aims at providing the use of this knockout animal and the tissue and cell derived from the said animal.

  The inventors of the present invention have intensively studied to solve the above problems. As a result, it was surprisingly found that knockout mice that have lost the function of the PI3K-C2α gene cause diseases / symptoms related to angiogenesis and angiogenesis. Furthermore, further investigations have found that C2α plays an essential role in angiogenesis “inhibition of vascular permeability by tight adhesion between endothelial cells”.

  The first form of the present invention completed based on the above findings has lost the function of all or part of the PI3K-C2α gene on the homologous chromosome, and is associated with angiogenesis and / or angiogenesis. Non-human knockout animals and their progeny animals exhibiting the disease or symptom.

  Moreover, the 2nd form of this invention is the tissue or cell obtained from said animal. This tissue or cell is derived from blood vessels, for example.

  Furthermore, a third aspect of the present invention is a method for screening a candidate substance for preventing and / or treating a disease or symptom related to angiogenesis and / or angiogenesis, using the above-mentioned animal or tissue / cell. is there. Similarly, the above-mentioned animals and tissues / cells can also be applied to screening methods for biomarkers of diseases or symptoms related to angiogenesis and / or angiogenesis.

  In addition, the fourth aspect of the present invention exhibits a disease or symptom related to angiogenesis and / or angiogenesis, which includes a knockout step of losing the function of all or part of the PI3K-C2α gene on the homologous chromosome. Methods for producing non-human knockout animals are provided. At this time, the knockout step includes, for example, substituting the region containing the start codon of exon 1 of the PI3K-C2α gene with another base sequence by homologous recombination.

  According to the findings of the present inventors for the first time in the present application, abnormalities in C2α function lead to abnormalities in vascular endothelial cells, in particular, increased vascular permeability, triggering an inflammatory reaction, and thus rupture of the vascular structure, It is suggested to result in the development of many angiogenesis (angiogenesis and / or angiogenesis) related diseases and / or symptoms such as aneurysm, arteriosclerosis, edema, anaphylactic shock. Therefore, the C2α knockout non-human animal provided by the present application will be a valuable biological resource not only for basic research but also clinically as a new model animal for grasping the pathophysiology of diseases and symptoms related to angiogenesis. . Further, by using this model animal, it becomes possible to screen a candidate substance capable of treating and / or preventing the disease / symptom at the individual level.

It is explanatory drawing which shows the strategy for producing the knockout (KO) mouse | mouth employ | adopted in the Example. In an Example, it is a figure which shows the result of having performed the whole mount dyeing | staining (DAB color development method) about each fetus of the C2 (alpha) homo KO mouse | mouth of a litter and the wild type mouse | mouth using the anti-CD31 antibody which is a vascular endothelial cell marker. In an Example, it is a figure which shows the result of having carried out the immunohistochemical dyeing | staining (DAB color development method) of the fetal tissue section about each of the C2 (alpha) homo KO mouse | mouth and the wild type mouse | mouth with the anti- alpha smooth muscle actin antibody which is a vascular smooth muscle marker. . It is a figure which shows the result of having observed the C2 (alpha) homo KO mouse | mouth shown in FIG. 3 in detail using the electron microscope. It is explanatory drawing which shows the strategy for producing the tissue-specific C2 (alpha) conditional knockout (CKO) mouse | mouth employ | adopted in the Example. In an Example, it is a figure which shows the result of having observed in detail about the C2 (alpha) -eCKO mouse | mouth. In Examples, fetal skin of C2α-eCKO mice was exfoliated and subjected to double fluorescence immuno whole mount staining with anti-CD31 antibody as a vascular endothelial cell marker and anti-α smooth muscle actin antibody as a vascular smooth muscle cell marker. It is a figure which shows the result. In an Example, it is a figure which shows the result of having evaluated the lumen formation ability with respect to VEGF using the Matrigel assay method about the C2 (alpha) knockdown HUVEC cell compared with a control HUVEC cell. In the Examples, mRFP-2XFYVE domain expression plasmid, which is an intracellular PtdIns (3) P fluorescent probe, was introduced into control HUVEC cells and C2α knockdown HUVEC cells by Amaxa electroporation method (Lonza), respectively, and living cells It is a figure which shows the result of having observed PtdIns (3) P localization in A with time using a real-time confocal laser microscope (made by Yokogawa Electric Corporation-Olympus). In an Example, it is a figure which shows the result of having observed the C2 (alpha) knockdown HUVEC cell by the immunofluorescence dyeing | staining method using the anti-trans Golgi network (TGN) antibody (BD company). In an Example, it is a figure which shows the result of having investigated the localization of adhesion | attachment molecule | numerator VE-cadherin between endothelial cells about the C2 (alpha) knockdown HUVEC cell by the immunofluorescence dyeing method using an anti-VE-cadherin antibody. In an Example, it is a figure which shows the result observed by the immunofluorescence dyeing method using the anti- GTP-RhoA antibody about the C2 (alpha) knockdown cell. In an Example, it is a figure which shows the result of having examined the blood-vessel permeability in a C2 (alpha) hetero KO mouse | mouth by applying the Miles assay method using the Evans blue pigment | dye with respect to the C2 (alpha) hetero KO mouse | mouth. In an Example, it is a graph which shows the change of the hematocrit value in each mouse | mouth when PAF was administered with respect to a KO mouse | mouth and a wild type mouse | mouth, and the anaphylactic shock was induced | guided | derived. In Examples, the survival rate of each mouse when angiotensin II was administered to KO mice and wild-type mice to induce aneurysms, the results of observation of the aorta of each mouse, and the aortic aneurysm in KO mice It is a figure which shows the observation result.

  Hereinafter, specific embodiments for carrying out the present invention will be described in detail, but the technical scope of the present invention is not limited to the following specific embodiments.

[PI3K-C2α gene]
Among the phosphatidylinositol 3-kinase (PI3K) family, genes encoding C2α isoforms belonging to class II (hereinafter also simply referred to as “C2α”) (in this application, collectively referred to as “PI3K-C2α gene”) are publicly known It is. For example, a PI3K-C2α gene (gene name: Pik3c2a) encoding a C2α protein in a mouse (Mus musculus) is located on mouse chromosome 7 (Location: 7 F1; 7 53.0 cM), and from this gene, 8042 bp Pik3c2a mRNA (Refseq Accession No. NM_011083.2) consisting of (33 exons) is transcribed. This Pik3c2a mRNA encodes a protein consisting of 1686 amino acids (mouse C2α protein; RefSeq Accession No. NP — 05213.2). Similarly, in humans (Homo sapiens), the PI3K-C2α gene (gene name: PIK3C2A) encoding the C2α protein is located on the short arm of human chromosome 11 (Location: 11p15.5-p14). , 8304 bp (32 exons) of PIK3C2A mRNA (Refseq Access No. NM_002645.2N) is transcribed. This PIK3C2A mRNA encodes a protein consisting of 1686 amino acids (human C2α protein; RefSeq Accession No. NP — 002636.2).

  In practicing the present invention, the PI3K-C2α gene can be obtained from a genomic library such as mouse or rat. For example, a target clone can be obtained from a bacterial artificial chromosome (BAC) library by a hybridization method. It is also possible to obtain the target clone by the PCR method.

[Non-human knockout animals]
The present invention provides non-human knockout animals that have lost the function of all or part of the PI3K-C2α gene on homologous chromosomes and exhibit diseases or symptoms associated with angiogenesis and / or angiogenesis. The present invention also provides a non-human knockout exhibiting a disease or symptom associated with angiogenesis and / or angiogenesis, including the step of losing the function of all or part of the PI3K-C2α gene on the homologous chromosome (knockout step). A method for producing an animal is provided. Examples of the technique for losing the function of all or part of the PI3K-C2α gene on the homologous chromosome include, for example, by disruption or mutation of the PI3K-C2α gene.

  In addition, in the present invention, “to lose the function of a gene” means in addition to the meaning “to completely lose the function of a gene”, “a state in which the function of the gene is reduced compared to the wild type” It is a concept that also includes the meaning of “to do”. In order to lose the function of a gene, the gene is simply destroyed or deleted, or a mutation is introduced into the gene so that the reading frame (ORF) is shifted at the translation stage. It may be modified.

  The “knockout animal” of the present invention can be prepared, for example, by the following method. In the following description, the case where the non-human animal used for producing the knockout animal is a mouse will be described as an example. However, it goes without saying that the same technique can be applied to other non-human animals.

  First, a modification of part or all of the base sequence of the PI3K-C2α gene is introduced into a totipotent cell, and a totipotent cell into which the modified PI3K-C2α gene has been introduced is selected. Subsequently, a tomatopotent cell having undergone the selected genetic modification (deletion, destruction, mutation, etc.) is introduced into a fertilized egg to produce a chimeric individual, and the resulting chimeric individual is mated to produce a homologous chromosome. An individual is created in which one or both PI3K-C2α genes are knocked out.

  The kind of animal used for this invention is not specifically limited. For example, non-human animals such as mice, rats, guinea pigs, rabbits, chickens, pigs, sheep, goats, dogs, cows, monkeys, chimpanzees and zebrafish can be used to create knockout animals. Of these, mice that are easy to handle and easy to breed are preferred.

  In order to lose the function of the PI3K-C2α gene in the knockout step, a part or all of the base sequence of the PI3K-C2α gene may be modified. Here, “modification” refers to adding a mutation that causes deletion, substitution, or addition to the DNA constituting the PI3K-C2α gene. Examples of these mutations include deletion (deletion) of part or all of the base sequence by genetic engineering techniques, or insertion or substitution of another gene or base sequence. For example, a PI3K-C2α-deficient gene can be prepared by shifting the codon reading frame (ORF) or disrupting the function of the promoter or exon. As a result, the function of the C2α protein, which is the expression product of the PI3K-C2α gene, is not expressed.

  The non-human knockout animal of the present invention can be produced by a known gene recombination method (gene targeting method). The gene targeting method is a well-known technique in this field, and its specific operation is disclosed in various experimental documents in this field.

  In designing the targeting vector, the site that causes a change in the structure of the PI3K-C2α gene is not particularly limited as long as the function of the PI3K-C2α gene is deleted. However, from the viewpoint of completely deleting the expression / function of the C2α protein, the knockout step includes replacing the region containing the start codon of exon 1 of the PI3K-C2α gene with another base sequence by homologous recombination. It is preferable. According to such a technique, mRNA encoding C2α protein is not transcribed at all, and the expression of C2α protein can be completely suppressed. However, the present invention is not limited to such a method. For example, an exon encoding an enzyme active site or activity regulatory site of C2α protein is deleted, and C2α protein that has lost enzyme activity or has reduced enzyme activity is expressed. The technique of making it also can be adopted.

  A recombinant into which a targeting vector has been introduced is preferably selected using a combination of screening using a drug resistance gene introduced by the targeting vector and screening using Southern blotting or PCR. As a marker gene for drug selection, neomycin resistance gene, hygromycin B phosphotransferase gene and the like can be used. Moreover, as a gene for negative selection, a diphtheria toxin A (DT-A) gene, an HSV thymidine kinase gene, etc. can be used.

  Subsequently, homologous recombination is performed using the targeting vector produced by the above method. In the present application, “homologous recombination” means that a modified PI3K-C2α gene is artificially recombined with the DNA region of the PI3K-C2α gene in the genome.

  In order to obtain the desired homologous recombinant, it is necessary to screen a large number of recombinants. However, it is technically difficult to perform a large number of screenings with fertilized eggs. Therefore, it is preferable to use cells that have pluripotency and can be cultured in vitro as in the case of a fertilized egg. Such totipotent cells include mice (Nature 292: 154-156, 1981), rats (Iannaccone, PM et al, Dev. Biol. 163 (1): 288-292, 1994), monkeys (Thomson, JA et al, Proc. Natl. Acad. Sci. USA 92 (17): 7844-7848, 1995), rabbit (Schoonjans, L. et al, Mol. Reprod. Dev. 45 (4): 439-443, 1996) ), Embryonic stem (ES) cells have been established. In addition, embryonic germ (EG) cells have been established for pigs (Shim H. et al, Biol. Reprod 57 (5): 1089-1095, 1997).

  Therefore, in the present invention, it is preferable to produce knockout animals for these animal species, but in particular, mice that have techniques for producing knockout animals are optimal. As mouse ES cells, several ES cell lines derived from mice have been established, for example, 129 / Ola E14-1 cell line, TT-2 cell line, AB-1 cell line, J1 cell line, R1 cell line Etc. can be used. Which ES cell line to use can be appropriately determined according to the purpose or technique of the experiment. When ES cells are established, blastocysts on the 3.5th day after fertilization are generally used, but in addition to this, 8-cell embryos are collected and cultured to blastocysts for efficient use. A large number of early embryos can be obtained. Although the ES cell line obtained in this way is usually very proliferative, it tends to lose its regenerative ability, so it needs to be subcultured carefully. For example, on a suitable feeder cell such as STO fibroblast, in the presence of leukemia inhibitory factor (LIF) (1 to 10,000 U / mL) in a carbon dioxide incubator (preferably 5% carbon dioxide / 95 % Air or 5% oxygen / 5% carbon dioxide gas / 90% air) at about 37 ° C., and at the time of passage, for example, it is made into single cells by treatment with trypsin / EDTA solution and seeded on newly prepared feeder cells. A method etc. may be adopted. Such passage is usually performed every 1 to 3 days, and it is preferable to observe the morphology of the cells.

  Gene introduction into ES cells can be performed by methods such as electroporation, calcium phosphate coprecipitation, lipofection, retrovirus infection, aggregation, microinjection, and particle gun. Among these, the electroporation method is preferably used in that a large number of cells can be easily treated.

  Next, the obtained recombinant ES cell is screened for homologous recombination to select a recombinant clone in which homologous recombination has occurred. Specifically, for example, when a neomycin resistance gene is introduced as a marker gene, a recombinant clone can be selected by culturing at about 37 ° C. in a medium containing an antibiotic such as G418.

  Further, for the obtained recombinant ES cells, Southern blot analysis using the DNA sequence on or near the PI3K-C2α gene as a probe, or the DNA sequence on the targeting vector and the PI3K- derived from the mouse used for the targeting vector By performing a PCR method using a DNA sequence in a neighboring region other than the C2α gene as a primer, it can be reliably screened whether or not homologous recombination has occurred.

  By the assay as described above, homologous recombination occurred correctly between the wild-type PI3K-C2α gene present on the chromosome and the introduced PI3K-C2α gene fragment, and the mutation was introduced into the PI3K-C2α gene on the chromosome. Recombinant clones can be selected.

  Subsequently, the ES cell in which the integration of the transgene is confirmed is returned into an embryo derived from a non-human animal of the same species, thereby forming a chimeric embryo integrated into the cell mass of the host embryo. As methods for introducing ES cells into embryos such as blastocysts, methods such as the microinjection method and the aggregation method are known, but any method can be used and can be selected as appropriate.

  In the case of mice, superovulation treatment is performed with hormone agents (for example, PSG (pregnant mare's serum gonadotropin) having FSH (follicle stimulating hormone) -like action and hCG (human chorionic gonadotropin) having LH (loteinizing hormone) action). The female mice subjected to are mated with male mice. Thereafter, early embryos are collected from the uterus on day 3.5 after fertilization when using blastocysts and on day 2.5 when using 8-cell stage embryos. The embryos thus recovered are injected in vitro with ES cells that have undergone homologous recombination using a targeting vector to produce chimeric embryos. Alternatively, the zona pellucida of the mouse 2-day embryo (8-cell stage embryo) is removed and cultured with ES cells to form aggregates. When the aggregate is cultured for one day, it becomes a blastocyst. A chimeric mouse can be obtained by transplanting these to a temporary parent and generating and growing them. A pseudopregnant female mouse for use as a foster parent can be obtained by mating a female mouse with 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 method into the uterus to the produced pseudopregnant mouse and allowing it to become pregnant / deliver. In order to ensure the implantation and pregnancy of the chimeric embryo, the female mouse that collects the fertilized egg and the pseudopregnant mouse that becomes the foster parent should be created from a group of female mice in the same sexual cycle. preferable.

  Then, a chimeric mouse is selected from the offspring born from the temporary parent. Once a mouse individual derived from an ES cell-transplanted embryo is obtained, this chimeric mouse is mated with a wild-type mouse, and it is confirmed whether or not an ES cell-derived trait appears in the next-generation individual. If a trait derived from an ES cell appears in a next generation individual, it can be considered that the ES cell has been introduced into the germ line of a chimeric mouse. In order to confirm that the ES cell has been introduced into the germ line, various traits can be used as an index. However, in consideration of ease of confirmation, it is desirable to use a coat color as an index. In mice, hair colors such as wild mouse color (agouti color), black color, ocher color, chocolate color and white color are known. It is also possible to perform selection by extracting chromosomal DNA from a part of the body (for example, the tip of the tail) and performing Southern blot analysis or PCR. Mice with high chimera contribution rates are likely to have ES cells introduced into the chimeric mouse germline. After selecting a chimeric mouse as described above, a mutant mouse strain can be established by crossing the chimeric mouse with a wild-type female to obtain F1.

  The chimeric animal obtained by the above method is obtained as a heterozygote having a gene deletion only in one of the homologous chromosomes. In order to obtain a knockout animal that is a homozygote in which both PI3K-C2α genes on the homologous chromosome are deleted, it is only necessary to cross heterozygotes having a gene defect only in one of the homologous chromosomes in the F1 animal.

  Confirmation that a knockout animal has been obtained can be performed by extracting chromosomal DNA from the tissue and performing Southern blot analysis or PCR. It can also be confirmed by observing abnormalities in various tissues and organs during dissection. Furthermore, it can be confirmed by extracting RNA from the tissue and analyzing the gene expression pattern by Northern blot analysis. Furthermore, blood can be collected as necessary and confirmed by performing a blood test or a serum biochemical test.

  Here, when a knockout animal that is a homozygote is produced, it may not be suitable as a model animal without growing to an adult, such as fetal lethality. From such a viewpoint, considering the case where the homozygote leads to embryonic lethality, the non-human knockout animal of the present invention is preferably a heterozygote.

  In addition, when the homozygote leads to embryonic lethality, it is preferable to knock out the PI3K-C2α gene only at a necessary time. Further, in order to examine the function of a gene in a specific tissue in vivo, it is preferable to knock out the gene in a tissue-specific manner. An animal knocked out only at a specific time and a specific cell line, and an animal knocked out only in a limited area of somatic cells are referred to as a conditional knockout animal (Bio-Manual Series 8, Gene Targeting: ES cells). Of a mutant mouse using Satoshi, Shinichi Aizawa, Yodosha, 1995).

  For example, Cre-loxP system (R. Kuhn. Et al., Science 269: 1427-1429) is a recombination system derived from bacteriophage P1 for gene targeting. 1995) 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, by inserting the gene to be targeted between the loxP sequence and the loxP sequence, and by incorporating the Cre recombinase gene downstream of the specific promoter, the site-specific and time-specific Cre is produced and the gene sandwiched by loxP It can be cut out (that is, the function of the target gene is lost at a specific site and time). In the present invention, when designing a targeting vector using the Cre-loxP system, the site that causes a change in the structure of the PI3K-C2α gene is not particularly limited as long as the function of the PI3K-C2α gene is deleted. However, as described above, it is particularly preferable to design so that the region containing the start codon of exon 1 of the PI3K-C2α gene is deleted.

  Progeny animals obtained by further mating the knockout animal of the present invention thus obtained, and tissues or cells isolated from these animals are also included in the technical scope of the present invention. Tissues isolated from knockout animals include all tissues, and are preferably blood vessel-derived tissues. Examples of cells isolated from knockout animals include cells isolated from the above tissues and cell lines established from these cells. Specifically, vascular endothelial cells, vascular smooth muscle cells, pericytes, blood vessels Endothelial progenitor cells, bone marrow-derived mesenchymal cells and the like are preferable. These tissues and cells can be isolated from knockout animals by methods well known to those skilled in the art.

  Non-human knockout animals provided by the present invention exhibit diseases or symptoms associated with angiogenesis and / or angiogenesis. Therefore, the non-human knockout animal of the present invention can be used as a model animal that can greatly contribute to the development of medicines for treating and / or preventing the above-mentioned diseases or symptoms.

  In the present invention, “diseases or symptoms associated with angiogenesis and / or angiogenesis” include aortic aneurysm (especially dissecting aortic aneurysm), arteriosclerosis, edema, anaphylactic shock, intimal thickening, vascular occlusion, Examples include vasculitis, tumor angiogenesis, post-ischemic angiogenesis, hypervascular pulmonary disorder, diabetic retinopathy, and the like, and these diseases or symptoms may be manifested alone or in combination.

  In addition, in the non-human knockout animal provided by the present invention or a progeny animal thereof, particularly in a hetero knockout body, the above-mentioned diseases / symptoms may not be sufficiently exhibited. Further, simply losing the function of the PI3K-C2α gene does not necessarily exhibit the desired disease / symptom. Accordingly, in some cases, a step of administering factors related to angiogenesis and / or angiogenesis to a non-human animal may be performed after the knockout step. By doing in this way, it becomes possible to fully express the above-mentioned disease / symptom also in a hetero knockout body. It is also possible to develop a desired disease / symptom by selecting a factor to be administered.

  Factors administered in this step include, for example, platelet activating factor (PAF), histamine, thrombin (above, anaphylactic shock-like pathogenic factor), fibroblast growth factor (FGF). Factor), vascular endothelial growth factor (VEGF; Vascular Endothelial Growth Factor), angiopoietin-1 / 2 (Angiopoietin-1 / 2), hepatocyte growth factor (HGF), platelet derived Examples include growth factor (PDGF), angiotensin II (aortic aneurysm inducing factor), cobalt chloride (ischemia inducing factor), streptozotocin (diabetes inducing factor), bradykinin (vascular permeability enhancing factor) and the like.

[Screening method]
Treatment of diseases or conditions associated with angiogenesis and / or angiogenesis and / or by using non-human knockout animals provided by the present invention or their progeny animals or tissues or cells isolated from these animals Candidate substances that can be used for prevention can be screened.

According to the present invention, for example, the following steps (a) to (c):
(A) a step of administering a test substance to the non-human knockout animal of the present invention or a progeny animal thereof;
(B) examining vascular tissue in an animal to which the test substance is administered and comparing with vascular tissue in an animal to which the test substance is not administered; and
(C) selecting a test substance capable of treating a disease or symptom associated with angiogenesis and / or angiogenesis based on the comparison result in step (b),
A method for screening a candidate substance for treating a disease or symptom associated with angiogenesis and / or angiogenesis (hereinafter also referred to as “screening method (1)”) is provided.

  In the step (a), the “non-human knockout animal or its progeny animal” is a concept including not only the whole body of the animal body but also a limited tissue or organ. Limited tissues or organs may include those removed from animals.

  In the step (a), the “test substance” may be any known substance or new substance, and is prepared using, for example, nucleic acid, carbohydrate, lipid, protein, peptide, low-molecular-weight organic compound, combinatorial chemistry technique. In addition to the prepared compound libraries, random peptide libraries prepared by solid phase synthesis or phage display methods, natural components derived from microorganisms, animals and plants, marine organisms, and the like can be mentioned. A mixture of two or more of these compounds can also be used as a test substance. These test substances may form a salt, and as a salt of the test substance, a salt with a physiologically acceptable acid (eg, inorganic acid) or a base (eg, organic acid) is used. In particular, physiologically acceptable acid addition salts are preferred. 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.

  In the step (a), the method for administering the test substance to the animal is not particularly limited, and it can be administered orally or parenterally. Examples of parenteral administration routes include systemic administration such as intravenous, intraarterial, and intramuscular, or local administration in the vicinity of target cells.

  The dose of the test substance in step (a) is appropriately set according to the type of active ingredient, the size of the molecule, the route of administration, the animal species to be administered, the drug acceptability of the administration subject, body weight, age, etc. sell.

  In the step (b), as a method for examining the vascular tissue in the animal administered with the test substance, for example, a method of staining the vascular tissue and observing with a microscope; expression of a specific target protein by Western blotting or immunoprecipitation Examples thereof include a confirmation method, a method for confirming the expression of a specific gene at the mRNA level by RT-PCR, real-time PCR, and the like, and a method for comparing the survival times of mice.

  In step (b), the vascular tissue in the animal to which the test substance is not administered is also examined simultaneously or separately, and the result of the administered animal is compared with the result of the non-administered animal.

  In step (c), based on the comparison result obtained in step (b), a test substance capable of treating a disease or symptom associated with angiogenesis and / or angiogenesis is selected. The criteria for selection can be appropriately set using as an index an animal that is not administered the test substance.

  The test substance thus selected is capable of treating a disease or symptom associated with angiogenesis and / or angiogenesis, and can be a candidate substance for a therapeutic agent for various of the above diseases / symptoms.

According to the present invention, the following steps (a) to (c):
(A) a step of administering a test substance to the non-human knockout animal of the present invention or a progeny animal thereof;
(B) examining vascular tissue in an animal to which the test substance is administered and comparing with vascular tissue in an animal to which the test substance is not administered; and
(C) selecting a test substance that can prevent or delay the onset of a disease or symptom related to angiogenesis and / or angiogenesis based on the comparison result in step (b);
A method for screening a candidate substance for prevention of a disease or symptom associated with angiogenesis and / or angiogenesis (hereinafter also referred to as “screening method (2)”).

  In the screening method (2), steps (a) to (b) are the same as the screening method (1) described above. However, the time when the non-human knockout animal of the present invention develops a disease or symptom related to angiogenesis and / or angiogenesis is examined in advance, and the above-mentioned disease / symptom in the knockout animal of the present invention in which the test substance is not administered. It is important to examine the vascular tissue after the administration of the test substance until the expression can be confirmed.

  In step (c) of screening method (2), based on the comparison result obtained in step (b), the disease or symptom related to angiogenesis and / or angiogenesis is not expressed or expressed. Select a test substance that can delay The criteria for selection can be appropriately set using as an index an animal that is not administered the test substance.

  The test substance thus selected is one that can prevent or delay the onset of a disease or symptom related to angiogenesis and / or angiogenesis. Can be a candidate substance for preventive drugs.

Furthermore, according to the present invention, the following steps (a) to (c):
(A) a step of collecting blood from each of the non-human knockout animal of the present invention or its progeny animal and the same-type wild-type animal of the animal;
(B) examining the protein in the blood obtained in step (a) and comparing the expression pattern of the protein in each animal; and
(C) based on the comparison result in step (b), selecting a serum protein that serves as an indicator of the development of a disease or condition associated with angiogenesis and / or angiogenesis;
A method for screening a biomarker for a disease or symptom associated with angiogenesis and / or angiogenesis (hereinafter also referred to as “screening method (3)”) is provided.

  In step (a) of screening method (3), blood can be collected from the animal by a method known per se. The amount of blood collected can be set to an appropriate amount depending on the type of animal. It is preferable to prepare serum from the collected blood by centrifugation or the like and use it in the step (b). In addition, the time for collecting blood is examined in advance for the time when the disease or symptom related to angiogenesis and / or angiogenesis is observed in the non-human knockout animal of the present invention. It is preferable to select a plurality of periods before and after the expression.

  In the step (b) of the screening method (3), the method for analyzing and comparing the expression pattern of the protein in the blood is not particularly limited and can be performed by a conventional method. For example, a method in which the blood proteins of non-human knockout animals and allogeneic wild-type animals are compared using methods such as peptide mass fingerprinting, and proteins that significantly increase or decrease in knockout animals are screened. Is done.

  In step (c) of screening method (3), based on the comparison results obtained in step (b), a serum protein that serves as an index of the expression of a disease or symptom related to angiogenesis and / or angiogenesis is selected. To do. Criteria to be selected can be used as an indicator that, among serum proteins, expression is significantly increased (preferably twice or more) in non-human knockout animals as compared to homologous wild-type animals. .

  The serum protein thus selected is useful as a biomarker for determining the presence or absence of a disease or symptom associated with angiogenesis and / or angiogenesis.

  EXAMPLES Hereinafter, although this invention is demonstrated in detail using an Example, the technical scope of this invention is not necessarily restrict | limited only to the form as described in the following Example.

[Preparation of knockout (KO) mouse: FIG. 1]
A knockout (KO) mouse in which the PI3K-C2α ( Pik3c2a ) gene was disrupted on a homologous chromosome was produced by the following procedure.

First, a 1.2 kbp DNA fragment was cloned from the 129 / Ola E14-1 mouse embryonic stem (ES) cell genome by PCR. This fragment has exon 1 which contains the translation initiation codon (ATG) of the mouse PI3K-C2α ( Pik3c2a ) gene.

The targeting vector was constructed according to a conventional method. At that time, the targeting vector was designed so as to incorporate the Cre-loxP system so as to be able to cope with the conditioning (conditional) KO mouse production. Specifically, based on a commercially available pBluescript-II vector (Invitrogen), an upstream intron region (5.6 kbp) of exon 1 of mouse PI3K-C2α ( Pik3c2a ) gene, bacteriophage-derived loxP sequence, exon of C2α gene 1 region, loxP sequence, neomycin resistance gene (Neo) cassette for positive selection, loxP sequence, downstream intron region of exon 1 of C2α gene (1.2 kbp), DT-A gene cassette for negative selection are appropriately linked Thus, a targeting vector was obtained.

The obtained targeting vector (20 μg) was linearized with SacII and introduced into 129 / Ola E14-1 mouse ES cells (1.25 × 10 ⁷ cells) by electroporation. The transfected cells were cultured at 37 ° C. in a medium for ES cells (15% fetal bovine serum (FBS) -DMEM) containing the antibiotic G418 (200 μg / mL), and recombinant clones were selected.

  The selected 450 G418-resistant clones were expanded and subjected to PCR under the following conditions to identify early mutant recombinant clones. That is, using a forward primer (F primer shown in Table 1 below) for the 5 ′ end region of the Neo cassette and a reverse primer (R primer shown in Table 1 below) for the 3 ′ end side sequence outside the homologous recombination region. In a PCR reaction (DNA polymerase: LA-Taq (Takara Bio Inc.)) in which 35 cycles of “94 ° C. 30 seconds, 62 ° C. 30 seconds, 72 ° C. 2 minutes 30 seconds” are performed, only a fragment derived from a homologous recombinant of 2 kbp is obtained. Amplified.

  An early mutant ES cell appropriately homologously recombined was infected with an adenovirus expressing Cre recombinase to obtain a knockout (KO) type recombinant ES cell lacking exon 1 and Neo expression cassette. The obtained KO type recombinant ES cells were identified by PCR under the following conditions. That is, the forward primer for the region in exon 1 of the C2α gene (F1 primer (SEQ ID NO: 3) shown in Table 2 below), the reverse primer for the region in the downstream intron of exon 1 of the C2α gene (in Table 2 below) R1 primer (SEQ ID NO: 4)) and a forward primer for the upstream Cre sequence of exon 1 of the C2α gene (F2 primer (SEQ ID NO: 5) shown in Table 2 below), “94 ° C. for 30 seconds, A 278 bp knockout fragment was amplified by PCR reaction (DNA polymerase: Go-Taq (Promega)) in which 35 cycles of “60 ° C. for 30 seconds and 72 ° C. for 30 seconds” were performed. On the other hand, since the exon 1 region of the C2α gene no longer exists in the early mutant ES cells, an amplification product (432 bp) that should be defined by the F1 primer and the R1 primer was not observed.

  Using the KO-type recombinant ES cells obtained above, chimeric embryos were obtained by the cell assembly (aggregation) method with 8-cell embryos obtained from C57BL / 6J mice (Jackson laboratories). The obtained chimeric embryo was implanted into a C57BL / 6J female female parental mouse to generate a chimeric mouse. By crossing a chimeric mouse male and a wild type C57BL / 6J mouse female, as a first generation (F1), a C2α heterozygous (+ / -) KO mice were obtained.

  For each fetal mouse and adult mouse, the genotype was identified using the three PCR primers (F1, R1, F2) (SEQ ID NOs: 3 to 5) shown in Table 2 above. In PCR using the set of F1 primer and R1 primer, a 432 bp C2α wild type band was detected. On the other hand, in the PCR using the F2 primer and the R1 primer, a 278 bp KO type band was amplified.

[Preparation of C2α systemic homo (− / −) KO mice]
Male and female of the first generation (F1) C2α hetero KO mice obtained above were mated to obtain a second generation (F2). Of the second generation, C2α hetero (+/−) KO mice and wild type (+ / +) mice were born normally. On the other hand, C2α homo (− / −) KO mice were embryonic lethal. Heterogeneous KO mice under normal breeding showed no difference in appearance, body weight, life span, or histopathological findings as compared with the wild type.

[Abnormalities of embryonic angiogenesis in C2α systemic homo-KO mice]
For each of the C2α systemic homo KO mice, C2α hetero KO mice and wild type mice obtained above, Western blot analysis was performed on fetal tissue samples using anti-C2α antibody (BD). As a result, no C2α protein band could be detected in the homo KO mice. In addition, it was confirmed that the expression level of C2α protein was about 50% in the hetero KO mouse compared to the wild type.

  Tissues of fetuses (embryonic day 11.5) of littermate C2α homo-KO mice and wild type mice were fixed with 4% paraformaldehyde solution. Subsequently, whole mount staining (DAB coloring method) was performed using an anti-CD31 antibody which is a vascular endothelial cell marker. The results are shown in FIG. As shown in FIG. 2, it was confirmed that the vascular network was constructed throughout the body in the wild type mouse. On the other hand, in the homo KO mouse, a remarkable abnormality in angiogenesis was observed.

  For each of C2α homo-KO mice and wild-type mice, the fetus was tissue fixed with 4% paraformaldehyde solution, embedded in paraffin, sliced, and the tissue section was immunized with anti-α smooth muscle actin antibody, which is a vascular smooth muscle marker. Chemical staining (DAB coloring method) was performed. The results are shown in FIG. As shown in FIG. 3, in the wild-type mouse, α-smooth muscle actin-positive cells were accumulated in the dorsal aorta so as to surround the blood vessels. On the other hand, it was hardly observed in homo KO mice.

  Furthermore, when analyzed in more detail using an electron microscope, in C2α homo-KO mice, abnormalities in the adhesion structure between endothelial cells were observed in addition to the failure of vascular smooth muscle cell accumulation (FIG. 4, arrowhead).

[Generation of tissue-specific C2a conditional knockout (CKO) mice: FIG. 5]
In order to analyze the cause of death of systemic C2α homo-KO mice in which embryonic lethality was confirmed and the physiological role of C2a, tissue-specific C2α CKO mice were prepared by the following method.

  A chimeric embryo is obtained by the cell aggregation (aggregation) method with an 8-cell embryo obtained from a C57BL / 6J mouse (Jackson Laboratories) using the early mutant (3LoxP type) recombinant ES cells described in FIG. It was. The obtained chimeric embryos were implanted into C57BL / 6J female mice to generate C2α-3LoxP type heterozygous mice that are chimeric mice. In this 3LoxP type heterozygous mouse, C2α expression was equivalent to that of the wild type mouse and grew normally.

  The 3LoxP-type heterozygous mouse (8-week-old female; first generation (F1)) obtained above was mated with male Ins-Cre transgenic (Tg) mice that are weak in sperm and express Cre recombinase. Thus, a Flox-type heterozygous mutant (C2αflox / +) mouse having a structure in which the exon 1 region of the C2α gene was inserted with two loxP sequences was generated. The Flox heterozygous mutant mice were identified using the three PCR primers (F1, R1, F2) (SEQ ID NOs: 3 to 5) shown in Table 2 above. A 486 bp Flox-type band and a 432 bp wild-type band were distinguished and detected by PCR using a set of F1 primer and R1 primer.

  The Flox-type heterozygous mice obtained above developed normally and grew without any difference from the wild type. The Flox heterozygous mutant mice were backcrossed for 10 generations by crossing with C57BL / 6J, and then males and females of Flox heterozygous mutants were mated to obtain Flox homozygous mutant (C2αflox / flox) mice.

  By crossing the resulting Flox-type homozygous mouse (8 weeks old) with a tissue-specific Cre recombinase-expressing Tg mouse, a tissue-specific CKO mouse in which C2α is knocked out in a tissue-specific manner can be produced. . In this example, tissue-specific CKO mice were prepared using Tg mice that specifically express Cre recombinase in vascular endothelial cells and vascular smooth muscle cells that constitute blood vessels, and cardiomyocytes that constitute the heart. . In these tissue-specific CKO mice, the C2α gene is selectively deleted in vascular endothelial cells, vascular smooth muscle cells, and cardiomyocytes, respectively.

[Observation of tissue-specific CKO mice]
Among the tissue-specific CKO mice prepared above, only vascular endothelial cell-specific CKO mice (C2α-eCKO mice) exhibited angiogenesis abnormalities from around the embryonic day 12, and died before and after birth. On the other hand, vascular smooth muscle cell-specific CKO mice and cardiomyocyte-specific CKO mice were born and developed in the same manner as littermate wild-type mice. There was no difference in histopathological findings from wild-type mice.

  When the C2α-eCKO mice were observed in more detail, bleeding from the skin blood vessels was observed around embryonic day 12.5 (FIGS. 6, ii, iv, vi, arrowheads). Further, the fetus of C2α-eCKO mouse was formalin-fixed and the paraffin sections were stained with hematoxylin and eosin. As a result, bleeding was observed in organs such as lung, liver and kidney in addition to skin blood vessels. In order to observe bleeding blood vessels with high resolution, mouse fetal skin was peeled off and fixed with 4% paraformaldehyde, and anti-CD31 antibody as a vascular endothelial cell marker and anti-vascular smooth muscle cell marker as an anti-vascular marker. Double fluorescence immuno whole mount staining with α-smooth muscle actin antibody was performed. As a result of observing the skin blood vessels of the C2α-eCKO mice using a high-resolution confocal laser microscope, the formation of incomplete microvascular networks was observed compared with the wild-type mice, and the blood vessels were smooth. Significant abnormalities were also observed in muscle accumulation (FIGS. 7, ii, iv). Furthermore, when the skin capillaries were observed by a high-resolution double fluorescent immuno-hole mount staining method (anti-VE cadherin antibody + anti-NG2 antibody double staining), localization of VE cadherin, which is an adhesion factor between endothelial cells, and pericytes were observed. An abnormality in the morphology was observed.

[In vitro analysis using C2α expression-inhibited vascular endothelial cells]
Using an RNA interference method, an in vitro experimental system capable of specifically suppressing C2α expression in human umbilical vein endothelial cells (HUVEC) was constructed.

  Specifically, using a commercially available interfering RNA synthesis kit (Ambion), an interfering RNA (C2α) targeting the sequence (5′-AAGGUUGGCACUACAAGAAU-3 ′ (SEQ ID NO: 6)) on the mRNA of the human C2α gene. -SiRNA) was synthesized. This synthetic C2α-siRNA (20 nM) was introduced into HUVEC cells using a commercially available gene introduction reagent Lipofectamine 2000 (Invitrogen) to construct C2α knockdown HUVEC cells. The C2α knockdown HUVEC cells were normally cultured for 48 hours, and then the C2α protein expression level was examined by Western blotting using an anti-C2α antibody. As a result, in the HUVEC cells into which C2α-siRNA was introduced, the expression level of C2α protein was suppressed by about 95% compared to HUVEC cells into which control interference RNA (scrambled sequence: sc-siRNA) was introduced (control HUVEC cells). It was.

  In C2α knockdown HUVEC cells, cell proliferation, cell adhesion and cell migration ability were significantly attenuated compared to control HUVEC cells. Furthermore, when the ability of VEGF to form a lumen was evaluated using the Matrigel assay, which is an in vitro angiogenesis evaluation method, the lumen length and the number of lumen branch points were significantly increased in C2α knockdown HUVEC cells. It decreased (FIG. 8).

  Here, it is known that C2α generates phosphatidylinositol (3) phosphate (PtdIns (3) P) on intracellular endosomes and trans-Golgi network (TGN). This PtdIns (3) P is known to be deeply involved in membrane transport in endosomes and TGNs. Endosomes transport newly produced proteins from the Golgi apparatus to cell membranes and other organelles, transport proteins to the lysosomal degradation system via endocytosis from the cell membrane, and to the cell membrane by recycling. It is known to function as a relay point for intracellular membrane transport, such as being involved in exocytosis responsible for the re-transport of hormones and the release of hormones.

  Therefore, mRFP-2XFYVE domain expression plasmids, which are intracellular PtdIns (3) P fluorescent probes, were introduced into control HUVEC cells and C2α knockdown HUVEC cells by Amaxa electroporation (Lonza), respectively, and PtdIns in living cells. (3) P localization was observed over time using a real-time confocal laser microscope (manufactured by Yokogawa Electric Corporation-Olympus). Further, as a positive control, a similar experiment was performed using HUVEC cells in which p110α (class I) or Vps34 (class III), which are PI3K families other than C2α, were knocked down. The results are shown in FIG. As shown in FIG. 9, a significant decrease in PtdIns (3) P positive endosomes was observed in C2α knockdown HUVEC cells. On the other hand, no changes were observed in control HUVEC cells or p110α (class I) or Vps34 (class III) knockdown HUVEC cells.

[Abnormal intracellular morphology of C2α expression-inhibited endothelial cells]
C2α knockdown HUVEC cells were fixed with 4% paraformaldehyde and then observed by immunofluorescence staining using an anti-trans Golgi network (TGN) antibody (BD). The results are shown in FIG. As shown in the upper part of FIG. 10, expansion of the TGN structure was observed in C2α knockdown HUVEC cells as compared to control HUVEC cells. In addition, since the knockdown HUVEC cells of p110α (class I) or Vps34 (class III) showed the same structure as the control HUVEC cells, it was considered that the structural change specifically occurred only in the C2α knockdown HUVEC cells. It is. This structural change of TGN was also observed in the detailed observation by electron microscope observation (FIG. 10, arrowhead). PtdIns (3) P on endosomes and TGN is an important lipid in the intracellular vesicle transport system, but in the cells where C2α expression is suppressed, the transport system between TGN and cell membrane via endosome is markedly inhibited. As a result, it is considered that the TGN structure is disturbed.

[VE cadherin localization abnormality in C2α knockdown HUVEC cells]
The HUVEC cells fixed with 4% paraformaldehyde were examined for the localization of the intercellular endothelial adhesion molecule VE-cadherin by immunofluorescence staining using an anti-VE-cadherin antibody. As a result, in C2α knockdown cells, localization of VE-cadherin among endothelial cells was remarkably impaired, and many cell gaps were observed (FIG. 11). As will be described later, vascular permeability was actually significantly enhanced in C2α knockdown cells.

  RhoA, which is a low molecular weight G protein, is known to play an important role in the formation and remodeling of actin fibers (F-actin) essential for constructing the cytoskeleton. From the observation by immunofluorescence staining using an anti-GTP-RhoA antibody recognizing active RhoA (GTP type-RhoA), activation of RhoA observed only under C2α knockdown cells under normal culture conditions (+ EBM2) Was significantly suppressed (FIG. 12, second photo from the left). In addition, it was confirmed by Alexa594-phalloidin staining that labeled F-actin that the morphology of actin bundles important for maintaining cell-cell adhesion was observed.

  It was found that the decrease in RhoA activity observed in C2α knockdown cells was due to attenuation of geranylation, which is a modification reaction essential for RhoA activation. Thus, when the inactive RhoA mutant was forcibly expressed in HUVEC cells, the intercellular localization of VE-cadherin completely disappeared. On the other hand, in C2α knockdown cells, the intercellular localization of VE-cadherin was partially restored by the introduction of activated RhoA mutant. Furthermore, by introducing an inactive C2α mutant (a 1268th aspartic acid to alanine point-mutated → mutant that does not bind ATP and loses phosphorylation activity) into HUVEC cells, an intercellular station of VE-cadherin I was disturbed. From these results, it was suggested that RhoA activity by C2α is involved in intercellular localization of VE-cadherin.

[Enhancement of cutaneous vascular permeability by VEGF]
As described above, C2α hetero KO mice (C2α +/−) having a C2α protein expression level of about 50% are born normally and grow normally under normal breeding. A Miles assay method using Evans Blue dye was applied to the C2α hetero KO mice to examine vascular permeability in the C2α hetero KO mice. Specifically, 50 μL of commercially available 2% Evans Blue dye (Fluka) dissolved in physiological saline was administered to the tail vein of the mouse, and 20 minutes later, commercially available VEGF (Peprotech, 100 ng / mL) was subcutaneously placed on the back of the mouse. ) 100 μL or PBS buffer 100 μL was injected as a control. Twenty minutes later, the skin containing the drug injection part on the back was excised, and Evans Blue dye was extracted overnight at 55 ° C. using formamide solution, and then the Evans Blue amount was quantified with a spectrophotometer. As a result, in the C2α hetero KO mice, it was confirmed that the leakage of Evans blue dye to the outside of the blood vessel increased about twice as much as that of the wild type mice, and an increase in vascular permeability was observed (FIG. 13).

[Anaphylactic shock model]
Anaphylactic shock is caused by histamine or platelet activating factor (PAF) released from mast cells activated by an abnormal immune reaction. As specific pathological conditions, vasodilation, leakage of plasma components to the outside of the blood vessels, and the like are observed, and as a result, blood pressure sharply decreases and various shock symptoms are exhibited.

  In order to induce this anaphylactic shock, PAF was administered at a dose of 12 μg / kg to the tail vein of C2α heterozygous KO mice or wild type mice under general anesthesia, respectively. As a result, only C2α hetero KO mice were shocked in all cases within about 35 minutes. Moreover, the hematocrit value in the C2α hetero KO mouse immediately after death was significantly higher than that in the wild type mouse (FIG. 14).

[Formation of aneurysm induced by angiotensin II]
Angiotensin II (AngII, 1.0 mg / kg / day), which is a vasoactive hormone, was systemically administered to C2α hetero KO mice continuously for 2 weeks by an osmotic pump (Alzette). As a result, formation of an aortic aneurysm was observed in about 48% of C2α hetero KO mice, and about 30% of hetero KO mice died of bleeding due to the rupture of the aortic aneurysm (the upper part of FIG. 15). On the other hand, in the wild type mouse, aortic aneurysm formation was about 11%, and the mortality rate was only 5%. Most of the aortic aneurysms observed in the hetero-KO mice were dissociative aortic aneurysms with pseudolumen (lower part of FIG. 15), and Mac3 positive macrophages were mobilized to the outer vascular membrane and inflammation was induced. Inflammatory macrophages in the extravascular membrane produce the proteolytic enzyme Matrix Metalloproteinase (MMP) and cause degradation of the extracellular matrix of the blood vessel wall. Also in the study by the present inventors, in C2α hetero KO mice, the enzyme activities of aorta MMP-2 and MMP-9 were significantly enhanced by AngII administration compared to wild type mice (not shown). ). Furthermore, in the C2α hetero KO mice administered with Ang II, abnormalities in the adhesion structure between endothelial cells composed of VE-cadherin were also observed. To support this, the observation by the Evans Blue dye method was also compared with the wild type mice. It was shown that vascular permeability was significantly enhanced.

[Discussion]
From the results of the above experiments, the physiological role of C2α in angiogenesis is as follows: 1) Regulation of intracellular membrane transport through PtdIns (3) P production in endothelial cells and TGN, 2) VE-cadherin membrane transport The involvement of two intracellular mechanisms of stabilization of VE-cadherin binding was suggested. That is, it is considered that C2α plays an essential role in “suppressing vascular permeability by tightening adhesion between endothelial cells” through the above two mechanisms.

  In embryonic angiogenesis, C2α is PI3K essential for angiogenesis such as endothelial cell branching and vascular maturation by wall cell recruitment, and is an enzyme that supports angiogenesis and homeostasis by a mechanism completely different from other PI3K families. is there. Abnormal C2α function results in abnormal vascular endothelial cell function, particularly increased vascular permeability, inflammatory reaction, and rupture of vascular structure. Many blood vessels such as aneurysm, arteriosclerosis, edema, anaphylactic shock, etc. It is likely to be involved in the development of diseases and / or symptoms associated with formation (angiogenesis and / or angiogenesis). The C2α knockout non-human animal provided by the present application is a valuable biological resource not only for basic research but also clinically, as a new model animal for grasping the pathophysiology of diseases and symptoms related to angiogenesis. Further, by using this model animal, it becomes possible to screen a candidate substance capable of treating and / or preventing the disease / symptom at an individual level.

Claims (14)

Phase on the same chromosome and lost all or part of the functions of the PI3K-C2α gene caused a disease or condition associated with angiogenesis and / or neovascularization, mice, rats, guinea pigs, rabbits, pigs, sheep A non-human knockout animal which is an animal selected from the group consisting of: goat, dog, cow, monkey and chimpanzee .   The non-human knockout animal according to claim 1, wherein the loss of the function of all or part of the PI3K-C2α gene is due to disruption or mutation of the PI3K-C2α gene.   It is a model animal for aortic aneurysm, arteriosclerosis, edema, anaphylactic shock, intimal thickening, vascular occlusion, vasculitis, tumor angiogenesis, post-ischemic neovascularization, hypervascular permeability lung disorder, or diabetic retinopathy The non-human knockout animal according to claim 1 or 2.   The non-human knockout animal according to any one of claims 1 to 3, which is a hetero knockout animal. The progeny animal of the non-human knockout animal according to any one of claims 1 to 4 . A tissue or cell isolated from the animal according to any one of claims 1 to 5 . The tissue or cell according to claim 6 , which is derived from a blood vessel. A disease or symptom associated with angiogenesis and / or angiogenesis is prevented and / or treated using the animal according to any one of claims 1 to 5 or the tissue or cell according to claim 6 or 7. A screening method for candidate substances. The following steps (a) to (c):
(A) a step of administering a test substance to the animal according to any one of claims 1 to 5 ;
(B) examining vascular tissue in an animal to which the test substance is administered and comparing with vascular tissue in an animal to which the test substance is not administered; and
(C) selecting a test substance capable of treating a disease or symptom associated with angiogenesis and / or angiogenesis based on the comparison result in step (b),
A method for screening a candidate substance for treatment of a disease or condition associated with angiogenesis and / or angiogenesis. The following steps (a) to (c):
(A) a step of administering a test substance to the animal according to any one of claims 1 to 5 ;
(B) examining vascular tissue in an animal to which the test substance is administered and comparing with vascular tissue in an animal to which the test substance is not administered; and
(C) selecting a test substance that can prevent or delay the onset of a disease or symptom related to angiogenesis and / or angiogenesis based on the comparison result in step (b);
A method for screening candidate substances for prevention of diseases or symptoms associated with angiogenesis and / or angiogenesis. The following steps (a) to (c):
(A) A step of collecting blood from each of the animal according to any one of claims 1 to 5 and the same species wild-type animal of the animal;
(B) examining the protein in the blood obtained in step (a) and comparing the expression pattern of the protein in each animal; and
(C) based on the comparison result in step (b), selecting a serum protein that serves as an indicator of the development of a disease or condition associated with angiogenesis and / or angiogenesis;
A method for screening a biomarker for a disease or condition associated with angiogenesis and / or angiogenesis. Knockout step of losing all or part of the function of the PI3K-C2α gene on the homologous chromosome of an animal selected from the group consisting of mouse, rat, guinea pig, rabbit, pig, sheep, goat, dog, cow, monkey and chimpanzee A method for producing a non-human knockout animal that exhibits a disease or condition associated with angiogenesis and / or angiogenesis. The production method according to claim 12 , wherein the knockout step comprises substituting a region containing the start codon of exon 1 of the PI3K-C2α gene of the non-human animal with another base sequence by homologous recombination. The production method according to claim 12 or 13 , further comprising a step of administering an agent associated with angiogenesis and / or angiogenesis to the non-human animal after the knockout step.